Identification and expression analysis of expansin gene family in Salvia miltiorrhiza.

BACKGROUND: Expansins (EXP) are important enzymes that are involved in the extension of plant cells and regulation of root configurations, which play important roles in resisting various stresses. As a model medicinal plant, Salvia miltiorrhiza is well recognized for treating coronary heart disease, myocardial infection, and other cardiovascular and cerebrovascular diseases; however, the SmEXP gene family has not yet been analyzed. METHODS: The SmEXP family was systematically analyzed using bioinformatics. Quantitative real-time PCR was employed to analyze the tissue expression patterns of the SmEXP family, as well as its expression under abscisic acid (ABA) treatment and abiotic stress. Subcellular localization assay revealed the localization of SmEXLA1, SmEXLB1, and SmEXPA2. RESULTS: This study identified 29 SmEXP that belonged to four different subfamilies. SmEXP promoter analysis suggested that it may be involved in the growth, development, and stress adaptation of S. miltiorrhiza. An analysis of the expression patterns of SmEXP revealed that ABA, Cu2+, and NaCl had regulatory effects on its expression. A subcellular localization assay showed that SmEXLA1 and SmEXLB1 were located on the nucleus and cell membrane, while SmEXPA2 was located on the cell wall. CONCLUSION: For this study, the SmEXP family was systematically analyzed for the first time, which lays a foundation for further elucidating its physiological and biological functionality.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Integrative analysis of transcriptome and metabolome reveals the sesquiterpenoids and polyacetylenes biosynthesis regulation in Atractylodes lancea (Thunb.) DC.

Atractylodes lancea (Thunb.) is a perennial medicinal herb, with its dry rhizomes are rich in various sesquiterpenoids and polyacetylenes components (including atractylodin, atractylon and ß-eudesmol). However, the contents of these compounds are various and germplasms specific, and the mechanisms of biosynthesis in A. lancea are still unknown. In this study, we identified the differentially expressed candidate genes and metabolites involved in the biosynthesis of sesquiterpenoids and polyacetylenes, and speculated the anabolic pathways of these pharmaceutical components by transcriptome and metabolomic analysis. In the sesquiterpenoids biosynthesis, a total of 28 differentially expressed genes (DEGs) and 6 differentially expressed metabolites (DEMs) were identified. The beta-Selinene is likely to play a role in the synthesis of atractylon and ß-eudesmol. Additionally, the polyacetylenes biosynthesis showed the presence of 3 DEGs and 4 DEMs. Notably, some fatty acid desaturase (FAB2 and FAD2) significantly down-regulated in polyacetylenes biosynthesis. The gamma-Linolenic acid is likely involved in the biosynthesis of polyacetylenes and thus further synthesis of atractylodin. Overall, these studies have investigated the biosynthetic pathways of atractylodin, atractylon and ß-eudesmol in A. lancea for the first time, and present potential new anchor points for further exploration of sesquiterpenoids and polyacetylenes compound biosynthesis pathways in A. lancea.

Systematic Identification and Functional Analysis of the Hypericum perforatum L. bZIP Gene Family Indicating That Overexpressed HpbZIP69 Enhances Drought Resistance.

Basic leucine zipper (bZIP) transcription factors play significant roles in plants' growth and development processes, as well as in response to biological and abiotic stresses. Hypericum perforatum is one of the world's top three best-selling herbal medicines, mainly used to treat depression. However, there has been no systematic identification or functional analysis of the bZIP gene family in H. perforatum. In this study, 79 HpbZIP genes were identified. Based on phylogenetic analysis, the HpbZIP gene family was divided into ten groups, designated A-I and S. The physicochemical properties, gene structures, protein conserved motifs, and Gene Ontology enrichments of all HpbZIPs were systematically analyzed. The expression patterns of all genes in different tissues of H. perforatum (i.e., root, stem, leaf, and flower) were analyzed by qRT-PCR, revealing the different expression patterns of HpbZIP under abiotic stresses. The HpbZIP69 protein is localized in the nucleus. According to the results of the yeast one-hybrid (Y1H) assays, HpbZIP69 can bind to the HpASMT2 (N-acetylserotonin O-methyltransferase) gene promoter (G-box cis-element) to activate its activity. Overexpressing HpbZIP69 in Arabidopsis wild-type lines enhanced their tolerance to drought. The MDA and H2O2 contents were significantly decreased, and the activity of superoxide dismutase (SOD) was considerably increased under the drought stress. These results may aid in additional functional studies of HpbZIP transcription factors, and in cultivating drought-resistant medicinal plants.

The Dynamic Changes in the Main Substances in Codonopsis pilosula Root Provide Insights into the Carbon Flux between Primary and Secondary Metabolism during Different Growth Stages.

The dried root of Codonopsis pilosula (Franch.) Nannf., referred to as Dangshen in Chinese, is a famous traditional Chinese medicine. Polysaccharides, lobetyolin, and atractylenolide III are the major bioactive components contributing to its medicinal properties. Here, we investigated the dynamic changes of the main substances in annual Dangshen harvested at 12 time points from 20 May to 20 November 2020 (from early summer to early winter). Although the root biomass increased continuously, the crude polysaccharides content increased and then declined as the temperature fell, and so did the content of soluble proteins. However, the content of total phenolics and flavonoids showed an opposite trend, indicating that the carbon flux was changed between primary metabolism and secondary metabolism as the temperature and growth stages changed. The changes in the contents of lobetyolin and atractylenolide III indicated that autumn might be a suitable harvest time for Dangshen. The antioxidant capacity in Dangshen might be correlated with vitamin C. Furthermore, we analyzed the expression profiles of a few enzyme genes involved in the polysaccharide biosynthesis pathways at different growth stages, showing that CpUGpase and CPPs exhibited a highly positive correlation. These results might lay a foundation for choosing cultivars using gene expression levels as markers.

SmJAZ4 interacts with SmMYB111 or SmMYC2 to inhibit the synthesis of phenolic acids in Salvia miltiorrhiza.

Jasmonic acid (JA), as an important plant hormone, can induce the synthesis of phenolic acids in Salvia miltiorrhiza Bunge, a model medicinal plant, but the specific mechanism remains to be further elucidated. JA-responsive SmMYB111 positively regulates the biosynthesis of salvianolic acid B (SalB), but the molecular mechanism is unclear. Here, we found that SmMYB111 directly binds to the promoters of SmTAT1 and SmCYP98A14 and activates their transcription. Yeast two hybrid and bimolecular fluorescent complementation assay indicated that SmMYB111 interacts with SmJAZ4. Furthermore, we systematically characterized the function of SmJAZ4, which was highly expressed in flowers and roots and located in the nucleus and cell membrane. The contents of phenolic acids in the SmJAZ4-overexpressed transgenic plantlets and SmJAZ4-overexpressed transgenic hairy roots decreased significantly. SmJAZ4 interacts with SmMYC2 or SmMYB111 to repress their transcriptional activation activity on target enzyme genes of the biosynthesis pathway of phenolic acids. Overall, the molecular mechanism of SmJAZ4-SmMYC2/SmMYB111 module participating in JA signaling regulation of SalB biosynthesis was elucidated, which give a clue for the molecular regulation of phenolic acids biosynthesis in S. miltiorrhiza.

A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi.

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 (SbMYB12), 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 SbMYB12 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.

Whole genome and transcriptome reveal flavone accumulation in Scutellaria baicalensis roots.

Scutellaria baicalensis Georgi is a medicinal plant in the Lamiaceae family that contains high levels of 4'-deoxyflavone and other flavonoids in its roots. Therefore, it has strong potential as a plant resource for researching the biosynthesis of specific flavonoids. In this study, we report on a chromosome-level S. baicalensis genome assembled to nine chromosomes (376.81M) using PacBio, HiSeq XTen, and Hi-C assisted assembly. The assembly ratio was 99.22%, the contig N50 was 1.80 million bases, and the scaffold N50 was 40.57 million bases, with 31896 genes being annotated. Comparative genome analysis revealed that S. baicalensis and Salvia miltiorrhiza belonged to the same branch, and diverged 36.3 million years ago. Other typically correlated species were Boea hygrometrica and Sesamum indicum. We investigated the structural genes involved in flavonoid synthesis in combination with transcriptome sequencing analysis for different tissues (roots, stems, flowers, leaves) of purple, pink, and white flowers. The results revealed that S.baiF6H is involved in the accumulation of baicalein and was significantly increased in both purple roots vs. pink roots and white roots vs. pink roots. S.baiMYB gene family expression pattern analysis and co-expression network analysis revealed that S.baiMYB transcription factors primarily regulated the production of flavonoids in S. baicalensis. S.baiMYB serves as a major factor regulating flavonoid synthesis in the roots, where yeast one-hybrid assays revealed that these transcription factors could bind to the promoter regions of structural genes to control the accumulation of flavonoids. Genome and transcriptome sequencing, co-expression analysis, and yeast one-hybrid experiments provided valuable genetic resources for understanding flavonoid biosynthesis in S. baicalensis. These findings contribute to a better understanding of the accumulation of metabolites in Lamiaceae.

SmMYB4 Is a R2R3-MYB Transcriptional Repressor Regulating the Biosynthesis of Phenolic Acids and Tanshinones in Salvia miltiorrhiza.

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.

Overexpression of SmLAC25 promotes lignin accumulation and decreases salvianolic acid content in Salvia miltiorrhiza.

Laccase (LAC) is a blue multicopper oxidase that contains four copper ions, which is involved in lignin polymerization and flavonoid biosynthesis in plants. Although dozens of LAC genes have been identified in Salvia miltiorrhiza Bunge (a model medicinal plant), most have not been functionally characterized. Here, we explored the expression patterns and the functionality of SmLAC25 in S. miltiorrhiza. SmLAC25 has a higher expression level in roots and responds to methyl jasmonate, auxin, abscisic acid, and gibberellin stimuli. The SmLAC25 protein is localized in the cytoplasm and chloroplasts. Recombinant SmLAC25 protein could oxidize coniferyl alcohol and sinapyl alcohol, two monomers of G-lignin and S-lignin. To investigate its function, we generated SmLAC25-overexpressed S. miltiorrhiza plantlets and hairy roots. The lignin content increased significantly in all SmLAC25-overexpressed plantlets and hairy roots, compared with the controls. However, the concentrations of rosmarinic acid and salvianolic acid B decreased significantly in all the SmLAC25-overexpressed lines. Further studies revealed that the transcription levels of some key enzyme genes in the lignin synthesis pathway (e.g., SmCCR and SmCOMT) were significantly improved in the SmLAC25-overexpressed lines, while the expression levels of multiple enzyme genes in the salvianolic acid biosynthesis pathway were inhibited. We speculated that the overexpression of SmLAC25 promoted the metabolic flux of lignin synthesis, which resulted in a decreased metabolic flux to the salvianolic acid biosynthesis pathway.

Systematic Analysis and Functional Characterization of R2R3-MYB Genes in Scutellaria baicalensis Georgi.

R2R3-MYB transcription factors participate in multiple critical biological processes, particularly as relates to the regulation of secondary metabolites. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine and possesses various bioactive attributes including anti-inflammation, anti-HIV, and anti-COVID-19 properties due to its flavonoids. In the current study, a total of 95 R2R3-MYB genes were identified in S. baicalensis and classified into 34 subgroups, as supported by similar exon-intron structures and conserved motifs. Among them, 93 R2R3-SbMYBs were mapped onto nine chromosomes. Collinear analysis revealed that segmental duplications were primarily responsible for driving the evolution and expansion of the R2R3-SbMYB gene family. Synteny analyses showed that the ortholog numbers of the R2R3-MYB genes between S. baicalensis and other dicotyledons had a higher proportion compared to that which is found from the monocotyledons. RNA-seq data indicated that the expression patterns of R2R3-SbMYBs in different tissues were different. Quantitative reverse transcriptase-PCR (qRT-PCR) analysis showed that 36 R2R3-SbMYBs from different subgroups exhibited specific expression profiles under various conditions, including hormone stimuli treatments (methyl jasmonate and abscisic acid) and abiotic stresses (drought and cold shock treatments). Further investigation revealed that SbMYB18/32/46/60/70/74 localized in the nucleus, and SbMYB18/32/60/70 possessed transcriptional activation activity, implying their potential roles in the regulatory mechanisms of various biological processes. This study provides a comprehensive understanding of the R2R3-SbMYBs gene family and lays the foundation for further investigation of their biological function.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Genome-Wide Identification and Characterization of the WRKY Gene Family in Scutellaria baicalensis Georgi under Diverse Abiotic Stress.

The WRKY gene family is an important inducible regulatory factor in plants, which has been extensively studied in many model plants. It has progressively become the focus of investigation for the secondary metabolites of medicinal plants. Currently, there is no systematic analysis of the WRKY gene family in Scutellaria baicalensis Georgi. For this study, a systematic and comprehensive bioinformatics analysis of the WRKY gene family was conducted based on the genomic data of S. baicalensis. A total of 77 WRKY members were identified and 75 were mapped onto nine chromosomes, respectively. Their encoded WRKY proteins could be classified into three subfamilies: Group I, Group II (II-a, II-b, II-c, II-d, II-e), and Group III, based on the characteristics of the amino acid sequences of the WRKY domain and genetic structure. Syntenic analysis revealed that there were 35 pairs of repetitive fragments. Furthermore, the transcriptome data of roots, stems, leaves, and flowers showed that the spatial expression profiles of WRKYs were different. qRT-PCR analysis revealed that 11 stress-related WRKYs exhibited specific expression patterns under diverse treatments. In addition, sub cellular localization analysis indicated that SbWRKY26 and SbWRKY41 were localized in nucleus. This study is the first to report the identification and characterization of the WRKY gene family in S. baicalensis, which is valuable for the further exploration of the biological function of SbWRKYs. It also provides valuable bioinformatics data for S. baicalensis and provides a reference for assessing the medicinal properties of the genus.

Genome-Wide Identification of the Hypericum perforatum WRKY Gene Family Implicates HpWRKY85 in Drought Resistance.

WRKY, named for its special heptapeptide conserved sequence WRKYGOK, is one of the largest transcription factor families in plants and is widely involved in plant responses to biotic, abiotic, and hormonal stresses, especially the important regulatory function in response to drought stress. However, there is no complete comprehensive analysis of this family in H. perforatum, which is one of the most extensively studied plants and is probably the best-known herbal medicine on the market today, serving as an antidepressant, neuroprotective, an antineuralgic, and an antiviral. Here, we identified 86 HpWRKY genes according to the whole genome database of H. perforatum, and classified them into three groups through phylogenetic analysis. Gene structure, conserved domain, motif, cis-elements, gene ontology, and expression profiling were performed. Furthermore, it was found that HpWRKY85, a homologous gene of AtWRKY75, showed obvious responses to drought treatment. Subcellular localization analysis indicated that this protein was localized in the nucleus by the Arabidopsis protoplasts transient transfection. Meanwhile, HpWRKY85-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. The results provide a reference for further understanding the role of HpWRKY85 in the molecular mechanism of drought resistance of H. perforatum.

Functional Characterization of Serotonin N-Acetyltransferase Genes (SNAT1/2) in Melatonin Biosynthesis of Hypericum perforatum.

Hypericum perforatum is a traditional medicinal plant that contains various secondary metabolites. As an active component in H. perforatum, melatonin plays important role in plant antioxidation, growth, and photoperiod regulation. Serotonin N-acetyltransferase (SNAT) is the key enzyme involved in the last or penultimate step of phytomelatonin biosynthesis. A total of 48 members of SNAT family were screened and analyzed based on the whole genome data of H. perforatum, and two SNAT genes (HpSNAT1 and HpSNAT2) were functionally verified to be involved in the biosynthesis of melatonin. It was found that HpSNAT1 and HpSNAT2 were highly expressed in the leaves and showed obvious responses to high salt and drought treatment. Subcellular localization analysis indicated that these two proteins were both localized in the chloroplasts by the Arabidopsis protoplasts transient transfection. Overexpression of HpSNAT1 and HpSNAT2 in Arabidopsis (SNAT) and H. perforatum (wild-type) resulted in melatonin content 1.9-2.2-fold and 2.5-4.2-fold higher than that in control groups, respectively. Meanwhile, SNAT-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. In this study, the complete transgenic plants of H. perforatum were obtained through Agrobacterium-mediated genetic transformation for the first time, which laid a significant foundation for further research on the function of key genes in H. perforatum.

R2R3-MYB Transcription Factor SmMYB52 Positively Regulates Biosynthesis of Salvianolic Acid B and Inhibits Root Growth in Salvia miltiorrhiza.

The dried root of Salvia miltiorrhiza is a renowned traditional Chinese medicine that was used for over 1000 years in China. Salvianolic acid B (SalB) is the main natural bioactive product of S. miltiorrhiza. Although many publications described the regulation mechanism of SalB biosynthesis, few reports simultaneously focused on S. miltiorrhiza root development. For this study, an R2R3-MYB transcription factor gene (SmMYB52) was overexpressed and silenced, respectively, in S. miltiorrhiza sterile seedlings. We found that SmMYB52 significantly inhibited root growth and indole-3-acetic acid (IAA) accumulation, whereas it activated phenolic acid biosynthesis and the jasmonate acid (JA) signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed that SmMYB52 suppressed the transcription levels of key enzyme-encoding genes involved in the IAA biosynthetic pathway and activated key enzyme-encoding genes involved in the JA and phenolic acid biosynthesis pathways. In addition, yeast one-hybrid (Y1H) and dual-luciferase assay showed that SmMYB52 directly binds to and activates the promoters of several key enzyme genes for SalB biosynthesis, including SmTAT1, Sm4CL9, SmC4H1, and SmHPPR1, to promote the accumulation of SalB. This is the first report of a regulator that simultaneously affects root growth and the production of phenolic acids in S. miltiorrhiza.

Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.

Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.

SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza.

Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA3), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.

MiR408-SmLAC3 Module Participates in Salvianolic Acid B Synthesis in Salvia miltiorrhiza.

MicroRNAs (miRNAs) are important regulators of gene expression involved in plant development and abiotic stress responses. Recently, miRNAs have also been reported to be engaged in the regulation of secondary plant metabolism. However, there are few functional studies of miRNAs in medicinal plants. For this study, we obtained Sm-miR408 interference lines to investigate the function of Sm-miR408 in a medicinal model plant (Salvia miltiorrhiza). It was found that inhibiting the expression of Sm-miR408 could increase the content of salvianolic acid B and rosmarinic acid in the roots. The SmLAC3 and Sm-miR408 expression patterns were analyzed by qRT-PCR. A 5' RLM-RACE assay confirmed that Sm-miR408 targets and negatively regulates SmLAC3. Moreover, the overexpression of SmLAC3 in S. miltiorrhiza promoted the accumulation of salvianolic acids in the roots. Furthermore, the lignin content of the roots in overexpressed SmLAC3 lines was decreased. Taken together, these findings indicated that Sm-miR408 modulates the accumulation of phenolic acids in S. miltiorrhiza by targeting SmLAC3 expression levels.

Transcription factor SmSPL7 promotes anthocyanin accumulation and negatively regulates phenolic acid biosynthesis in Salvia miltiorrhiza.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play critical regulatory roles during plant growth and development. However, the functions of SPLs in Salvia miltiorrhiza (SmSPLs; a model medicinal plant) have not been reported. Here, the expression patterns and functions of SmSPL7 were characterized in S. miltiorrhiza. SmSPL7 was expressed in all parts of S. miltiorrhiza, with the highest expression level in the leaves, and could be inhibited by multiple hormones, including methyl jasmonate, auxin, abscisic acid, and gibberellin. SmSPL7 is localized within the nucleus and exhibits robust transcriptional activation activity. Transgenic lines overexpressing SmSPL7 demonstrated pronounced growth inhibition, accompanied by increased anthocyanin accumulation via the genetic activation of the anthocyanin biosynthesis pathway. However, SmSPL7 overexpression significantly decreased salvianolic acid B (SalB) production by inhibiting the transcripts of genes implicated in its biosynthesis pathway. Further analysis indicated that SmSPL7 directly binds to SmTAT1 and Sm4CL9 promoters and blocks their expression to inhibit the biosynthesis of SalB. Taken together, these results indicate that SmSPL7 is a negative regulator of SalB biosynthesis but positively regulates anthocyanin accumulation in S. miltiorrhiza. These findings provide new insights into the functionality of the SPL family while establishing an important foundation for further uncovering the crucial roles of SmSPL7 in the growth of S. miltiorrhiza.