The discovery of a key prenyltransferase gene assisted by a chromosome-level Epimedium pubescens genome.

Epimedium pubescens is a species of the family Berberidaceae in the basal eudicot lineage, and a main plant source for the traditional Chinese medicine "Herba Epimedii". The current study achieved a chromosome-level genome assembly of E. pubescens with the genome size of 3.34 Gb, and the genome guided discovery of a key prenyltransferase (PT) in E. pubescens. Our comparative genomic analyses confirmed the absence of Whole Genome Triplication (WGT-γ) event shared in core eudicots and further revealed the occurrence of an ancient Whole Genome Duplication (WGD) event approximately between 66 and 81 Million Years Ago (MYA). In addition, whole genome search approach was successfully applied to identify 19 potential flavonoid PT genes and an important flavonoid PT (EpPT8) was proven to be an enzyme for the biosynthesis of medicinal compounds, icaritin and its derivatives in E. pubescens. Therefore, our results not only provide a good reference genome to conduct further molecular biological studies in Epimedium genus, but also give important clues for synthetic biology and industrial production of related prenylated flavonoids in future.

Genome-wide analysis of UGT gene family identified key gene for the biosynthesis of bioactive flavonol glycosides in Epimedium pubescens Maxim.

Epimedium pubescens Maxim. is a well-known traditional Chinese medicinal herb with flavonol glycosides as the major pharmaceutically active compounds. UDP-glycosyltransferases (UGTs) are a group of enzymes responsible for the glycosylation of flavonoid glycosides. In this study, a genome-wide analysis was performed to identify UGT family genes in E. pubescens. As a result, a total of 339 putative UGT genes were identified, which represents the largest UGT gene family known thus far, implying a significant expansion of the UGT gene family in E. pubescens. All EpUGTs were unevenly distributed across six chromosomes, and they were classified into 17 major groups. The expression profiles showed that UGT genes were differentially expressed in roots, leaves, flowers, shoots and fruits. In particular, several EpUGTs were highly induced by high light intensity, which was consistent with the accumulation level of bioactive flavonoids in E. pubescens. Six UGT79 genes that were preferentially expressed in roots or leaves were successfully expressed in E. coli, and only the recombinant EpGT60 protein was found to be active toward 8-prenylkaempferol and icaritin to produce the key bioactive compounds baohuoside II and baohuoside I. The optimal temperature, pH, k m and V max were determined for the recombinant EpGT60 protein. In addition, expression of recombinant EpGT60 in E. coli cell culture led to successful production of baohuoside II when fed 8-prenylkaempferol. Our study provides a foundation for further functional characterization of UGT genes in E. pubescens and provides key candidate genes for bioengineering bioactive flavonoids in E. pubescens.

[Content correlation of eight phenolic acids and flavonoids in different medicinal parts of PA-type Perilla germplasms].

In this study, 10 PA-type Perilla germplasms were selected to detect the content of two phenolic acids, i.e., rosmarinic acid(RA) and caffeic acid(CA), and six flavonoids, including scutellarin-7-O-diglucuronoside(SDG), luteolin-7-O-diglucuronoside(LDG), apigenin-7-O-diglucuronoside(ADG), scutellarin-7-O-glucuroside(SG), luteolin-7-O-glucuroside(LG), and apigenin-7-O-glucuroside(AG) in leaves, stems, and fruits. The total content of phenolic acids and flavonoids in leaves was 3.991-12.028 mg·g~(-1) and 12.309-25.071 mg·g~(-1), respectively, which was much higher than that in stems(0.586-2.015 mg·g~(-1) and 0.879-1.413 mg·g~(-1), respectively) and fruits(0.004-2.222 mg·g~(-1) and 0.651-1.936 mg·g~(-1), respectively). RA was detected in five fruit samples, and RA content between leaves and fruits showed a significant negative correlation in the other five samples. For flavonoids, only LG and LDG could be detected in stems, and SG and SDG were not detected in fruits, while other flavonoids were not detected in some samples. The content of total flavonoids and LG in leaves and fruits was significantly positively correlated, and the content of LG in stems and fruits was significantly positively correlated. In 10 stem samples, seven met the standard that the content of RA in the stem should be not less than 0.1% specified in the Chinese Pharmacopoeia(2020 edition). Only one fruit sample reached the standard of RA content in the fruit not less than 0.25% specified in the Chinese Pharmacopoeia.

[Contents determination of eight phenolic compounds in Perilla frutescens leaves of different cultivation years and harvesting periods].

A method was established for content determination of two kinds of phenolic acids, including rosmarinic acid)(RA) and caffeic acid(CA), and six kinds of flavonoids including scutellarein-7-O-diglucuronide(SDG), luteolin-7-O-diglucuronide(LDG), apigenin-7-O-diglucuronide(ADG), scutellarin-7-O-glucuronide(SG), luteolin-7-O-glucuronide(LG), and apigenin-7-O-glucuronide(AG) in Perilla frutescens leaves. The content of eight chemical components was measured based on ten P. frutescens germplasms of different chemotypes of volatile oil, different cultivated years, and different harvesting periods. The results showed that there was a great difference between the two kinds of constituents of different germplasms. The total content of the two phenolic acids was 2.24-34.44 mg·g~(-1), and the total content of the six flavonoids was 11.55-34.71 mg·g~(-1). Then according to content from most to least, the order of each component was RA(2.13-33.97 mg·g~(-1)), LDG(1.31-14.80 mg·g~(-1)), SG(1.97-8.45 mg·g~(-1)), ADG(2.68-7.60 mg·g~(-1)), SDG(1.16-5.87 mg·g~(-1)), LG(0.78-1.91 mg·g~(-1)), AG(0.56-1.00 mg·g~(-1)), and CA(0.11-0.68 mg·g~(-1)). The chemical contents of the 5 PA-type germplasms in 2017 were mostly higher than those in 2018 showing a large variation with the cultivation years. These contents of two kinds of phenolic acids of 9 germplasms fluctuated with the harvesting time. The content decreased before early flower spike(the 3~(rd) to 18~(th) in August) at first and began to increase in flowering and fruiting period(the 18~(th) in August to 2~(nd) in September). However, these contents had slowly decreasing trend after 2~(nd) in September till 17~(th) in the same month. Interestingly, the content raised again in the maturity of fruits. The variation tendency of contents in six kinds of flavonoids components was inconsistent in different germplasms with the variation of harvesting time. The content of flavonoids in part of germplasms was negatively correlated with the fluctuation of phenolic acids. There was no correlation between phenolic acids and chemical type of the volatile oil. This paper may provide a reference for the high-quality germplasm of P. frutescens cultivation.

The complete chloroplast genome of Epimedium wushanense T. S. Ying. (Berberidaceae), a traditional Chinese medicinal herb.

Epimedium wushanense is a well-known medicinal plant in Berberidaceae in China. In this study, we sequenced the complete chloroplast (cp) genome of E. wushanense. The results showed that the cp genome of E. wushanense was 157,283 bp in length, which is composed of a large single-copy region (LSC, 88,579 bp) and a small single-copy region (SSC, 17,082 bp) that were separated by a pair of inverted repeat regions (IRa and IRb, 25,811 bp). The chloroplast genome of E. wushanense contains 112 unique genes, of which are 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The overall GC content was 38.78%. The phylogenetic tree analysis showed that E. wushanense was closely related to E. pseudowushanense, E. lishihchenii, and E. sagittatum.

The complete chloroplast genome of Epimedium brevicornu (Berberidaceae), a traditional Chinese medicinal herb.

Epimedii Folium has been used as a common traditional Chinese medicine for more than 2000 years in China. In this study, we assembled the complete chloroplast (cp) genome of Epimedium brevicornu. The whole cp genome of E. brevicornu is 158,658 bp in length, comprising a pair of inverted repeat (IR) regions (27,699 bp) separated by a large single copy (LSC) region (86,558 bp) and a small single copy (SSC) region (16,702bp). The E. brevicornu cp genome contains 129 genes, of which 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis shows that E. brevicornu is closely clustered with E. wushanense, E. lishihchenii, and E. sagittatum. The published E. brevicornu chloroplast genome will provide useful information for the phylogenetic and evolutionary study on Epimedium family of Berberidaceae.

[Effects of light quality on growth and icariin flavonoid content of Epimedium pseudowushanense under different light intensity].

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) µmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) µmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.