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Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF ( Sb MYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that Sb MYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4 , SbCHI-2 , and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis .

Phenolic acids and tanshinones are active principles in Salvia miltiorrhiza Bunge administered for cardiovascular and cerebrovascular diseases. Jasmonic acid (JA) promotes secondary metabolite accumulation, but the regulatory mechanism is unknown in S. miltiorrhiza . We identified and characterized the JA-responsive gene SmMYB97 . Multiple sequence alignment and phylogenetic tree analyses showed that SmMYB97 was clustered with AtMYB11, AtMYB12, and ZmP1 in the subgroup S7 regulating flavonol biosynthesis. SmMYB97 was highly expressed in S. miltiorrhiza leaves and induced by methyl jasmonate (MeJA). SmMYB97 was localized in the nucleus and had strong transcriptional activation activity. SmMYB 97 overexpression increased phenolic acid and tanshinone biosynthesis and upregulated the genes implicated in these processes. Yeast one-hybrid and transient transcriptional activity assays disclosed that SmMYB97 binds the PAL1 , TAT1 , CPS1 , and KSL1 promoter regions. SmJAZ8 interacts with SmMYB97 and downregulates the genes that it controls. This study partially clarified the regulatory network of MeJA-mediated secondary metabolite biosynthesis in S. miltiorrhiza .

The R2R3-MYB family is one of the largest families of plant transcription factor playing significant roles in plant growth. Although this gene family has been studied in many species, the R2R3-MYBs in Hypericum perforatum which is the first sequenced species in Malpighiales have not been analyzed. A total of 109 R2R3-MYB genes were identified in H. perforatum and clustered into 36 clades. Gene Ontology analysis revealed that most of the R2R3-MYB genes were involved in biological processes. Four kinds of cis-acting elements were found within the promoter regions, the majority of which were related to the stress responses and plant growth/development. The transcriptome data of different tissues (root, stems, leaves, and flowers) showed that the spatial expression profiles of R2R3-MYBs were different. Also, real-time quantitative PCR analysis revealed that eleven stress-related R2R3-MYB genes showed specific expression patterns under diverse treatments. In addition, sub-cellular localization analysis indicated that five significant proteins HpMYB45, HpMYB48, HpMYB55, HpMYB63, and HpMYB70 were all localized in the nucleus. This study was the first report on identification and characterization of R2R3-MYB gene family in H. perforatum. It facilitated the identification of tissue-preferential and stress-related genes and provided deep insights into the function of R2R3-MYBs in H. perforatum., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Transcription factors that include myeloblastosis (MYB), basic helix-loop-helix (bHLH), and tryptophan-aspartic acid (WD)-repeat protein often form a ternary complex to regulate the phenylpropanoid pathway. However, only a few MYB and bHLH members involved in the biosynthesis of salvianolic acid B (Sal B) have been reported, and little is known about Sal B pathway regulation by the WD40 protein transparent testa glabra 1 (TTG1)-dependent transcriptional complexes in Salvia miltiorrhiza. We isolated SmTTG1 from that species for detailed functional characterization. Enhanced or reduced expression of SmTTG1 was achieved by gain- or loss-of-function assays, respectively, revealing that SmTTG1 is necessary for Sal B biosynthesis. Interaction partners of the SmTTG1 protein were screened by yeast two-hybrid (Y2H) assays with the cDNA library of S. miltiorrhiza. A new R2R3-MYB transcription factor, SmMYB111, was found through this screening. Transgenic plants overexpressing or showing reduced expression of SmMYB111 upregulated or deregulated, respectively, the yields of Sal B. Both Y2H and bimolecular fluorescent complementation experiments demonstrated that SmMYB111 interacts with SmTTG1 and SmbHLH51, a positive regulator of the phenolic acid pathway. Our data verified the function of SmTTG1 and SmMYB111 in regulating phenolic acid biosynthesis in S. miltiorrhiza. Furthermore, ours is the first report of the potential ternary transcription complex SmTTG1-SmMYB111-SmbHLH51, which is involved in the production of Sal B in that species.

The dried root of Gentiana macrophylla is a well-known traditional Chinese herbal medicine for treating jaundice, hepatitis, and stomachic and choleretic ailments. However, natural sources are now in short supply. A lack of information about its genetic background has been a great hindrance to producing its active constituents via genetic engineering. We performed RNA-seq to obtain 42,918 unigenes (average length = 667 bp) in its transcriptome. Of these, 32,141 (74.89 %) were annotated and 2,339 unigenes were assigned to secondary-metabolite pathways. In all, 114 putative unigenes involved in secoiridoid biosynthesis were identified in our transcriptome library. A Blast X search against the Arabidopsis gene regulatory information server showed that 4,413 unigenes are homologous to transcription factor genes from Arabidopsis. Organ-specific genes and candidate gene expression profiles were also investigated with digital gene expression technology. Quantitative PCR was used to verify the expression patterns of several novel transcripts involved in secoiridoid biosynthesis. Our results not only enrich the gene resource but will also benefit research into the molecular genetics and functional genomics of this species.

Lotus corniculatus L. is used worldwide as a forage crop due to its abundance of secondary metabolites and its ability to grow in severe environments. Although the entire genome of L. corniculatus var. japonicus R. is being sequenced, the differences in morphology and production of secondary metabolites between these two related species have led us to investigate this variability at the genetic level, in particular the differences in flavonoid biosynthesis. Our goal is to use the resulting information to develop more valuable forage crops and medicinal materials. Here, we conducted Illumina/Solexa sequencing to profile the transcriptome of L. corniculatus. We produced 26,492,952 short reads that corresponded to 2.38 gigabytes of total nucleotides. These reads were then assembled into 45,698 unigenes, of which a large number associated with secondary metabolism were annotated. In addition, we identified 2,998 unigenes based on homology with L. japonicus transcription factors (TFs) and grouped them into 55 families. Meanwhile, a comparison of four tag-based digital gene expression libraries, built from the flowers, pods, leaves, and roots, revealed distinct patterns of spatial expression of candidate unigenes in flavonoid biosynthesis. Based on these results, we identified many key enzymes from L. corniculatus which were different from reference genes of L. japonicus, and five TFs that are potential enhancers in flavonoid biosynthesis. Our results provide initial genetics resources that will be valuable in efforts to manipulate the flavonoid metabolic pathway in plants.

Basic leucine zipper (bZIP) transcription factors (TFs) are one of the largest families involved in plant physiological processes such as biotic and abiotic responses, growth, and development, etc. In this study, 66 members of the bZIP family were identified in Bletilla striata, which were divided into 10 groups based on their phylogenetic relationships with AtbZIPs. A structural analysis of BsbZIPs revealed significant intron–exon differences among BsbZIPs. A total of 63 bZIP genes were distributed across 16 chromosomes in B. striata. The tissue-specific and germination stage expression patterns of BsbZIPs were based on RNA-seq. Stress-responsive expression analysis revealed that partial BsbZIPs were highly expressed under low temperatures, wounding, oxidative stress, and GA treatments. Furthermore, subcellular localization studies indicated that BsbZIP13 was localized in the nucleus. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays suggested that BsbZIP13 could interact with multiple BsSnRK2s. The results of this study provide insightful data regarding bZIP TF as one of the stress response regulators in B. striata, while providing a theoretical basis for transgenic and functional studies of the bZIP gene family in B. striata. [ABSTRACT FROM AUTHOR]

B-box (BBX) is a type of zinc finger transcription factor that contains a B-box domain. BBX transcription factors play important roles in plant photomorphogenesis, signal transduction, as well as abiotic and biological stress responses. However, the BBX gene family of Salvia miltiorrhiza has not been systematically investigated to date. For this study, based on the genomic data of Salvia miltiorrhiza, 27 SmBBXs genes were identified and clustered into five evolutionary branches according to phylogenetic analysis. The promoter analysis suggested that SmBBXs may be involved in the regulation of the light responses, hormones, stress signals, and tissue-specific development. Based on the transcriptome data, the expression patterns of SmBBXs under different abiotic stresses and plant hormones were analyzed. The results revealed that the expressions of the SmBBXs genes varied under different conditions and may play essential roles in growth and development. The transient expression analysis implied that SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were in the nucleus. A transcriptional activation assay showed SmBBX1, SmBBX4, SmBBX20, and SmBBX24 had transactivation activities, while SmBBX27 had none. These results provided a basis for further research on the role of SmBBXs in the development of Salvia miltiorrhiza. [ABSTRACT FROM AUTHOR]

Salvia miltiorrhiza Bunge is one of the most famous traditional Chinese medicinal plants. The two most important classes of pharmaceutically relevant compounds in S. miltiorrhiza are phenolic acids and tanshinones. The MYB family of transcription factors may efficiently regulate the secondary metabolism in plants. In this study, a subgroup 4 R2R3MYB transcription factor gene, SmMYB4, was isolated from S. miltiorrhiza and functionally characterized using overexpression and a RNAi-mediated silencing. We achieved a total of six overexpressions and eight RNAi transgenic lines from the Agrobacterium leaf disc method. The content of the total phenolics, rosmarinic acid, and salvianolic acid B markedly decreased in the SmMYB4-overexpressing lines but increased in the SmMYB4-RNAi lines. The content of the total tanshinones, cryptotanshinone, and tanshinone IIA decreased in the SmMYB4-overexpressing transgenic lines but increased in the SmMYB4-RNAi lines. A gene expression analysis demonstrated that SmMYB4 negatively regulated the transcription of the critical enzyme genes involved in the phenolic acid and tanshinone biosynthesis. The genetic control of this transcriptional repressor may be used to improve the content of these bioactive compounds in the cultivated S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Gentiana macrophylla, a medicinal plant with significant pharmacological properties, contains the bioactive compound gentiopicroside. Methyl jasmonate (MeJA) is an effective elicitor for enhancing the production of such compounds. However, little is known about MeJA-mediated biosynthesis of gentiopicroside. We investigated this phenomenon as well as gene expression profiles to determine the molecular mechanisms for MeJA-mediated gentiopicroside biosynthesis and regulation in G. macrophylla. Our HPLC results showed that Gentiana macrophylla seedlings exposed to MeJA had significantly higher concentrations of gentiopicroside when compared with control plants. We used RNA sequencing to compare transcriptional profiles in seedlings treated for 5 d with either 0 μmol L-1 MeJA (C) or 250 μmol L-1 MeJA (M5) and detected differentially expressed genes (DEGs). In total, 77,482 unique sequences were obtained from approximately 34 million reads. Of these, 48,466 (57.46%) sequences were annotated based on BLASTs performed against public databases. We identified 5,206 DEGs between the C and M5 samples, including genes related to the α-lenolenic acid degradation pathway, JA signaling pathway, and gentiopicroside biosynthesis. Expression of numerous enzyme genes in the glycolysis pathway was significantly up-regulated. Many genes encoding transcription factors (e.g. ERF, bHLH, MYB, and WRKY) also responded to MeJA elicitation. Rapid acceleration of the glycolysis pathway that supplies precursors for IPP biosynthesis and up-regulates the expression of enzyme genes in that IPP pathway are probably most responsible for MeJA stimulation of gentiopicroside synthesis. Our qRT-PCR results showed that the expression profiles of 12 gentiopicroside biosynthesis genes were consistent with the RNA-Seq data. These results increase our understanding about how the gentiopicroside biosynthesis pathway in G. macrophylla responds to MeJA. [ABSTRACT FROM AUTHOR]

Abstract: Phenolic acids are universally distributed in plant species, where they participate in numerous bioactivities. However, their low abundance in Salvia miltiorrhiza is a perplexing problem. We examined the heterologous expression of two transcription factors, Delila (DEL) and Rosea1 (ROS1), from Antirrhinum majus in genetically modified S. miltiorrhiza. Productions of rosmarinic acid (RA) and salvianolic acid B (Sal B) were significantly elevated during normal growth stages. Contents of other phenylpropanoid metabolites were enhanced and antioxidant activity was markedly stronger. Both SmCHS and SmRAS were stimulated by co-expression of DEL and ROS1. Because levels of phenylpropanoid metabolites were increased while both were co-expressed in S. miltiorrhiza, we believe that this will be a useful strategy for improving the contents of phenolic acids (especially RA and Sal B) in this species. [Copyright &y& Elsevier]