Production of species-specific anthocyanins through an inducible system in plant hairy roots.

Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a ß-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.

Characterization of UDP-glycosyltransferase family members reveals how major flavonoid glycoside accumulates in the roots of Scutellaria baicalensis.

BACKGROUND: Flavonoid glycosides extracted from roots of Scutellaria baicalensis exhibit strong pharmaceutical antitumor, antioxidative, anti-inflammatory, and antiviral activities. UDP glycosyltransferase (UGT) family members are responsible for the transfer of a glycosyl moiety from UDP sugars to a wide range of acceptor flavonoids. Baicalin is the major flavonoid glycoside found in S. baicalensis roots, and its aglycone baicalein is synthesized from a specially evolved pathway that has been elucidated. However, it is necessary to carry out a genome-wide study of genes involved in 7-O-glucuronidation, the final biosynthesis step of baicalin, which might elucidate the relationship between the enzymes and the metabolic accumulation patterns in this medicinal plant. RESULTS: We reported the phylogenetic analysis, tissue-specific expression, biochemical characterization and evolutionary analysis of glucosyltransferases (SbUGTs) and glucuronosyltransferases (SbUGATs) genes based on the recently released genome of S. baicalensis. A total of 124 UGTs were identified, and over one third of them were highly expressed in roots. In vitro enzyme assays showed that 6 SbUGTs could use UDP-glucose as a sugar donor and convert baicalein to oroxin A (baicalein 7-O-glucoside), while 4 SbUGATs used only UDP-glucuronic acid as the sugar donor and catalyzed baicalein to baicalin. SbUGAT4 and SbUGT2 are the most highly expressed SbUGAT and SbUGT genes in root tissues, respectively. Kinetic measurements revealed that SbUGAT4 had a lower Km value and higher Vmax/Km ratio to baicalein than those of SbUGT2. Furthermore, tandem duplication events were detected in SbUGTs and SbUGATs. CONCLUSIONS: This study demonstrated that glucosylation and glucuronidation are two major glycosylated decorations in the roots of S. baicalensis. Higher expression level and affinity to substrate of SbUGAT4, and expansion of this gene family contribute high accumulation of baicalin in the root of S. baicalensis.

Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4'-deoxyflavones in Scutellaria baicalensis Georgi.

The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4'-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4'-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3'-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4'-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.

Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism.

BACKGROUND: The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites. RESULTS: In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes' behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones. CONCLUSION: The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Overexpression of SmANS Enhances Anthocyanin Accumulation and Alters Phenolic Acids Content in Salvia miltiorrhiza and Salvia miltiorrhiza Bge f. alba Plantlets.

Flavonoids play multiple roles in plant coloration and stress resistance and are closely associated with human health. Flavonoids and non-flavonoids (such as phenolic acids) are produced via the phenylpropanoid-derived pathway. Anthocyanidin synthase (ANS) catalyzes the synthesis of anthocyanins from leucoanthocyanidin in the flavonoids branched pathway. In this study, SmANS from Salvia miltiorrhiza was cloned and mainly localized in the endoplasmic reticulum (ER), plastids, Golgi, plasma membrane, and nucleus of tobacco epidermal cells, and was most highly expressed in purple petals in S. miltiorrhiza, whereas it showed almost no expression in white petals, green calyxes, and pistils in S. miltiorrhiza Bge f. alba. Overexpressed SmANS enhanced anthocyanin accumulation but reduced salvianolic acid B (SAB) and rosmarinic acid (RA) biosynthesis in S. miltiorrhiza and S. miltiorrhiza Bge f. alba plantlets, meanwhile, it restored the purple-red phenotype in S. miltiorrhiza Bge f. alba. These changes were due to reallocation of the metabolic flow, which was influenced by the SmANS gene. These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in S. miltiorrhiza, but also acts as a "switch" for the coloration of S. miltiorrhiza Bge f. alba. This study provides baseline information for further research on flavonoids metabolism and improvement of anthocyanin or phenolic acid production by genetic engineering.

[A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus].

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots.

Jasmonates (JAs) are important plant hormones that regulate a variety of plant development and defense processes, including biosynthesis of secondary metabolites. The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the JA signaling pathways of plants. We first verified that methyl jasmonate (MeJA) enhanced the accumulation of both salvianolic acids and tanshinones in Salvia miltiorrhiza (Danshen) hairy roots by inducing the expression of their biosynthetic pathway genes. Nine JAZ genes were cloned from Danshen and their expression levels in hairy roots were all increased by treatment with MeJA. When analyzed in detail, however, SmJAZ8 showed the strongest expression in the induced hairy roots. Overexpression or RNAi of SmJAZ8 deregulated or up-regulated the yields of salvianolic acids and tanshinones in the MeJA-induced transgenic hairy roots, respectively, and transcription factors and biosynthetic pathway genes showed an expression pattern that mirrored the production of the compounds. Genetic transformation of SmJAZ8 altered the expression of other SmJAZ genes, suggesting evidence of crosstalk occurring in JAZ-regulated secondary metabolism. Furthermore, the transcriptome analysis revealed a primary-secondary metabolism balance regulated by SmJAZ8. Altogether, we propose a novel role for SmJAZ8 as a negative feedback loop controller in the JA-induced biosynthesis of salvianolic acids and tanshinones.

Functional evolution and diversification of the CYP82D subfamily members shape flavonoid diversification in the genus Scutellaria.

Flavonoids, the largest class of polyphenols, exhibit substantial structural and functional diversity, yet their evolutionary diversification and specialized functions remain largely unexplored. The genus Scutellaria is notable for its rich flavonoid diversity, particularly the 6/8-hydroxylated variants biosynthesized by the cytochrome P450 subfamily CYP82D. Our study analyzes metabolic differences between Scutellaria baicalensis and Scutellaria barbata, suggesting that CYP82Ds have acquired a broad range of catalytic functions over their evolution. By integrating analyses of metabolic networks and gene evolution across 22 Scutellaria species, we rapid identified 261 flavonoids and delineated five clades associated with various catalytic functions of CYP82Ds. This approach uncovered a unique catalytic mode for 6/8-hydroxylated function under flavanone substrates and the first instance of 7-O-demethylation of flavonoid substrates catalyzed by cytochrome P450. Ancestral sequence reconstruction and functional validation demonstrated that gradual neofunctionalization of CYP82Ds has driven the chemical diversity of flavonoids in Scutellaria throughout its evolutionary history. Our study enhances the understanding of flavonoid diversity, elucidates the intricate roles of CYP82Ds in Scutellaria plants, and underscores the extensive catalytic versatility of cytochrome P450 members within plant taxa.

SbMYB3 transcription factor promotes root-specific flavone biosynthesis in Scutellaria baicalensis.

Scutellaria baicalensis Georgi produces abundant root-specific flavones (RSFs), which provide various benefits to human health. We have elucidated the complete biosynthetic pathways of baicalein and wogonin. However, the transcriptional regulation of flavone biosynthesis in S. baicalensis remains unclear. We show that the SbMYB3 transcription factor functions as a transcriptional activator involved in the biosynthesis of RSFs in S. baicalensis. Yeast one-hybrid and transcriptional activation assays showed that SbMYB3 binds to the promoter of flavone synthase II-2 (SbFNSII-2) and enhances its transcription. In S. baicalensis hairy roots, RNAi of SbMYB3 reduced the accumulation of baicalin and wogonoside, and SbMYB3 knockout decreased the biosynthesis of baicalein, baicalin, wogonin, and wogonoside, whereas SbMYB3 overexpression enhanced the contents of baicalein, baicalin, wogonin, and wogonoside. Transcript profiling by qRT-PCR demonstrated that SbMYB3 activates SbFNSII-2 expression directly, thus leading to more abundant accumulation of RSFs. This study provides a potential target for metabolic engineering of RSFs.

Gap-free genome assembly and CYP450 gene family analysis reveal the biosynthesis of anthocyanins in Scutellaria baicalensis.

Scutellaria baicalensis Georgi, a member of the Lamiaceae family, is a widely utilized medicinal plant. The flavones extracted from S. baicalensis contribute to numerous health benefits, including anti-inflammatory, antiviral, and anti-tumor activities. However, the incomplete genome assembly hinders biological studies on S. baicalensis. This study presents the first telomere-to-telomere (T2T) gap-free genome assembly of S. baicalensis through the integration of Pacbio HiFi, Nanopore ultra-long and Hi-C technologies. A total of 384.59 Mb of genome size with a contig N50 of 42.44 Mb was obtained, and all sequences were anchored into nine pseudochromosomes without any gap or mismatch. In addition, we analysed the major cyanidin- and delphinidin-based anthocyanins involved in the determination of blue-purple flower using a widely-targeted metabolome approach. Based on the genome-wide identification of Cytochrome P450 (CYP450) gene family, three genes (SbFBH1, 2, and 5) encoding flavonoid 3'-hydroxylases (F3'Hs) and one gene (SbFBH7) encoding flavonoid 3'5'-hydroxylase (F3'5'H) were found to hydroxylate the B-ring of flavonoids. Our studies enrich the genomic information available for the Lamiaceae family and provide a toolkit for discovering CYP450 genes involved in the flavonoid decoration.

Specific Flavonoids and Their Biosynthetic Pathway in Scutellaria baicalensis.

Scutellaria baicalensis, is one of the most traditional medicinal plants in the Lamiaceae family, and has been widely used to treat liver and lung complaints and as a complementary cancer treatment in traditional Chinese medicine. The preparation from its roots, called "Huang Qin," is rich in specialized flavones such as baicalein, wogonin, and their glycosides which lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones), with anti-tumor, antioxidant, and antiviral activities. Baicalein has recently been reported to inhibit the replication of the COVID-19 virus. These 4'-deoxyflavones are found only in the order Lamiales and were discovered in the genus Scutellaria, suggesting that a new metabolic pathway synthesizing 4'-deoxyflavones evolved recently in this genus. In this review, we focus on the class of 4'-deoxyflavones in S. baicalensis and their pharmacological properties. We also describe the apparent evolutionary route taken by the genes encoding enzymes involved in the novel, root-specific, biosynthetic pathway for baicalein and wogonin, which provides insights into the evolution of specific flavone biosynthetic pathways in the mint family.

Functional pleiotropism, diversity, and redundancy of Salvia miltiorrhiza Bunge JAZ family proteins in jasmonate-induced tanshinone and phenolic acid biosynthesis.

Jasmonate (JA) signaling regulates plant growth and development, biotic and abiotic stress tolerance, and primary and secondary metabolism biosynthesis. It is extensively modulated by JA-ZIM-domain (JAZ) family genes. In previous work, we obtained nine SmJAZ genes of Salvia miltiorrhiza and proved that SmJAZ8 was the core repressor of JA-induced tanshinone and phenolic acid biosynthesis. Here, we demonstrate that SmJAZ3 and SmJAZ4 act as repressors of JA-induced biosynthesis of tanshinones and salvianolic acid B (Sal B). This suggests that SmJAZ3/4 are functionally redundant in tanshinone and Sal B biosynthesis. SmJAZ1/2/5/6/9 are activators of JA-induced tanshinone biosynthesis and repressors of JA-induced Sal B biosynthesis. This demonstrates the redundancy and diversity of SmJAZ1/2/5/6/9 functions. Besides, SmJAZ10 inhibited JA-induced Sal B synthesis, but had no effect on the synthesis of tanshinone. Two-hybrid screening (Y2H) showed that SmJAZs formed homologous or heterogeneous dimers. Y2H and firefly luciferase complementation imaging (LCI) assays revealed that SmJAZs also formed a complex regulatory network with SmMYC2a, SmMYC2b, SmMYB39, and SmPAP1. Quantitative reverse transcription-PCR (qRT-PCR) indicated that SmJAZs regulated each other at the transcriptional level. Herein, we prove that SmJAZs have functional pleiotropism, diversity, and redundancy in JA-induced tanshinone and phenolic acid biosynthesis. This study provides an important clue for further understanding the inherent biological significance and molecular mechanisms of the JAZ family as the gene number increases during plant evolution.

The genome of Tripterygium wilfordii and characterization of the celastrol biosynthesis pathway.

Tripterygium wilfordii is a vine from the Celastraceae family that is used in traditional Chinese medicine (TCM). The active ingredient, celastrol, is a friedelane-type pentacyclic triterpenoid with putative roles as an antitumor, immunosuppressive, and anti-obesity agent. Here, we report a reference genome assembly of T. wilfordii with high-quality annotation using a hybrid sequencing strategy. The total genome size obtained is 340.12 Mb, with a contig N50 value of 3.09 Mb. We successfully anchored 91.02% of sequences into 23 pseudochromosomes using high-throughput chromosome conformation capture (Hi-C) technology. The super-scaffold N50 value was 13.03 Mb. We also annotated 31,593 structural genes, with a repeat percentage of 44.31%. These data demonstrate that T. wilfordii diverged from Malpighiales species approximately 102.4 million years ago. By integrating genome, transcriptome and metabolite analyses, as well as in vivo and in vitro enzyme assays of two cytochrome P450 (CYP450) genes, TwCYP712K1 and TwCYP712K2, it is possible to investigate the second biosynthesis step of celastrol and demonstrate that this was derived from a common ancestor. These data provide insights and resources for further investigation of pathways related to celastrol, and valuable information to aid the conservation of resources, as well as understand the evolution of Celastrales.

SmGRAS1 and SmGRAS2 Regulate the Biosynthesis of Tanshinones and Phenolic Acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most widely used traditional Chinese medicinal plants because of its excellent performance in treating heart diseases. Tanshinones and phenolic acids are two important classes of effective metabolites, and their biosynthesis has attracted widespread interest. Here, we functionally characterized SmGRAS1 and SmGRAS2, two GRAS family transcription factors from S. miltiorrhiza. SmGRAS1/2 were highly expressed in the root periderm, where tanshinones mainly accumulated in S. miltiorrhiza. Overexpression of SmGRAS1/2 upregulated tanshinones accumulation and downregulated GA, phenolic acids contents, and root biomass. However, antisense expression of SmGRAS1/2 reduced the tanshinones accumulation and increased the GA, phenolic acids contents, and root biomass. The expression patterns of biosynthesis genes were consistent with the changes in compounds accumulation. GA treatment increased tanshinones, phenolic acids, and GA contents in the overexpression lines, and restored the root growth inhibited by overexpressing SmGRAS1/2. Subsequently, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays (EMSA) showed SmGRAS1 promoted tanshinones biosynthesis by directly binding to the GARE motif in the SmKSL1 promoter and activating its expression. Yeast two-hybrid assays showed SmGRAS1 interacted physically with SmGRAS2. Taken together, the results revealed that SmGRAS1/2 acted as repressors in root growth and phenolic acids biosynthesis but as positive regulators in tanshinones biosynthesis. Overall, our findings revealed the potential value of SmGRAS1/2 in genetically engineering changes in secondary metabolism.

SmMYB36, a Novel R2R3-MYB Transcription Factor, Enhances Tanshinone Accumulation and Decreases Phenolic Acid Content in Salvia miltiorrhiza Hairy Roots.

Phenolic acids and tanshinones are two major bioactive components in Salvia miltiorrhiza Bunge. A novel endogenous R2R3-MYB transcription factor, SmMYB36, was identified in this research. This transcript factor can simultaneously influence the content of two types of components in SmMYB36 overexpression hairy roots. SmMYB36 was mainly localized in the nucleus of onion epidermis and it has transactivation activity. The overexpression of SmMYB36 promoted tanshinone accumulation but inhibited phenolic acid and flavonoid biosynthesis in Salvia miltiorrhiza hairy roots. The altered metabolite content was due to changed metabolic flow which was regulated by transcript expression of metabolic pathway genes. The gene transcription levels of the phenylpropanoid general pathway, tyrosine derived pathway, methylerythritol phosphate pathway and downstream tanshinone biosynthetic pathway changed significantly due to the overexpression of SmMYB36. The wide distribution of MYB binding elements (MBS, MRE, MBSI and MBSII) and electrophoretic mobility shift assay results indicated that SmMYB36 may be an effective tool to regulate metabolic flux shifts.

The Protein Kinase SmSnRK2.6 Positively Regulates Phenolic Acid Biosynthesis in Salvia miltiorrhiza by Interacting with SmAREB1.

Subclass III members of the sucrose non-fermenting-1-related protein kinase 2 (SnRK2) play essential roles in both the abscisic acid signaling and abiotic stress responses of plants by phosphorylating the downstream ABA-responsive element (ABRE)-binding proteins (AREB/ABFs). This comprehensive study investigated the function of new candidate genes, namely SmSnRK2.3, SmSnRK2.6, and SmAREB1, with a view to breeding novel varieties of Salvia miltiorrhiza with improved stress tolerance stresses and more content of bioactive ingredients. Exogenous ABA strongly induced the expression of these genes. PlantCARE predicted several hormones and stress response cis-elements in their promoters. SmSnRK2.6 and SmAREB1 showed the highest expression levels in the leaves of S. miltiorrhiza seedlings, while SmSnRK2.3 exhibited a steady expression in their roots, stems, and leaves. A subcellular localization assay revealed that both SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane, cytoplasm, and nucleus, whereas SmAREB1 was exclusive to the nucleus. Overexpressing SmSnRK2.3 did not significantly promote the accumulation of rosmarinic acid (RA) and salvianolic acid B (Sal B) in the transgenic S. miltiorrhiza hairy roots. However, overexpressing SmSnRK2.6 and SmAREB1 increased the contents of RA and Sal B, and regulated the expression levels of structural genes participating in the phenolic acid-branched and side-branched pathways, including SmPAL1, SmC4H, Sm4CL1, SmTAT, SmHPPR, SmRAS, SmCHS, SmCCR, SmCOMT, and SmHPPD. Furthermore, SmSnRK2.3 and SmSnRK2.6 interacted physically with SmAREB1. In summary, our results indicate that SmSnRK2.6 is involved in stress responses and can regulate structural gene transcripts to promote greater metabolic flux to the phenolic acid-branched pathway, via its interaction with SmAREB1, a transcription factor. In this way, SmSnRK2.6 contributes to the positive regulation of phenolic acids in S. miltiorrhiza hairy roots.