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.

Main Conclusion: GbMYBR1, a new type of R2R3-MYB repressor from Ginkgo biloba, displayed pleiotropic effects on plant growth, phenylpropanoid accumulation, by regulating multiple related genes at different levels. Ginkgo biloba is a typical gymnosperm that has been thriving on earth for millions of years. MYB transcription factors (TFs) play important roles in diverse processes in plants. However, the role of MYBs remains largely unknown in Ginkgo. Here, an MYB TF gene from Ginkgo, designated as GbMYBR1, was found to act as a repressor in multiple processes. GbMYBR1 was mainly expressed in the leaves of Ginkgo. Over-expression of GbMYBR1 in Arabidopsis thaliana led to growth retardation, decreases in lignin content, reduced trichome density, and remarkable reduction in anthocyanin and flavonol contents in leaves. Proanthocyanidin content was decreased in the seeds of transgenic Arabidopsis, which led to light-brown seed color. Both qPCR and transcriptome sequencing analyses demonstrated that the transcript levels of multiple genes related to phenylpropanoid biosynthesis, trichome formation, and pathogen resistance were down-regulated in the transgenic Arabidopsis. In particular, we found that GbMYBR1 directly interacts with the bHLH cofactor GL3 as revealed by yeast two-hybrid assays. Our work indicated that GbMYBR1 has pleiotropic effects on plant growth, phenylpropanoid accumulation, and trichome development, mediated by interaction with GL3 or direct suppression of key pathway genes. Thus, GbMYBR1 represents a novel type of R2R3 MYB repressor.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest K_m value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.

Plant β-galactosidases (BGAL) function in various cell wall biogeneses and modifications, and they belong to the glycoside hydrolase family. However, the roles of BGAL family members in Medicago truncatula cell wall remodeling remain unclear. In this study, a total of 25 MtBGAL members of the glycoside hydrolase gene family 35 were identified, and they were clustered into nine sub-families. Many cis-acting elements possibly related to MeJA and abscisic acid responses were identified in the promoter region of the MtBGAL genes. Transcript analyses showed that these MtBGAL genes exhibited distinct expression patterns in various tissues and developing stem internodes. Furthermore, a stem-specific expression module associated with cell wall metabolic pathways was identified by weighted correlation network analysis (WGCNA). In particular, MtBGAL1 and MtBGAL23 within the stem-specific expression module were highly expressed in mature stems. In addition, several genes involved in lignin, cellulose, hemicellulose and pectin pathways were co-expressed with MtBGAL1 and MtBGAL23. It was also found that MtBGAL1 and MtBGAL23 were localized to the cell wall at the subcellular level, indicating their roles in the modification of cell wall metabolites in Medicago. As a whole, these results will be useful for further functional characterization and utilization of BGAL genes in cell wall modifications aiming to improve the quality of legume forage crops.

Epimedium sagittatum is an important traditional medicinal plant in China. In this study, we assembled the complete chloroplast (cp) genome of E. sagittatum. The whole cp genome of E. sagittatum is 157,114 bp in length, comprising a pair of inverted repeat (IR) regions (25,775 bp) separated by a large single copy (LSC) region (88,507 bp) and a small single copy (SSC) region (17,057 bp). The E. sagittatum cp genome contains 133 genes, of which 82 protein-coding genes, 38 tRNA genes, 8 rRNA genes and 5 pseudogenes. Phylogenetic analysis shows that E. sagittatumis closely clustered with E. lishihchenii. This genome provides a wealth of information for distinguishing of Epimedium species.