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1.
BMC Plant Biol ; 24(1): 798, 2024 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-39179969

RESUMO

BACKGROUND: In the past two decades, the impacts of Helium-Neon (He-Ne) laser on stress resistance and secondary metabolism in plants have been studied, but the signaling pathway which by laser regulates this process remains unclear. Therefore, the current study sought to explore the role of RBOH-dependent signaling in He-Ne laser-induced salt tolerance and elicitation of secondary metabolism in Salvia officinalis. Seeds were primed with He-Ne laser (6 J cm- 2) and peroxide hydrogen (H2O2, 5 mM) and 15-old-day plants were exposed to two salinity levels (0, 75 mM NaCl). RESULTS: Salt stress reduced growth parameters, chlorophyll content and relative water content (RWC) and increased malodialdehyde (MDA) and H2O2 contents in leaves of 45-old-day plants. After 48 h of salt exposure, higher transcription levels of RBOH (encoding NADPH oxidase), PAL (phenylalanine ammonia-lyase), and RAS (rosmarinic acid synthase) were recorded in leaves of plants grown from seeds primed with He-Ne laser and/or H2O2. Despite laser up-regulated RBOH gene in the early hours of exposing to salinity, H2O2 and MDA contents were lower in leaves of these plants after 30 days. Seed pretreatment with He-Ne laser and/or H2O2 augmented the accumulation of anthocyanins, total phenol, carnasol, and rosmarinic acid and increased total antioxidant capacity under non-saline and more extensively at saline conditions. Indeed, these treatments improved RWC, and K+/Na+ ratio, enhanced the activities of superoxide dismutase and ascorbate peroxidase and proline accumulation, and significantly decreased membrane injury and H2O2 content in leaves of 45-old-day plants under salt stress. However, applying diphenylene iodonium (DPI as an inhibitor of NADPH oxidase) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) after laser priming reversed the aforementioned effects which in turn resulted in the loss of laser-induced salt tolerance and secondary metabolism. CONCLUSIONS: These findings for the first time deciphered that laser can induce a transient RBOH-dependent H2O2 burst, which might act as a downstream signal to promote secondary metabolism and salt stress alleviation in S. officinalis plants.


Assuntos
Cinamatos , Depsídeos , Ácido Rosmarínico , Tolerância ao Sal , Salvia officinalis , Transdução de Sinais , Salvia officinalis/metabolismo , Salvia officinalis/fisiologia , Salvia officinalis/efeitos dos fármacos , Salvia officinalis/genética , Depsídeos/metabolismo , Cinamatos/metabolismo , Abietanos/metabolismo , Peróxido de Hidrogênio/metabolismo , Lasers , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas
2.
Plant Signal Behav ; 19(1): 2391659, 2024 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-39145499

RESUMO

Salvia miltiorrhiza, known as Danshen, is a traditional Chinese medicinal plant with significant cardiovascular benefits, attributed to its secondary metabolites, particularly tanshinones. Despite their medicinal value, tanshinones occur in low natural abundance, necessitating research to increase their content. This study explores the role of the ARF transcription factor (SmARF1) in tanshinone accumulation in Danshen. Overexpressing SmARF1 in hairy roots significantly increased tanshinone levels. EMSA and Dual-LUC assays revealed that SmMYB36, a transcription factor interacting with SmMAPK3, binds to and regulates the SmARF1 promoter. SmMYB36 alone inhibited the expression of SmARF1 gene, while its interaction with SmMAPK3 enhanced SmARF1 promoter activity. This MAPK3-MYB36-ARF1 module elucidates a complex regulatory mechanism for tanshinone biosynthesis, offering insights for targeted enhancement of tanshinone content through advanced biotechnological approaches.


Assuntos
Abietanos , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Salvia miltiorrhiza , Salvia miltiorrhiza/metabolismo , Salvia miltiorrhiza/genética , Abietanos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regiões Promotoras Genéticas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/genética
3.
Plant Physiol Biochem ; 214: 108932, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39018777

RESUMO

Understanding the regulatory biosynthesis mechanisms of active compounds in herbs is vital for the preservation and sustainable use of natural medicine resources. Diterpenoids, which play a key role in plant growth and resistance, also serve as practical products for humans. Tanshinone, a class of abietane-type diterpenes unique to the Salvia genus, such as Salvia miltiorrhiza, is an excellent model for studying diterpenoids. In this study, we discovered that a transcription factor, SmERF106, responds to MeJA induction and is located in the nucleus. It exhibits a positive correlation with the expression of SmKSL1 and SmIDI1, which are associated with tanshinone biosynthesis. We performed DNA affinity purification sequencing (DAP-seq) to predict genes that may be transcriptionally regulated by SmERF106. Our cis-elements analysis suggested that SmERF106 might bind to GCC-boxes in the promoters of SmKSL1 and SmIDI1. This indicates that SmKSL1 and SmIDI1 could be potential target genes regulated by SmERF106 in the tanshinone biosynthesis pathway. Their interaction was then demonstrated through a series of in vitro and in vivo binding experiments, including Y1H, EMSA, and Dual-LUC. Overexpression of SmERF106 in the hairy root of S. miltiorrhiza led to a significant increase in tanshinone content and the transcriptional levels of SmKSL1 and SmIDI1. In summary, we found that SmERF106 can activate the transcription of SmKSL1 and SmIDI1 in response to MeJA induction, thereby promoting tanshinone biosynthesis. This discovery provides new insights into the regulatory mechanisms of tanshinones in response to JA and offers a potential gene tool for tanshinone metabolic engineering strategy.


Assuntos
Abietanos , Acetatos , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Oxilipinas , Proteínas de Plantas , Salvia miltiorrhiza , Fatores de Transcrição , Salvia miltiorrhiza/metabolismo , Salvia miltiorrhiza/genética , Abietanos/metabolismo , Abietanos/biossíntese , Oxilipinas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Acetatos/metabolismo , Acetatos/farmacologia , Regiões Promotoras Genéticas/genética
4.
Plant Physiol Biochem ; 211: 108671, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38703500

RESUMO

Salvia castanea Diels, a close wild relative to the medicinal plant, Salvia miltiorrhiza Bunge, primarily grows in high-altitude regions. While the two species share similar active compounds, their content varies significantly. WRKY transcription factors are key proteins, which regulate plant growth, stress response, and secondary metabolism. We identified 46 ScWRKY genes in S. castanea and found that ScWRKY35 was a highly expressed gene associated with secondary metabolites accumulation. This study aimed to explore the role of ScWRKY35 gene in regulating the accumulation of secondary metabolites and its response to UV and cadmium (Cd) exposure in S. miltiorrhiza. It was found that transgenic S. miltiorrhiza hairy roots overexpressing ScWRKY35 displayed upregulated expression of genes related to phenolic acid synthesis, resulting in increased salvianolic acid B (SAB) and rosmarinic acid (RA) contents. Conversely, tanshinone pathway gene expression decreased, leading to lower tanshinone levels. Further, overexpression of ScWRKY35 upregulated Cd transport protein HMA3 in root tissues inducing Cd sequestration. In contrast, the Cd uptake gene NRAMP1 was downregulated, reducing Cd absorption. In response to UV radiation, ScWRKY35 overexpression led to an increase in the accumulation of phenolic acid and tanshinone contents, including upregulation of genes associated with salicylic acid (SA) and jasmonic acid (JA) synthesis. Altogether, these findings highlight the role of ScWRKY35 in enhancing secondary metabolites accumulation, as well as in Cd and UV stress modulation in S. miltiorrhiza, which offers a novel insight into its phytochemistry and provides a new option for the genetic improvement of the plants.


Assuntos
Cádmio , Depsídeos , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Salvia miltiorrhiza , Salvia miltiorrhiza/genética , Salvia miltiorrhiza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cádmio/metabolismo , Depsídeos/metabolismo , Metabolismo Secundário/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Benzofuranos/metabolismo , Ácido Rosmarínico , Cinamatos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/genética , Raios Ultravioleta , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Abietanos/metabolismo , Abietanos/biossíntese , Hidroxibenzoatos/metabolismo
5.
Microbiol Res ; 285: 127740, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38795408

RESUMO

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.


Assuntos
Abietanos , Cálcio , Endófitos , Peróxido de Hidrogênio , Raízes de Plantas , Salvia miltiorrhiza , Salvia miltiorrhiza/metabolismo , Salvia miltiorrhiza/microbiologia , Peróxido de Hidrogênio/metabolismo , Abietanos/biossíntese , Abietanos/metabolismo , Endófitos/metabolismo , Endófitos/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Lantânio/farmacologia , Lantânio/metabolismo , Regulação da Expressão Gênica de Plantas , Hypocreales/metabolismo , Hypocreales/genética
6.
Plant Cell Rep ; 42(11): 1733-1744, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37615706

RESUMO

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.


Assuntos
Produtos Biológicos , Diterpenos , Abietanos/metabolismo , Tripterygium/genética , Hidroxilação , Benzeno/metabolismo , Simulação de Acoplamento Molecular , Diterpenos/química , Diterpenos/metabolismo , Produtos Biológicos/metabolismo
7.
Mol Plant ; 16(3): 549-570, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36639870

RESUMO

The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.


Assuntos
Diterpenos Clerodânicos , Diterpenos , Plantas Medicinais , Scutellaria , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/metabolismo , Scutellaria/genética , Scutellaria/química , Scutellaria/metabolismo , Abietanos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismo
8.
Plant Cell Rep ; 42(2): 297-308, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36459184

RESUMO

KEY MESSAGE: Overexpression and antisense expression of Sm4CL2 re-directed the biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a widely used traditional Chinese medicine and its main active ingredients are water-soluble phenolic acids and lipophilic diterpenoids which are produced through the phenylpropanoid pathway and terpenoid pathway, respectively. 4-Coumaric acid: Coenzyme A ligase (4CL) is a key enzyme in the phenylpropanoid metabolism. We had obtained Sm4CL2-overexpressing (Sm4CL2-OE) and antisense Sm4CL2-expressing (anti-Sm4CL2) danshen hairy roots over ten years ago. In the follow-up study, we found that total salvianolic acids in Sm4CL2-OE-4 hairy roots increased to 1.35 times of the control-3, and that in anti-Sm4CL2-1 hairy roots decreased to 37.32% of the control-3, but tanshinones in anti-Sm4CL2-1 was accumulated to 1.77 ± 0.16 mg/g of dry weight, compared to undetectable in Sm4CL2-OE-4 and the control-3 hairy roots. Interestingly, Sm4CL2-OE-4 hairy roots contained more lignin, 1.36 times of the control-3, and enhanced cell wall and xylem lignification. Transcriptomic analysis revealed that overexpression of Sm4CL2 caused the upregulation of other phenylpropanoid pathway genes and antisense Sm4CL2 expression resulted in the downregulation of other phenylpropanoid pathway genes but activated the expression of terpenoid pathway genes like SmCYP76AK5, SmGPPS.SSUII.1 and SmDXS2. Protein-protein interaction analysis suggested that Sm4CL2 might interact with PAL, PAL4, CSE, CCoAOMT and SmCYP84A60, and appeared to play a key role in the interaction network. The tracking work in this study proved that Sm4CL2 could redirect both salvianolic acids and tanshinones biosynthesis possibly through synergistically regulating other pathway genes. It also indicated that genetic modification of plant secondary metabolism with biosynthetic gene might cause other responses through protein-protein interactions.


Assuntos
Diterpenos , Salvia miltiorrhiza , Salvia miltiorrhiza/genética , Salvia miltiorrhiza/metabolismo , Seguimentos , Abietanos/metabolismo , Diterpenos/metabolismo , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
9.
Plant J ; 113(4): 819-832, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36579923

RESUMO

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11 Gb in size; the genome has a scaffold N50 value of 95.5 Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.


Assuntos
Diterpenos , Rosmarinus , Salvia , Rosmarinus/genética , Rosmarinus/metabolismo , Salvia/genética , Salvia/metabolismo , Abietanos/metabolismo , Diterpenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Cromossomos
10.
Molecules ; 27(19)2022 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-36235219

RESUMO

Abietic acid (AA), dehydroabietic acid (DHA) and triptoquinones (TQs) are bioactive abietane-type diterpenoids, which are present in many edible vegetables and medicinal herbs with health-promoting properties. Evidence suggests that beneficial effects of diterpenes operate, at least in part, through effects on cells in the immune system. Dendritic cells (DCs) are a key type of leukocyte involved in the initiation and regulation of the immune/inflammatory response and natural or synthetic compounds that modulate DC functions could be potential anti-inflammatory/immunomodulatory agents. Herein, we report the screening of 23 known semisynthetic AA and DHA derivatives, and TQs, synthesized previously by us, in a multi-analyte DC-based assay that detects inhibition of pro-inflammatory cytokine production. Based on the magnitude of the inhibitory effect observed and the number of cytokines inhibited, a variety of activities among compounds were observed, ranging from inactive/weak to very potent inhibitors. Structurally, either alcohol or methyl ester substituents on ring A along with the introduction of aromaticity and oxidation in ring C in the abietane skeleton were found in compounds with higher inhibitory properties. Two DHA derivatives and two TQs exhibited a significant inhibition in all pro-inflammatory cytokines tested and were further investigated. The results confirmed their ability to inhibit, dose dependently, LPS-stimulated expression of the co-stimulatory molecules CD40 and/or CD86 and the production of the pro-inflammatory cytokines IL-1ß, IL-6, IL-12 and TNFα. Our results demonstrate that DC maturation process can be targeted by semisynthetic DHA derivatives and TQ epimers and indicate the potential of these compounds as optimizable anti-inflammatory/immunomodulatory agents.


Assuntos
Abietanos , Fator de Necrose Tumoral alfa , Abietanos/metabolismo , Abietanos/farmacologia , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Células Dendríticas , Ésteres/farmacologia , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
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