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Summary: Salvia miltiorrhiza holds significant importance in traditional Chinese medicine. Stress‐associated proteins (SAP), identified by A20/AN1 zinc finger structural domains, play crucial roles in regulating plant growth, development, resistance to biotic and abiotic stress, and hormone responses.Herein, we conducted a genome‐wide identification of the SAP gene family in S. miltiorrhiza. The expression analysis revealed a significant upregulation of SmSAP4 under methyl jasmonate (MeJA) and salt stress. Overexpressing SmSAP4 in S. miltiorrhiza hairy roots increased tanshinones content while decreasing salvianolic acids content, while RNAi‐silencing SmSAP4 had the opposite effect. SmSAP4 overexpression in both Arabidopsis thaliana and S. miltiorrhiza hairy roots decreased their salt stress tolerance, accompanied by increased activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and a hindered ability to maintain the Na+ : K+ ratio.Further investigations demonstrated that MeJA alleviated the inhibitory effect of SmJAZ3 on SmSAP4 activation by SmbHLH37 and SmERF73. However, MeJA did not affect the inhibition of SmSAP4 activation by SmJAZ8 through SmbHLH37.In summary, our research reveals that SmSAP4 negatively regulates the accumulation of salvianic acid through the SmJAZs‐SmbHLH37/SmERF73‐SmSAP4 module and positively impacting the accumulation of tanshinones. Additionally, it functions as a negative regulator under salt stress. [ABSTRACT FROM AUTHOR]

SUMMARY: The jasmonic acid (JA) signaling pathway plays an important role in promoting the biosynthesis of tanshinones. While individual transcription factors have been extensively studied in the context of tanshinones biosynthesis regulation, the influence of methyl jasmonate (MeJA)‐induced transcriptional complexes remains unexplored. This study elucidates the positive regulatory role of the basic helix–loop–helix protein SmMYC2 in tanshinones biosynthesis in Salvia miltiorrhiza. SmMYC2 not only binds to SmGGPPS1 promoters, activating their transcription, but also interacts with SmMYB36. This interaction enhances the transcriptional activity of SmMYC2 on SmGGPPS1, thereby promoting tanshinones biosynthesis. Furthermore, we identified three JA signaling repressors, SmJAZ3, SmJAZ4, and SmJAZ8, which interact with SmMYC2. These repressors hindered the transcriptional activity of SmMYC2 on SmGGPPS1 and disrupted the interaction between SmMYC2 and SmMYB36. MeJA treatment triggered the degradation of SmJAZ3 and SmJAZ4, allowing the SmMYC2–SmMYB36 complex to subsequently activate the expression of SmGGPPS1, whereas SmJAZ8 inhibited MeJA‐mediated degradation due to the absence of the LPIARR motif. These results demonstrate that the SmJAZ‐SmMYC2–SmMYB36 module dynamically regulates the JA‐mediated accumulation of tanshinones. Our results reveal a new regulatory network for the biosynthesis of tanshinones. This study provides valuable insight for future research on MeJA‐mediated modulation of tanshinones biosynthesis. Significance Statement: The results demonstrate that the SmJAZ‐SmMYC2–SmMYB36 module dynamically regulates the jasmonic acid‐mediated accumulation of tanshinones. [ABSTRACT FROM AUTHOR]

Phenolic acids are the main active ingredients in Salvia miltiorrhiza , which can be used for the treatment of many diseases, particularly cardiovascular diseases. It is known that salicylic acid (SA) can enhance phenolic acid content, but the molecular mechanism of its regulation is still unclear. Nonexpresser of PR genes 1 (NPR1) plays a positive role in the SA signaling pathway. In this study, we identified a SmNPR1 gene that responds to SA induction and systematically investigated its function. We found that SmNPR1 positively affected phenolic acid biosynthesis. Then, we identified a novel TGA transcription factor, SmTGA2, which interacts with SmNPR1. SmTGA2 positively regulates phenolic acid biosynthesis by directly up-regulating SmCYP98A14 expression. After double-gene transgenic analysis and other biochemical assays, it was found that SmNPR1 and SmTGA2 work synergistically to regulate phenolic acid biosynthesis. In addition, SmNPR4 forms a heterodimer with SmNPR1 to inhibit the function of SmNPR1, and SA can alleviate this effect. Collectively, these findings elucidate the molecular mechanism underlying the regulation of phenolic acid biosynthesis by SmNPR1–SmTGA2/SmNPR4 modules and provide novel insights into the SA signaling pathway regulating plant secondary metabolism. [ABSTRACT FROM AUTHOR]

DNA methylation plays important roles through the methyl-CpG-binding domain (MBD) to realize epigenetic modifications. Thirteen AtMBD proteins have been identified from the Arabidopsis thaliana genome, but the functions of some members are unclear. AtMBD3 was found to be highly expressed in pollen and seeds and it preferably binds methylated CG, CHG, and unmethylated DNA sequences. Then, two mutant alleles at the AtMBD3 locus were obtained in order to further explore its function using CRISPR/Cas9. When compared with 92.17% mature pollen production in the wild type, significantly lower percentages of 84.31% and 78.91% were observed in the mbd3-1 and mbd3-2 mutants, respectively. About 16–21% of pollen from the mbd3 mutants suffered a collapse in reproductive transmission, whereas the other pollen was found to be normal. After pollination, about 16% and 24% of mbd3-1 and mbd3-2 mutant seeds underwent early or late abortion, respectively. Among all the late abortion seeds in mbd3-2 plants, 25% of the abnormal seeds were at the globular stage, 31.25% were at the transition stage, and 43.75% were at the heart stage. A transcriptome analysis of the seeds found 950 upregulated genes and 1128 downregulated genes between wild type and mbd3-2 mutants. Some transcriptional factors involved in embryo development were selected to be expressed, and we found significant differences between wild type and mbd3 mutants, such as WOXs, CUC1, AIB4, and RGL3. Furthermore, we found a gene that is specifically expressed in pollen, named PBL6. PBL6 was found to directly interact with AtMBD3. Our results provide insights into the function of AtMBD3 in plants, especially in sperm fertility. [ABSTRACT FROM AUTHOR]

Tanshinone and phenolic acids are the most important active substances of Salvia miltiorrhiza, and the insight into their transcriptional regulatory mechanisms is an essential process to increase their content in vivo. SmMYB36 has been found to have important regulatory functions in the synthesis of tanshinone and phenolic acid; paradoxically, its mechanism of action in S. miltiorrhiza is not clear. Here, we demonstrated that SmMYB36 functions as a promoter of tanshinones accumulation and a suppressor of phenolic acids through the generation of SmMYB36 overexpressed and chimeric SmMYB36-SRDX (EAR repressive domain) repressor hairy roots in combination with transcriptomic-metabolomic analysis. SmMYB36 directly down-regulate the key enzyme gene of primary metabolism, SmGAPC, up-regulate the tanshinones biosynthesis branch genes SmDXS2, SmGGPPS1, SmCPS1 and down-regulate the phenolic acids biosynthesis branch enzyme gene, SmRAS. Meanwhile, SmERF6, a positive regulator of tanshinone synthesis activating SmCPS1, was up-regulated and SmERF115, a positive regulator of phenolic acid biosynthesis activating SmRAS, was down-regulated. Furthermore, the seven acidic amino acids at the C-terminus of SmMYB36 are required for both self-activating domain and activation of target gene expression. As a consequence, this study contributes to reveal the potential relevance of transcription factors synergistically regulating the biosynthesis of tanshinone and phenolic acid. [ABSTRACT FROM AUTHOR]

Background: Previous phytochemical studies showed that extracts from leaves and seed oil of Catalpa plants showed antioxidant and antitumor activities. However, the active components and their roles and molecular mechanisms were still incompletely identified. Objective: We aimed to extract and identify the active fraction from Catalpa bungei leaves and investigate its underlying mechanism in suppressing cervical cancer cell survival. Materials and Methods: Extracts from C. bungei leaves with 80% methanol were separated into different fractions. The component with optimal inhibitory effect on HeLa cell growth was isolated, identified, and examined. Results: Extracts from C. bungei leaves with methanol were separated into petroleum ether, ethyl acetate (EA), n-butanol, and water fractions. The chemicals in EA fraction exhibited optimal inhibition effects, which were further separated into 29 fractions by the silica column chromatography. The compound with optimal antitumor activity was eventually determined to be ursolic acid (UA) based on the results of Sephadex column chromatography and1H nuclear magnetic resonance analysis. UA at 5.0 and 10.0 μg/mL substantially inhibited the growth and migration of HeLa cells. UA also retarded cell cycle at G0/G1 stage and promoted cell apoptosis through activating death receptor and mitochondria-associated pathways. Conclusion: UA isolated from C. bungei leaves could inhibit growth and migration and induce apoptosis in HeLa cells. Abbreviations used: UA: Ursolic acid; EA: Ethyl acetate; HPV: Human papillomavirus, PARP: Poly ADP-ribose polymeras; ROS: Reactive oxygen species; PBS: Phosphate buffered saline; SRB: Sulfonyl rhodamine B; DMSO: Dimethyl sulfoxide; TLC: Thin layer chromatography; NMR: Nuclear magnetic resonance; CAS: Chemical abstracts service. [ABSTRACT FROM AUTHOR]

Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca2+ signaling is essential for the LPS-triggered immune response, Ca2+ channel blocker LaCl3 and CaM antagonist W-7 were used to investigate the role of Ca2+ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl3 and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca2+ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca2+ signaling. Ca2+ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Eucommia ulmoides Oliv. is widely regarded in China as a precious medicinal and commercial endemic tree. Due to cross-breeding or natural variation of E. ulmoides, the metabolite composition may vary significantly, making control of the medical quality difficult. In order to improve the rational development and utilization, the quality of seven varieties of E. ulmoides were evaluated based on metabolite profiles (total phenolic, total flavonoid, gutta-percha, aucubin, geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside, rutin, hyperoside, and astragalin), bioactivities (in vitro, in vivo antioxidant activities, and antibacterial activities) and HPLC fingerprint combined with chemometrics analysis. On this basis, the differences of medicinal parts (leaf and bark) were further carried out. For the traditional use of bark, Purple-leaf E. ulmoides was the most suitable. For the use of leaf, Qinzhong 1 and Purple-leaf E. ulmoides were appropriate. HPLC fingerprint analysis showed that significant differences in metabolite profiles exist among seven varieties of E. ulmoides. Combined with chemometrics analysis, seven varieties of E. ulmoides were divided into three groups from the use of leaf and bark. The analysis not only evaluated quality of seven varieties of E. ulmoides, but also could distinguish different varieties and different regions of origin. The results can provide theoretical basis for E. ulmoides resources utilization and cultivation of fine varieties. [ABSTRACT FROM AUTHOR]

In order to screen the Catalpa plant with high antioxidant activity and confirm the corresponding active fractions from Catalpa ovata G. Don, C. fargesii Bur., and C. bungei C. A. Mey., total flavonoid contents and antioxidant activities of the extracts/fractions of Catalpa plant leaves were determined. The determined total flavonoid content and antioxidant activity were used as assessment criteria. Those compounds with antioxidant activity were isolated with silica gel column chromatography and ODS column chromatography. Our results showed that the total flavonoid content in C. bungei C. A. Mey. (30.07 mg/g · DW) was the highest, followed by those in C. fargesii Bur. (25.55 mg/g · DW) and C. ovata G. Don (24.96 mg/g · DW). According to the determination results of total flavonoid content and antioxidant activity in 3 clones of leaves of C. bungei C. A. Mey., the total flavonoid content and antioxidant activity in crude extracts from C. bungei C. A. Mey. 6 (CA6) leaves were the highest. Moreover, the results showed that the total flavonoid content and antioxidant activities of ethyl acetate (EA) fraction in ethanol crude extracts in CA6 leaves were the highest, followed by n-butanol, petroleum ether (PE), and water fractions. Two flavonoid compounds with antioxidant activity were firstly isolated based on EA fraction. The two compounds were luteolin (1) and apigenin (2), respectively. [ABSTRACT FROM AUTHOR]

Teosinte Branched1/Cycloidea/Proliferating cell factor (TCP) genes constitute a unique and small class of transcription factors in plants, and are highly important in hormone response and secondary metabolite accumulation. However, Senna tora L. (S. tora) TCP genes have not been studied. In this study, the whole S. tora TCP genome was analyzed to explore its evolution and function. A protein interaction experiment was conducted to further analyze the involvement of TCP in jasmonic acid (JA) signal transduction. A total of 24 TCP TFs were identified in the S. tora genome, and were divided into 2 classes and 3 subfamilies. We found that 16 StTCP gene pairs with collinearity were distributed on 10 different chromosomes, with only one tandem repeat. The expression profiles at different seed developmental stages and tissue specificity analysis showed that StTCP4.1 and S tTCP11 may regulate the synthesis of anthraquinone compounds in S. tora. Gene expression levels determined under methyl jasmonate (MeJA) treatment and gene network correlation analysis showed that StTCP4.1 and StTCP11 may be involved in jasmonic acid (JA) response signaling. In addition, subcellular localization analysis showed that StTCP4.1 and StTCP11 were expressed and elicited their functions in the nucleus. Yeast two-hybrid (Y2H) and luciferase complementation (LCI) assay findings showed that StTCP4.1 could directly interact with StMYC2a. In summary, these findings lay a new foundation for improving understanding on the complex mechanism underlying StTCP function in response to JA-induced signaling pathways and on the regulation of secondary metabolite synthesis in S. tora. ● A total of 24 TCP genes were identified in the Senna tora genome, which was divided into 3 subfamilies. ● Only one tandem repeat events occurred in StTCP genes. ● The heatmap of 24 StTCPs in different stages of seed development was established. ● StTCP4.1 and StTCP11 were highly expressed in seeds, and functioned in the nucleus. ● StTCP4.1 and StTCP11 directly interact with StMYC2a, which could regulate the biosynthesis of anthraquinone compounds in the JA signaling pathway of Senna tora. [ABSTRACT FROM AUTHOR]