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.     

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza

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.     

The Mixture of Salvianolic Acids from Salvia miltiorrhiza and Total Flavonoids from Anemarrhena asphodeloides Attenuate Sulfur Mustard-Induced Injury

Sulfur mustard (SM) is a vesicating chemical warfare agent used in numerous military conflicts and remains a potential chemical threat to the present day. Exposure to SM causes the depletion of cellular antioxidant thiols, mainly glutathione (GSH), which may lead to a series of SM-associated toxic responses. MSTF is the mixture of salvianolic acids (SA) of Salvia miltiorrhiza and total flavonoids (TFA) of Anemarrhena asphodeloides. SA is the main water-soluble phenolic compound in Salvia miltiorrhiza. TFA mainly includes mangiferin, isomangiferin and neomangiferin. SA and TFA possess diverse activities, including antioxidant and anti-inflammation activities. In this study, we mainly investigated the therapeutic effects of MSTF on SM toxicity in Sprague Dawley rats. Treatment with MSTF 1 h after subcutaneous injection with 3.5 mg/kg (equivalent to 0.7 LD₅₀) SM significantly increased the survival levels of rats and attenuated the SM-induced morphological changes in the testis, small intestine and liver tissues. Treatment with MSTF at doses of 60 and 120 mg/kg caused a significant (p < 0.05) reversal in SM-induced GSH depletion. Gene expression profiles revealed that treatment with MSTF had a dramatic effect on gene expression changes caused by SM. Treatment with MSTF prevented SM-induced differential expression of 93.8% (973 genes) of 1037 genes. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 36 pathways, such as the MAPK signaling pathway, pathways in cancer, antigen processing and presentation. These data suggest that MSTF attenuates SM-induced injury by increasing GSH and targeting multiple pathways, including the MAPK signaling pathway, as well as antigen processing and presentation. These results suggest that MSTF has the potential to be used as a potential therapeutic agent against SM injuries.     

Yeast α-Glucosidase Inhibitory Phenolic Compounds Isolated from Gynura medica Leaf

Gynura medica leaf extract contains significant amounts of flavonols and phenolic acids and exhibits powerful hypoglycemic activity against diabetic rats in vivo. However, the hypoglycemic active constituents that exist in the plant have not been fully elaborated. The purpose of this study is to isolate and elaborate the hypoglycemic activity compounds against inhibition the yeast α-glucosidase in vitro. Seven phenolic compounds including five flavonols and two phenolic acids were isolated from the leaf of G. medica. Their structures were identified by the extensive NMR and mass spectral analyses as: kaempferol (1), quercetin (2), kaempferol-3-O-β-D-glucopyranoside (3), kaempferol-3-O-rutinoside (4), rutin (5), chlorogenic acid (6) and 3,5-dicaffeoylquinic acid methyl ester (7). All of the compounds except 1 and 3 were isolated for the first time from G. medica. Compounds 1–7 were also assayed for their hypoglycemic activity against yeast α-glucosidase in vitro. All of the compounds except 1 and 6 showed good yeast α-glucosidase inhibitory activity with the IC₅₀ values of 1.67 mg/mL, 1.46 mg/mL, 0.38 mg/mL, 0.10 mg/mL and 0.53 mg/mL, respectively.     

Facile synthesis of Sm2S3 diffused nanoflakes and their pseudocapactive behavior

The Sm₂S₃ thin films with diffused nanoflakes morphology are prepared by an environment-friendly facile chemical synthesis method and used in electrochemical supercapacitors. The structural, elemental and surface morphological characterization are carried out using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM) and wettability techniques. The FESEM images show tree root like distribution of flakes with average flake width of about 80 nm. The film surface is lyophilic with propylene carbonate contact angle of 21°. The supercapacitive measurements are carried out through cyclic voltammetry, galvanostatic charge-discharge and electrochemical impedance spectroscopy (EIS) techniques. The Sm₂S₃ film electrode exhibited a highest specific capacitance (C_s) of 213 Fg⁻¹ at 5 mVs⁻¹ scan rate in LiClO₄-propylene carbonate electrolyte. Asymmetric nature of charge–discharge curves confirmed pseudocapacitive behavior of electrode with energy and power densities of 39.39 Whkg⁻¹ and 4.33 kWkg⁻¹, respectively. An equivalent series resistance of 0.44 Ωcm⁻² indicated negligible ohmic losses in charge storage. An electrochemical stability of 81.47% is retained after 1000 cycles indicating that Sm₂S₃ is a promising candidate for supercapacitor application.     

Transport properties of solid electrolytes based on <Emphasis Type="Italic">Me</Emphasis>Sm<Subscript>2</Subscript>S<Subscript>4</Subscript>

The solid-solution regions in the MeSm₂S₄-MeS and MeSm₂S₄-Sm₂S₃ (Me = Ca, Ba) systems are revealed. The average ion, cation, and anion transport number of the synthesized solid electrolytes xSm₂S₃[Ca(Ba)S] · (100 ? x)Ca(Ba)Sm₂S₄ (x = 1?10 mol %) are determined by the electromotive force (emf) method with the use of concentration cells with and without transfer. In the phases under investigation, the ion transfer in the temperature range 673–723 K is provided by sulfide ions (\(t_{S^2 } \) = 1.00±0.02). The diffusion coefficients of S^2? ions in the solid electrolytes are determined by potentiostatic chronoamperometry. A vacancy mechanism of defect formation is proposed. It is demonstrated that the transport characteristics of the solid electrolytes based on the CaSm₂S₄ compound are worse than those of the solid electrolytes based on the BaSm₂S₄ compound.     

Biosynthesis of Oxylipins by Rhizoctonia solani with Allene Oxide and Oleate 8S,9S‐Diol Synthase Activities

Oxylipin biosynthesis by fungi is catalyzed by both the lipoxygenase (LOX) family and the linoleate diol synthase (LDS) family of the peroxidase‐cyclooxygenase superfamily. Rhizoctonia solani, a pathogenic fungus, infects staple crops such as potato and rice. The genome predicts three genes with 9–13 introns, which code for tentative dioxygenase (DOX)–cytochrome P450 fusion enzymes of the LDS family, and one gene, which might code for a 13‐LOX. The objective was to determine whether mycelia or nitrogen powder of mycelia oxidized unsaturated C₁₈ fatty acids to LDS‐ or LOX‐related metabolites. Mycelia converted 18:2n‐6 to 8R‐hydroxy‐9Z,12Z‐octadecadienoic acid and to an α‐ketol, 9S‐hydroxy‐10‐oxo‐12Z‐octadecenoic acid. In addition to these metabolites, nitrogen powder of mycelia oxidized 18:2n‐6 to 9S‐hydroperoxy‐10E, 12Z‐octadecadienoic, and 13S‐hydroperoxy‐9Z,11E‐octadecadienoic acids; the latter was likely formed by the predicted 13‐LOX. 18:1n‐9 was transformed into 8S‐hydroperoxy‐9Z‐octadecenoic and into 8S,9S‐dihydroxy‐10E‐octadecenoic acids, indicating the expression of 8,9‐diol synthase. The allene oxide, 9S(10)epoxy‐10,12Z‐octadecadienoic acid, is unstable and decomposes rapidly to the α‐ketol above, indicating biosynthesis by 9S‐DOX‐allene oxide synthase. This allene oxide and α‐ketol are also formed by potato stolons, which illustrates catalytic similarities between the plant host and fungal pathogen.     

ArabidopsisKCS5 and KCS6 Play Redundant Roles in Wax Synthesis

3-ketoacyl-CoA synthases (KCSs), as components of a fatty acid elongase (FAE) complex, play key roles in determining the chain length of very-long-chain fatty acids (VLCFAs). KCS6, taking a predominate role during the elongation from C26 to C28, is well known to play an important role in wax synthesis. KCS5 is one paralog of KCS6 and its role in wax synthesis remains unknown. Wax phenotype analysis showed that in kcs5 mutants, the total amounts of wax components derived from carbon 32 (C32) and C34 were apparently decreased in leaves, and those of C26 to C32 derivatives were obviously decreased in flowers. Heterologous yeast expression analysis showed that KCS5 alone displayed specificity towards C24 to C28 acids, and its coordination with CER2 and CER26 catalyzed the elongation of acids exceeding C28, especially displaying higher activity towards C28 acids than KCS6. BiLC experiments identified that KCS5 physically interacts with CER2 and CER26. Wax phenotype analysis of different organs in kcs5 and kcs6 single or double mutants showed that KCS6 mutation causes greater effects on the wax synthesis than KCS5 mutation in the tested organs, and simultaneous repression of both protein activities caused additive effects, suggesting that during the wax biosynthesis process, KCS5 and KCS6 play redundant roles, among which KCS6 plays a major role. In addition, simultaneous mutations of two genes nearly block drought-induced wax production, indicating that the reactions catalyzed by KCS5 and KCS6 play a critical role in the wax biosynthesis in response to drought.     

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.