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Periploca forrestii, a medicinal plant of the family Apocynaceae, is known as an effective and widely used clinical prescription for the treatment of rheumatoid diseases. In this study, we de novo sequenced and assembled the completement chloroplast (cp) genome of P. forrestii based on combined Oxford Nanopore PromethION and Illumina data. The cp genome was 153 724 bp in length and had four subregions. Moreover, an 84 433 bp large single-copy and a 17 731 bp small single-copy were separated by 25 780 bp inverted repeats (IRs). The cp genome included 132 genes with 18 duplicates in the IRs. A total of 45 repeat structures and 183 simple sequence repeats were detected. Codon usage showed a bias toward A/T-ending codons. A comparative study of Apocynaceae revealed that an IR expansion occurred on P. forrestii. The Ka/Ks values of eight species of Apocynaceae suggested that positive selection was exerted on the psaI and ycf2 genes, which might reflect specific adaptions to the P. forrestii particular growth environment. Phylogenetic analysis indicated that Periplocoideae was a sister to Asclepiadoideae, forming a monophyletic group in the family Apocynaceae. This study provided an important P. forrestii genomic resource for future evolutionary studies and the phylogenetic reconstruction of the family Apocynaceae.

Medicinal trees provide a main resource for diverse medicinal compounds. However, the biosynthesis of tree metabolites and their pathway evolution has gained limited understanding. Horse chestnut (Aesculus chinensis) is an important medicinal tree and its seeds are rich in aescins, barrigenol-type triterpenoid saponins (BAT), and aesculin, a coumarin glycoside, which are effective in the therapy of chronic venous insufficiency and asthenopia (eye strain). To understand the biosynthesis of these compounds, herein, we assembled a 470.04-Mb high-quality horse chestnut genome and characterized an Aesculus-specific whole-genome duplication (WGD) event. Spatial metabolome imaging, co-expression, and biosynthetic gene cluster analyses indicated that the Aesculus-specific WGD event led to the formation of two gene clusters (BGCs) including oxidosqualene cyclase, cytochrome P450 monooxygenase, cellulose synthase-derived glycotransferases, and BADH acyltransferases. Further biochemical investigation revealed the roles of AcOCS6, AcCYP716A278, AcCYP716A275, AcCSL1, and AcBAHD3 genes distributed between these two BGCs in catalyzing the formation of aescins. To understand the evolution of BAT pathways, the collinearity analysis showed the collinear BGC segments could be traced back to early-diverging angiosperms, then the essential gene-encoding enzymes necessary for the BAT biosynthesis were recruited before the split of Aesculus, Acer, and Xanthoceras. Meanwhile, we identified three UDP-glucosyltransferases and demonstrated their involvement in the biosynthesis of aesculin via a de novo synthesis. Taken together, these findings provide important information in understanding the evolution of gene clusters associated with medicinal tree metabolites.

Laportea bulbifera, a Miao medicine grown in karst areas, has exerts a unique curative effect on skin itching in the elderly, with an annual sales of > 100 million Yuan. Owing to the shortage of resources and large morphological variations in L. bulbifera, it is difficult to identify the species correctly using only traditional methods, which seriously affects the safety of drug usage for patients. This study obtained the complete high-quality L. bulbifera chloroplast (cp) genome, using second- and third-generation high-throughput sequencing. The cp genome was 149,911 bp in length, with a typical quadripartite structure. A total of 127 genes were annotated, including 83 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. There was an inverted small single copy (SSC) structure in the L. bulbifera cp genome, one large-scale rearrangement of ~ 39 kb excised in the SSC and IR regions. The complete cp genome sequence used as a super-barcode and the highly variable regions (ycf1, matK, and ndhD), especially matK, can be used as specific barcodes to accurately distinguish L. bulbifera from counterfeits and closely related species. This study is important for the identification of L. bulbifera and lays a theoretical foundation for elucidating the phylogenetic relationship of the species.

As auxins are among the most important phytohormones, the regulation of auxin homeostasis is complex. Generally, auxin conjugates, especially IAA glucosides, are predominant at high auxin levels. Previous research on terminal glucosylation focused mainly on the O-position, while IAA-N-glucoside and IAA-Asp-N-glucoside have been neglected since their discovery in 2001. In our study, IAA-Asp-N-glucoside was found to be specifically abundant (as high as 4.13 mg/g) in the seeds of 58 ginkgo cultivars. Furthermore, a novel N-glucosyltransferase, termed GbNGT1, was identified via differential transcriptome analysis and in vitro enzymatic testing. It was found that GbNGT1 could catalyze IAA-Asp and IAA to form their corresponding N-glucosides. The enzyme was demonstrated to possess a specific catalytic capacity toward the N-position of the IAA-amino acid or IAA from 52 substrates. Docking and site-directed mutagenesis of this enzyme confirmed that the E15G mutant could almost completely abolish its N-glucosylation ability toward IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. The effect of GbNGT1 on IAA distribution promotes root growth in Arabidopsis thaliana.

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

As auxins are among the most important phytohormones, the regulation of auxin homeostasis is complex. Generally, auxin conjugates, especially IAA glucosides, are predominant at high auxin levels. Previous research on terminal glucosylation focused mainly on the O-position, while IAA-N-glucoside and IAA-Asp-N-glucoside have been neglected since their discovery in 2001. In our study, IAA-Asp-N-glucoside was found to be specifically abundant (as high as 4.13 mg/g) in the seeds of 58 ginkgo cultivars. Furthermore, a novel N-glucosyltransferase, termed GbNGT1, was identified via differential transcriptome analysis and in vitro enzymatic testing. It was found that GbNGT1 could catalyze IAA-Asp and IAA to form their corresponding N-glucosides. The enzyme was demonstrated to possess a specific catalytic capacity toward the N-position of the IAA-amino acid or IAA from 52 substrates. Docking and site-directed mutagenesis of this enzyme confirmed that the E15G mutant could almost completely abolish its N-glucosylation ability toward IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. The effect of GbNGT1 on IAA distribution promotes root growth in Arabidopsis thaliana.

Ginseng contains a variety of flavonol glycosides that possess diverse biological activities; however, scant information of flavonoid glycosylation was reported in ginseng. We found that panasenoside and kaempferol 3-O-glucoside were commonly accumulated along with cultivation years in leaves. In order to explore the procedure of flavonol glycosylation in ginseng, 50 UDP-glycosyltransferases (UGTs) were screened out using differentiated data-independent acquisition (DIA) proteomics and phylogenetic analysis. UGT92A10 and UGT94Q4 were found contributing to the formation of kaempferol 3-O-glucoside. UGT73A18, UGT74T4, and UGT75W1 could catalyze galactosylation of kaempferol 3-O-glucoside. Ser278, Trp335, Gln338, and Val339 were found forming hydrogen bonds with UDP-galactose in UGT75W1 by docking. MeJA induced transcripts of UGT73A18 and UGT74T4 by over fourfold, consistent with the decrease of kaempferol 3-O-glucoside, which indicated that these genes may be related to resisting adversity stress in ginseng. These results highlight the significance of integrative metabolite profiles, proteomics, and phylogenetic analysis for exploring flavonol glycosylation in ginseng.

As a group of the most important phytohormone, auxin homeostasis is regulated in a complex manner. Generally, auxin conjugations especially IAA glucosides are dominant on high auxin level conditions. Former terminal glucosylation researches mainly focus on O-position, while IAA-N-glucoside or IAA-Asp-N-glucoside has been neglected since their found in 2001. In our study, IAA-Asp-N-glucoside was firstly found specifically abundant (as high as 4.13 mg/g) in ginkgo seeds of 58 cultivars from Ginkgo Resource Nursery built in 1990. Furthermore, a novel N-glucosyltransferase GbNGT1, which could catalyze IAA-Asp and IAA to form their corresponding N-glucoside, was identified through differential transcriptome analysis and in vitro enzymatic test. The enzyme was demonstrated to possess specific catalyze capacity toward the N-position of IAA-amino acid or IAA among 52 substrates, and was typical of acid tolerance, metal ion independence and high temperature sensitivity. Docking and site-directed mutagenesis of this enzyme confirmed that E15G mutant could almost abolish enzyme catalytic activity towards IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. In conclusion, our results complement the terminal metabolic pathway of auxin, and the specific catalytic function of GbNGT1 towards IAA-amino acid provide a new way to biosynthesis indole-amide compounds.HighlightThe N-glucosylation of IAA or IAA-amino acids in auxin metabolism had been neglected over decades, our work for GbNGT1 redeems the missing chain of auxin metabolic pathway.

GmMYB58 and GmMYB205 are key positive regulators that are involved in isoflavonoid biosynthesis in seeds of Glycine max, and they activate the expression of several structural genes in the isoflavonoid pathway. MYB transcription factors (TFs) are major regulators involved in flavonoid/isoflavonoid biosynthesis in many plant species. However, functions of most MYB TFs remain unknown in flavonoid/isoflavonoid pathway in Glycine max. In this study, we identified 321 MYB TFs by genome-wide searching, and further isolated and functionally characterized two MYB TFs, GmMYB58 and GmMYB205. The deduced GmMYB58 and GmMYB205 proteins contain highly conserved R2R3 repeat domain at the N-terminal region that is the signature motif of R2R3-type MYB TFs. GmMYB58 and GmMYB205 were highly expressed in early seed development stages than in the other tested organs. GmMYB58 and GmMYB205 GFP fusion proteins were found to be localized in the nucleus when they were transiently expressed in Arabidopsis thaliana mesophyll protoplast. Both GmMYB58 and GmMYB205 can activate the promoter activities of GmCHS, GmIFS2, and GmHID in the transient trans-activation assays, and the activation of GmHID by both GmMYB58 and GmMYB205 was further confirmed by yeast one-hybrid assay. In addition, over-expression of GmMYB58 and GmMYB205 resulted in significant increases in expression levels of several pathway genes in soybean hairy roots, in particular, IFS2 by more than fivefolds in GmMYB205-over-expressing lines. Moreover, isoflavonoid contents were remarkably enhanced in the GmMYB58 and GmMYB205 over-expressing hairy roots than in the control. Our results suggest that GmMYB58 and GmMYB205 are seed-specific TFs, and they can enhance isoflavonoid biosynthesis mainly through the regulation of GmIFS2 and GmHID in G. max.

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.

With people's increasing needs for health concern, rutin and emodin in tartary buckwheat have attracted much attention for their antioxidant, anti-diabetic and reducing weight function. However, the biosynthesis of rutin and emodin in tartary buckwheat is still unclear; especially their later glycosylation contributing to make them more stable and soluble is uncovered. Based on tartary buckwheat' genome, the gene structures of 106 UGTs were analyzed; 21 candidate FtUGTs were selected to enzymatic test by comparing their transcript patterns. Among them, FtUGT73BE5 and other 4 FtUGTs were identified to glucosylate flavonol or emodin in vitro; especially rFtUGT73BE5 could catalyze the glucosylation of all tested flavonoids and emodin. Furthermore, the identical in vivo functions of FtUGT73BE5 were demonstrated in tartary buckwheat hairy roots. The transcript profile of FtUGT73BE5 was consistent with the accumulation trend of rutin in plant; this gene may relate to anti-adversity for its transcripts were up-regulated by MeJA, and repressed by ABA.

Andrographis paniculata is a herbaceous dicot plant widely used for its anti-inflammatory and anti-viral properties across its distribution in China, India and other Southeast Asian countries. A. paniculata was used as a crucial therapeutic treatment during the influenza epidemic of 1919 in India, and is still used for the treatment of infectious disease in China. A. paniculata produces large quantities of the anti-inflammatory diterpenoid lactones andrographolide and neoandrographolide, and their analogs, which are touted to be the next generation of natural anti-inflammatory medicines for lung diseases, hepatitis, neurodegenerative disorders, autoimmune disorders and inflammatory skin diseases. Here, we report a chromosome-scale A. paniculata genome sequence of 269 Mb that was assembled by Illumina short reads, PacBio long reads and high-confidence (Hi-C) data. Gene annotation predicted 25 428 protein-coding genes. In order to decipher the genetic underpinning of diterpenoid biosynthesis, transcriptome data from seedlings elicited with methyl jasmonate were also obtained, which enabled the identification of genes encoding diterpenoid synthases, cytochrome P450 monooxygenases, 2-oxoglutarate-dependent dioxygenases and UDP-dependent glycosyltransferases potentially involved in diterpenoid lactone biosynthesis. We further carried out functional characterization of pairs of class-I and -II diterpene synthases, revealing the ability to produce diversified labdane-related diterpene scaffolds. In addition, a glycosyltransferase able to catalyze O-linked glucosylation of andrograpanin, yielding the major active product neoandrographolide, was also identified. Thus, our results demonstrate the utility of the combined genomic and transcriptomic data set generated here for the investigation of the production of the bioactive diterpenoid lactone constituents of the important medicinal herb A. paniculata.

Highlight CHIL is essential for the accumulation of proanthocyanidins and flavonols in seeds of Arabidopsis and it functions as a unique enhancer in the flavonoid pathway., 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.

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.

Highlight The UGT72 family is a major group of UDP-glycosyltransferases with diverse substrates. UGT72AD1, UGT72AH1, and UGT72Z2 may be involved in flavonol rhamnoside biosynthesis in seeds of Lotus japonicus., 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.