Characterization of the horse chestnut genome reveals the evolution of aescin and aesculin biosynthesis.

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.

Cannabis sativa: origin and history, glandular trichome development, and cannabinoid biosynthesis.

Is Cannabis a boon or bane? Cannabis sativa has long been a versatile crop for fiber extraction (industrial hemp), traditional Chinese medicine (hemp seeds), and recreational drugs (marijuana). Cannabis faced global prohibition in the twentieth century because of the psychoactive properties of ∆9-tetrahydrocannabinol; however, recently, the perspective has changed with the recognition of additional therapeutic values, particularly the pharmacological potential of cannabidiol. A comprehensive understanding of the underlying mechanism of cannabinoid biosynthesis is necessary to cultivate and promote globally the medicinal application of Cannabis resources. Here, we comprehensively review the historical usage of Cannabis, biosynthesis of trichome-specific cannabinoids, regulatory network of trichome development, and synthetic biology of cannabinoids. This review provides valuable insights into the efficient biosynthesis and green production of cannabinoids, and the development and utilization of novel Cannabis varieties.

Characterization and co-expression analysis of ATP-binding cassette transporters provide insight into genes related to cannabinoid transport in Cannabis sativa L.

Plant ATP-binding cassette (ABC) transporters contribute the transport of diverse secondary metabolites. However, their roles in cannabinoid trafficking are still unsolved in Cannabis sativa. In this study, 113 ABC transporters were identified and characterized in C. sativa from their physicochemical properties, gene structure, and phylogenic relationship, as well as spatial gene expression patterns. Eventually, seven core transporters were proposed including one member in ABC subfamily B (CsABCB8) and six ABCG members (CsABCG4, CsABCG10, CsABCG11, CsABCG32, CsABCG37, and CsABCG41), harboring potential in participating cannabinoid transport, by combining phylogenetic and co-expression analysis from the gene and metabolite level. The candidate genes exhibited a high correlation with cannabinoid biosynthetic pathway genes and the cannabinoid content, and they were highly expressed where cannabinoids appropriately biosynthesized and accumulated. The findings underpin further research on the function of ABC transporters in C. sativa, especially in unveiling the mechanisms of cannabinoid transport to boost systematic and targeted metabolic engineering.

Genetic and molecular analysis of the anthocyanin pigmentation pathway in Epimedium.

Introduction: Flower color is an ideal trait for studying the molecular basis for phenotypic variations in natural populations of species. Epimedium (Berberidaceae) species exhibit a wide range of flower colors resulting from the varied accumulation of anthocyanins and other pigments in their spur-like petals and petaloid sepals. Methods: In this work, the anthocyanidins of eight different Epimedium species with different floral pigmentation phenotypes were analyzed using HPLC. Twelve genes involved in anthocyanin biosynthesis were cloned and sequenced, and their expression was quantified. Results: The expression levels of the catalytic enzyme genes DFR and ANS were significantly decreased in four species showing loss of floral pigmentation. Complementation of EsF3'H and EsDFR in corresponding Arabidopsis mutants together with overexpression of EsF3'5'H in wild type Arabidopsis analysis revealed that these genes were functional at the protein level, based on the accumulation of anthocyanin pigments. Discussion: These results strongly suggest that transcriptional regulatory changes determine the loss of anthocyanins to be convergent in the floral tissue of Epimedium species.

A transcriptional complex of FtMYB102 and FtbHLH4 coordinately regulates the accumulation of rutin in Fagopyrum tataricum.

Tartary buckwheat is rich in flavonoids, which not only play an important role in the plant-environment interaction, but are also beneficial to human health. Rutin is a therapeutic flavonol which is massively accumulated in Tartary buckwheat. It has been demonstrated that transcription factors control rutin biosynthesis. However, the transcriptional regulatory network of rutin is not fully clear. In this study, through transcriptome and target metabolomics, we validated the role of FtMYB102 and FtbHLH4 TFs at the different developmental stages of Tartary buckwheat. The elevated accumulation of rutin in the sprout appears to be closely associated with the expression of FtMYB102 and FtbHLH4. Yeast two-hybrid, transient luciferase activity and co-immunoprecipitation demonstrated that FtMYB102 and FtbHLH4 can interact and form a transcriptional complex. Moreover, yeast one-hybrid showed that both FtMYB102 and FtbHLH4 directly bind to the promoter of chalcone isomerase (CHI), and they can coordinately induce CHI expression as shown by transient luciferase activity assay. Finally, we transferred FtMYB102 and FtbHLH4 into the hairy roots of Tartary buckwheat and found that they both can promote the accumulation of rutin. Our results indicate that FtMYB102 and FtbHLH4 can form a transcriptional complex by inducing CHI expression to coordinately promote the accumulation of rutin.

Genome-wide identification of key enzyme-encoding genes and the catalytic roles of two 2-oxoglutarate-dependent dioxygenase involved in flavonoid biosynthesis in Cannabis sativa L.

BACKGROUND: Flavonoids are necessary for plant growth and resistance to adversity and stress. They are also an essential nutrient for human diet and health. Among the metabolites produced in Cannabis sativa (C. sativa), phytocannabinoids have undergone extensive research on their structures, biosynthesis, and biological activities. Besides the phytocannabinoids, C. sativa is also rich in terpenes, alkaloids, and flavonoids, although little research has been conducted in this area. RESULTS: In this study, we identified 11 classes of key enzyme-encoding genes, including 56 members involved in the flavonoid biosynthesis in C. sativa, from their physical characteristics to their expression patterns. We screened the potentially step-by-step enzymes catalyzing the precursor phenylalanine to the end flavonoids using a conjoin analysis of gene expression with metabolomics from different tissues and chemovars. Flavonol synthase (FLS), belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, catalyzes the dihydroflavonols to flavonols. In vitro recombinant protein activity analysis revealed that CsFLS2 and CsFLS3 had a dual function in converting naringenin (Nar) to dihydrokaempferol (DHK), as well as dihydroflavonols to flavonols with different substrate preferences. Meanwhile, we found that CsFLS2 produced apigenin (Api) in addition to DHK and kaempferol when Nar was used as the substrate, indicating that CsFLS2 has an evolutionary relationship with Cannabis flavone synthase I. CONCLUSIONS: Our study identified key enzyme-encoding genes involved in the biosynthesis of flavonoids in C. sativa and highlighted the key CsFLS genes that generate flavonols and their diversified functions in C. sativa flavonoid production. This study paves the way for reconstructing the entire pathway for C. sativa's flavonols and cannflavins production in heterologous systems or plant culture, and provides a theoretical foundation for discovering new cannabis-specific flavonoids.

Comparative chloroplast genome analyses of Paraboea (Gesneriaceae): Insights into adaptive evolution and phylogenetic analysis.

Paraboea (Gesneriaceae) distributed in the karst areas of South and Southwest China and Southeast Asia, is an ideal genus to study the phylogeny and adaptive evolution of karst plants. In this study, the complete chloroplast genomes of twelve Paraboea species were sequenced and analyzed. Twelve chloroplast genomes ranged in size from 153166 to 154245 bp. Each chloroplast genome had a typical quartile structure, and relatively conserved type and number of gene components, including 131 genes which are composed of 87 protein coding genes, 36 transfer RNAs and 8 ribosomal RNAs. A total of 600 simple sequence repeats and 389 non-overlapped sequence repeats were obtained from the twelve Paraboea chloroplast genomes. We found ten divergent regions (trnH-GUG-psbA, trnM-CAU, trnC-GCA, atpF-atpH, ycf1, trnK-UUU-rps16, rps15, petL, trnS-GCU-trnR-UCU and psaJ-rpl33) among the 12 Paraboea species to be potential molecular markers. In the phylogenetic tree of 31 Gesneriaceae plants including twelve Paraboea species, all Paraboea species clustered in a clade and confirmed the monophyly of Paraboea. Nine genes with positive selection sites were detected, most of which were related to photosynthesis and protein synthesis, and might played crucial roles in the adaptability of Paraboea to diverse karst environments. These findings are valuable for further study of the phylogeny and karst adaptability of Gesneriaceae plants.

Genome-Wide Identification of MAPK, MAPKK, and MAPKKK Gene Families in Fagopyrum tataricum and Analysis of Their Expression Patterns Under Abiotic Stress.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signal transduction pathway, ubiquitous in eukaryotes, such as animals and plants. The MAPK cascade has a dominant role in regulating plant adaptation to the environment, such as through stress responses, osmotic adjustment, and processes that modulate pathogenicity. In the present study, the MAPK cascade gene family was identified in Fagopyrum tataricum (Tartary buckwheat), based on complete genome sequence data. Using phylogenetic tree, conservative motif, and chromosome location analyses, a total of 65 FtMAPK cascade genes, distributed on five chromosomes, were classified into three families: MAPK (n = 8), MAPKK (n = 1), and MAPKKK (n = 56). Transcriptome data from Tartary buckwheat seedlings grown under different light conditions demonstrated that, under blue and red light, the expression levels of 18 and 36 FtMAPK cascade genes were up-regulated and down-regulated, respectively. Through qRT-PCR experiments, it was observed that FtMAPK5, FtMAPKK1, FtMAPKKK8, FtMAPKKK10, and FtMAPKKK24 gene expression levels in the Tartary buckwheat seedlings increased under three types of abiotic stress: drought, salt, and high temperature. A co-expression network of FtMAPK cascade genes was constructed, based on gene expression levels under different light conditions, and co-expressed genes annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, which identified numerous transcription factors related to plant abiotic stress. The authors conclude that FtMAPK cascade genes have important roles in the growth and development of Tartary buckwheat, as well as its responses to abiotic stress.

Genome-Wide Identification and Expression Profiles of bZIP Genes in Cannabis sativa L.

Background: The bZIP gene family plays roles in biotic and abiotic stress, secondary metabolism, and other aspects in plants. They have been reported in Arabidopsis thaliana, Oryza sativa, Artemisia annua, and other plants, but their roles in Cannabis sativa have not been determined. Materials and Methods: In this study, we analyzed the genome-wide identification and expression profile of the bZIP gene family in C. sativa. Results: A total of 51 members of the bZIP gene family were identified based on the C. sativa genome and numbered in order from CsbZIP1 to CsbZIP51. Their phylogenetic relationships, cis-elements in promoter region, gene structures and motif compositions, physicochemical properties, chromosome locations, and expression profiles, were analyzed. The results showed that the 51 CsbZIPs were unevenly distributed on 10 chromosomes and could be clustered into 11 subfamilies. Furthermore, CsbZIPs located in the same subfamilies presented similar intron/exon organization and motif composition. The expression levels of CsbZIPs in various tissues (flowers, bracts, vegetative leaves, stems, and seeds) were determined using reverse transcription quantitative polymerase chain reaction. The expression levels of CsbZIPs were higher in flowers and bracts. The 51 CsbZIPs were explored, and their structure, evolution, and expression pattern in different tissues of C. sativa were characterized synthetically. The findings indicated that CsbZIPs are essential for the growth and development of C. sativa. Conclusions: These results provide a theoretical basis for subsequent research on hemp bZIP transcription factors and the cultivation of high-cannabidiol and low-tetrahydrocannabinol high-quality cannabis varieties.

Identification of the Histone Deacetylases Gene Family in Hemp Reveals Genes Regulating Cannabinoids Synthesis.

Histone deacetylases (HDACs) play crucial roles nearly in all aspects of plant biology, including stress responses, development and growth, and regulation of secondary metabolite biosynthesis. The molecular functions of HDACs have been explored in depth in Arabidopsis thaliana, while little research has been reported in the medicinal plant Cannabis sativa L. Here, we excavated 14 CsHDAC genes of C. sativa L that were divided into three relatively conserved subfamilies, including RPD3/HDA1 (10 genes), SIR2 (2 genes), and HD2 (2 genes). Genes associated with the biosynthesis of bioactive constituents were identified by combining the distribution of cannabinoids with the expression pattern of HDAC genes in various organs. Using qRT-PCR and transcription group analysis, we verified the expression of candidate genes in different tissues. We found that the histone inhibitor Trichostatin A (TSA) affected the expression of key genes in the cannabinoid metabolism pathway and the accumulation of synthetic precursors, which indirectly indicates that histone inhibitor may regulate the synthesis of active substances in C. sativa L.

Liquid chromatography-mass spectrometry-based metabolomics analysis of flavonoids and anthraquinones in Fagopyrum tataricum L. Gaertn. (tartary buckwheat) seeds to trace morphological variations.

Polyphenols (flavonoids and anthraquinones) are one of the most important phytochemicals in Fagopyrum tataricum L. Gaertn. (tartary buckwheat). However, the relationship between the polyphenols of tartary buckwheat seeds and their morphological variations is unclear. We developed a liquid chromatography-mass spectrometry-based targeted metabolomics method to study the chemical profiles of 60 flavonoids and 11 anthraquinones in 40 seed cultivars (groats and hulls). Both flavonoids and anthraquinones were related to variations in seed color; the fold change from yellowish-brown to black seeds was 1.24-1.55 in groats and 0.26-0.76 in hulls. Only flavonoids contributed to significant differences in seed shape; the fold change from long to short seeds was 1.29-1.78 in groats and 1.39-1.44 in hulls. Some differential metabolites were identified at higher concentrations in hulls than in groats. This study provides new insights into differences in polyphenols among tartary buckwheat seeds with different color and shape.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum).

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7-10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p < 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

[Advances in biosynthesis of indigo in plants].

Natural indigo, as one of the oldest dyes, is also a pivotal dye utilized in cotton fabrics today. A diversity of plants rich in indigo compounds belong to traditional Chinese herbal medicines. Indigo compounds have a variety of biological and pharmacological activities, including anticonvulsant, antibacterial, antifungal, antiviral and anticancer activities. A substantial progress in indigo biosynthesis has been made lately. This paper summarizes the value of indigo from the aspects of cultural history, biosynthetic pathways and the medicinal activities of its related derivatives involved in the pathways. In addition, the latest research advancements in indigo biosynthetic pathways is demonstrated in this paper, which would lay the theoretical foundation for the exploration and utilization of natural indigo.

[Application of MALDI-mass spectrometry imaging in spatial distribution of secondary metabolites in medicinal plants -- a case study of Lepdium meyenii root].

Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) is a novel technique for in-situ distribution of various substances in tissue without labeling. This technique is increasingly applied to the study of medicinal plants owing to its high spatial resolution and its potential of in-situ analysis in small molecules. In this study, the structural information and their fragmentation patterns of the midazole alkaloids(1,3-dibenzyl-4,5-dimethylimidazolium chloride and 1,3-dibenzyl-2,4,5-trimethylimi-dazolium chloride) and benzylglucosinolate in the medicinal plant Maca(Lepdium meyeni) root were analyzed by ultra-high-performance liquid phase combined with LTQ-Orbitrap mass spectrometry(UHPLC-HR-MS). The localization of these active ingredients in the cross-sections of Maca root was performed by MALDI-MSI. These results demonstrated that the two types of imidazole alkaloids had a similar distributed pattern. They were located more in the cortex and the periderm than those in the medulla of a lateral root, while the localization of benzylglucosinolate was concentrated in the center of the root rather than in the cortex and the periderm. The precise spatial distribution of various secondary metabolites in tissue provides an important scientific basis for the accumulation of medicinal plant active ingredients in tissues. In addition, this imaging method is a promising technique for the rapid evaluation and identification of the active ingredients of traditional Chinese medicine in plant tissues, as well as assisting the research on the processing of medicinal plants.

Assessing the Identity of Commercial Herbs From a Cambodian Market Using DNA Barcoding.

In Cambodia, medicinal plants are often used to treat various illnesses. However, the identities of many medicinal plants remain unknown. In this study, we collected 50 types of traditional Cambodian medicinal plants that could not be identified by their appearance from a domestic market. We utilized the DNA barcoding technique, combined with the literature survey, to trace their identities. In the end, 33 species were identified at the species level and 7 species were identified at the genus level. The ethnopharmacological information of 33 medicinal plants was documented. The DNA barcoding technique is useful in the identification of medicinal plants with no previous information.

Genomic Characterization of WRKY Transcription Factors Related to Andrographolide Biosynthesis in Andrographis paniculata.

Andrographolide, which is enriched in the leaves of Andrographis paniculata, has been known as "natural antibiotic" due to its pharmacological activities such as anti-inflammatory, antimicrobial and antioxidant effects. Several key enzymes in andrographolide biosynthetic pathway have been studied since the genome sequences were released, but its regulatory mechanism remains unknown. WRKY transcription factors proteins have been reported to regulate plant secondary metabolism, development as well as biotic and abiotic stresses. Here, WRKY transcription factors related to andrographolide biosynthesis were systematically identified, including sequences alignment, phylogenetic analysis, chromosomal distribution, gene structure, conserved motifs, synteny, alternative splicing event and Gene ontology (GO) annotation. A total of 58 WRKYs were identified in Chuanxinlian genome and phylogenetically classified into three groups. Moreover, nine WRKY genes underwent alternative splicing events. Furthermore, the combination of binding site prediction, gene-specific expression patterns, and phylogenetic analysis suggested that 7 WRKYs (ApWRKY01, ApWRKY08, ApWRKY12, ApWRKY14, ApWRKY19, ApWRKY20, and ApWRKY50) might regulate andrographolide biosynthesis. This study laid a foundation for understanding the regulatory mechanism of andrographolide biosynthesis and the improvement and breeding of Andrographis paniculata varieties.