Rosmarinic Acid Synthase Research Articles

Analysis of rosmarinic acid synthase (RAS) gene family and functional study of SmRAS1/2/4 in Salvia miltiorrhiza

Rosmarinic acid synthase is an essential enzyme involved in the biosynthesis of rosmarinic acid (RA), which facilitates the coupling of phenylpropanoid and tyrosine-derived pathway products. Our study identified six SmRAS genes in Salvia miltiorrhiza, with SmRAS1 being the only one functionally characterized to date. Real-time quantitative PCR was employed to analyze the expression profiles of the SmRAS gene family, revealing that SmRAS1/2/4 are predominantly expressed in the roots, which are the medicinal components of S. miltiorrhiza. SmRAS2 and SmRAS4 exhibited significant responses to abscisic acid (ABA), gibberellin (GA3), and methyl jasmonate (MeJA) stimuli, while SmRAS1 had notable responses to GA3 and MeJA. β-glucuronidase (GUS) staining in transgenic Arabidopsis thaliana confirmed a spatiotemporal expression pattern of SmRAS1/2/4 that was consistent with the qRT-PCR results. SmRAS1/2/4 are primarily localized to the cytoplasm and plasma membrane. Our findings suggested that the overexpressions of SmRAS1 or SmRAS4 led to increased levels of salvianolic acid B (SalB) and RA, with a concomitant decrease in the Danshensu (DSS) content, which served as a substrate. In contrast, RNA interference lines exhibited a downward trend in the content of these substances. Interestingly, no significant changes were detected in the SalB, RA, or DSS contents due to the overexpression of SmRAS2 or RNA interference lines. Collectively, our study demonstrated that SmRAS1 and SmRAS4 are key regulators of RA and SalB biosynthesis in S. miltiorrhiza, while SmRAS2′s role appears less impactful, suggesting a complex regulatory network that influences the medicinal properties of this plant.

RBOH-dependent signaling is involved in He-Ne laser-induced salt tolerance and production of rosmarinic acid and carnosol in Salvia officinalis

BackgroundIn 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).ResultsSalt 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.ConclusionsThese 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.

Salvia multicaulis × Salvia officinalis hybridization: Changes in the phenolic and essential oil profiles, gene expression and biological activity under Saint Catherine conditions—Egypt

The Salvia genus, particularly the wild genotypes, received significant attention due to its antibacterial, antioxidant, antiviral, and anticancer properties. Crossbreeding wild and cultivated plants can conserve the genetic resources of wild plants and develop new hybrids with high yields and high resistance to climatic changes. In this study, Salvia multicaulis (P1, wild) and Salvia officinalis (P2, cultivated) have been crossed to produce a novel hybrid (F1 hybrid) in the Saint Catherin protectorate - Egypt. In P1, P2, and F1 hybrid, heterosis, phytochemical composition, gene expression of PAL (Phenylalanine ammonia-lyase), RAS1 (Rosmarinic acid synthase I), CYP (Cytochrome P450), and CS (1,8-Cineole synthase), besides antioxidant, anticancer, anti-inflammatory, and antibacterial effects were assessed. The F1 hybrid recorded the highest means of plant height in the two seasons. Heterosis over the mid-parents was positively associated with various traits, including stem thickness, number of branches, fresh yield, and percentage of oil in both seasons. Total phenolic and flavonoid content were found in the following order: P2 > P1 > F1. DPPH antioxidant effect was found in the following order: P2 > F1 > P1. The F1 hybrid had the highest content of rosmarinic acid (12.379 mg/g), quercetin (0.595 mg/g), and naringenin (0.152 mg/g), followed by P1 and P2. The 1,8-cineol and α-pinene were the main compounds in the essential oils of P1, P2, and F1. The 1,8-cineol was found in the following order: P2 > F1 > P1, while α-pinene was found in the following order: P1 > F1 > P2. The RAS1 expression showed a weak correlation with the concentration of rosmarinic acid, while the expressions of PAL and CYP were strongly correlated with the concentrations of main phenolics, including rosmarinic acid, quercetin, and naringenin. The 1,8-cineol content was negatively correlated with the expression of the CS gene. The P2 extract and the F1 hybrid essential oil showed potent anticancer, anti-inflammatory, and antibacterial properties. Crossbreeding S. multicaulis (P1, wild) and S. officinalis (P2, cultivated) in this study developed a new hybrid (F1) with good growth characteristics, high essential oil yields, high levels of bioactives, and good bioactivity. This makes it suitable for large-scale production for use in food and pharmaceutical applications.

Unraveling the transcriptional regulators of polyphenolic variation in Thymus daenensis Celak

Due to its economic value and antioxidant phenolic compositions, there is a growing interest for Thymus daenensis in the food, cosmetic, and pharmaceutical industries. The current study was motivated by the paucity of studies on the molecular regulation of polyphenols and flavonoids in T. daenensis. From breeding materials, we selected and explored the transcriptome profiles of two chemotypes (‘Zagheh-11’ and ‘Malayer-21’). Tandem mass spectrometry identified 14 phenolic and flavonoid compounds in T. daenensis. Quantitative HPLC-PDA also revealed that ‘Zagheh-11’ contains higher amounts of flavonoids (luteolin, apigenin, kaempferol, quercetin, and catechin derivatives), while ‘Malayer-21’ was a rich source of rosmarinic acid. RNA-seq data mining indicated that 1840 unigenes have differential expression (DE) patterns between these chemotypes. Mapping of unigenes in flavonoids and phenylpropanoids pathways identified a total of 74% and 45% of structural genes, respectively. Four out of 15 DE unigenes in the aforementioned pathways played a key role in the metabolic divergence of these genotypes. RNA-seq and qRT-PCR indicated that the richness of flavonoids in ‘Zagheh-11’ was driven by higher expression of chalcone isomerase (CHI) and flavonoid 3′-hydroxlase (F3′H). In contrast, overexpression of phenylalanine ammonia lyase (PAL) and rosmarinic acid synthase (RAS) coincided with enhanced accumulation of rosmarinic acid in ‘Malayer-21’. To further understanding of these four key genes, we performed bioinformatic and structural analysis on their complete coding sequences. These findings should improve knowledge on how specialized metabolites are governed by transcriptional regulators and which are suitable for targeted metabolic engineering.

Analysing a Group of Homologous BAHD Enzymes Provides Insights into the Evolutionary Transition of Rosmarinic Acid Synthases from Hydroxycinnamoyl-CoA:Shikimate/Quinate Hydroxycinnamoyl Transferases.

The interplay of various enzymes and compounds gives rise to the intricate secondary metabolic networks observed today. However, the current understanding of their formation and expansion remains limited. BAHD acyltransferases play important roles in the biosynthesis of numerous significant secondary metabolites. In plants, they are widely distributed and exhibit a diverse range of activities. Among them, rosmarinic acid synthase (RAS) and hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) have gained significant recognition and have been extensively investigated as prominent members of the BAHD acyltransferase family. Here, we conducted a comprehensive study on a unique group of RAS homologous enzymes in Mentha longifolia that display both catalytic activities and molecular features similar to HCT and Lamiaceae RAS. Subsequent phylogenetic and comparative genome analyses revealed their derivation from expansion events within the HCT gene family, indicating their potential as collateral branches along the evolutionary trajectory, leading to Lamiaceae RAS while still retaining certain ancestral vestiges. This discovery provides more detailed insights into the evolution from HCT to RAS. Our collective findings indicate that gene duplication is the driving force behind the observed evolutionary pattern in plant-specialized enzymes, which probably originated from ancestral enzyme promiscuity and were subsequently shaped by principles of biological adaptation.

Versatile CYP98A enzymes catalyse meta-hydroxylation reveals diversity of salvianolic acids biosynthesis.

Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester-forming products all were determined in SA-rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C-3' hydroxylation, and SmCYP98A14 preferentially catalyses C-3 hydroxylation in RA generation. In addition, relative to C-3' hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA-abundant species and versatility of CYP98A enzymes catalytic preference in meta-hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.

Accumulation of Polyphenols and Associated Gene Expression in Hairy Roots of Salvia viridis Exposed to Methyl Jasmonate.

Methyl jasmonate (MJA), a signaling molecule in stress pathways, can be used to induce secondary metabolite synthesis in plants. The present study examines its effects on the growth of Salvia viridis hairy roots, and the accumulation of bioactive compounds, and correlates it with the expression of genes involved in the phenylpropanoid pathway. To our knowledge, this study represents the first exploration of elicitation in S. viridis culture and the first comprehensive analysis of MJA's influence on such a wide array of genes within the polyphenol metabolic pathway in the Salvia genus. Plants were treated with 50 and 100 µM MJA, and samples were collected at intervals of one, three, five, and seven days post-elicitation. HPLC analysis revealed that MJA stimulated the accumulation of all tested compounds, with a 30% increase (38.65 mg/g dry weight) in total polyphenol content (TPC) on day five. Quantitative real-time polymerase chain reaction (RT-PCR) analysis demonstrated a significant increase in the expression of the phenylpropanoid pathway genes-TAT (tyrosine aminotransferase), HPPR (4-hydroxyphenylpyruvate reductase), PAL (phenylalanine ammonia-lyase), C4H (cinnamic acid 4-hydroxylase), 4CL (4-coumarate-CoA ligase), and RAS (rosmarinic acid synthase)-following MJA treatment. For the majority of the genes, this increase was observed after the first day of treatment. Importantly, our present results confirm strong correlations of the analyzed gene expression with polyphenol biosynthesis. These findings support the notion that hairy roots provide a promising biotechnological framework for augmenting polyphenol production. Additionally, the combination of elicitor treatment and transgenic technology emerges as a viable strategy to enhance the biosynthesis of these valuable metabolites.

Changes in Rosmarinic Acid Accumulation and Rosmarinic Acid Synthase Gene Expression in Thymus vulgaris L. Influenced by Phenology and Environmental Factors

Changes in Rosmarinic Acid Accumulation and Rosmarinic Acid Synthase Gene Expression in Thymus vulgaris L. Influenced by Phenology and Environmental Factors

The Influence of Methyl Jasmonate on Expression Patterns of Rosmarinic Acid Biosynthesis Genes, and Phenolic Compounds in Different Species of Salvia subg. Perovskia Kar L.

Salvia yangii B.T. Drew and Salvia abrotanoides Kar are two important fragrant and medicinal plants that belong to the subgenus Perovskia. These plants have therapeutic benefits due to their high rosmarinic acid (RA) content. However, the molecular mechanisms behind RA generation in two species of Salvia plants are still poorly understood. As a first report, the objectives of the present research were to determine the effects of methyl jasmonate (MeJA) on the rosmarinic acid (RA), total flavonoid and phenolic contents (TFC and TPC), and changes in the expression of key genes involved in their biosynthesis (phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and rosmarinic acid synthase (RAS)). The results of High-performance liquid chromatography (HPLC) analysis indicated that MeJA significantly increased RA content in S. yungii and S. abrotanoides species (to 82 and 67 mg/g DW, respectively) by 1.66- and 1.54-fold compared with untreated plants. After 24 h, leaves of Salvia yangii and Salvia abrotanoides species treated with 150 M MeJA had the greatest TPC and TFC (80 and 42 mg TAE/g DW, and 28.11 and 15.14 mg QUE/g DW, respectively), which was in line with the patterns of gene expression investigated. Our findings showed that MeJA dosages considerably enhanced the RA, TPC, and TFC contents in both species compared with the control treatment. Since increased numbers of transcripts for PAL, 4CL, and RAS were also detected, the effects of MeJA are probably caused by the activation of genes involved in the phenylpropanoid pathway.

System network analysis of Rosmarinus officinalis transcriptome and metabolome-Key genes in biosynthesis of secondary metabolites.

Medicinal plants contain valuable compounds that have attracted worldwide interest for their use in the production of natural drugs. The presence of compounds such as rosmarinic acid, carnosic acid, and carnosol in Rosmarinus officinalis has made it a plant with unique therapeutic effects. The identification and regulation of the biosynthetic pathways and genes will enable the large-scale production of these compounds. Hence, we studied the correlation between the genes involved in biosynthesis of the secondary metabolites in R. officinalis using proteomics and metabolomics data by WGCNA. We identified three modules as having the highest potential for the metabolite engineering. Moreover, the hub genes highly connected to particular modules, TFs, PKs, and transporters were identified. The TFs of MYB, C3H, HB, and C2H2 were the most likely candidates associated with the target metabolic pathways. The results indicated that the hub genes including Copalyl diphosphate synthase (CDS), Phenylalanine ammonia lyase (PAL), Cineole synthase (CIN), Rosmarinic acid synthase (RAS), Tyrosine aminotransferase (TAT), Cinnamate 4-hydroxylase (C4H), and MYB58 are responsible for biosynthesis of important secondary metabolites. Thus, we confirmed these results using qRT-PCR after treating R. officinalis seedlings with methyl jasmonate. These candidate genes may be employed for genetic and metabolic engineering research to increase R. officinalis metabolite production.

Transcriptomic analysis of Salvia miltiorrhiza hairy roots in response to smoke-isolated karrikinolide (KAR1) using RNA-seq

• KAR 1 might participated in the biosynthesis of phenylpropanoid by up-regulating genes involved in relative pathway in S. miltiorrhiza hairy roots. • The expression of 4CL, C4H, TAT, HPPR and RAS genes in S. miltiorrhiza hairy roots were up-regulated by treatment with KAR 1 , indicating that the salvianolic acids synthesis pathway was activated. • The genes related to the salvianolic acids biosynthesis were regulated by KAR 1 in S. miltiorrhiza hairy roots. The effect of karrikinolide (KAR 1 ) on the salvianolic acid biosynthesis in hairy roots of Salvia miltiorrhiza has been studied by applying transcriptomics technology. Application of Illumina RNA-seq provides insights into the transcriptome profiles 0, 24 and 48 h after treatment with KAR 1 . The transcriptome was assembled with Trinity software, producing 138,993 unigenes with an average length of 1711 bp and an N50 value of 2523 bp. These included generous secondary metabolite biosynthesis and genes defense-related. Our investigations indicated significant differences in the responses of S. miltiorrhiza to KAR 1 treatment. A total of 9411 and 9406 significant differentially expressed genes (DEGs) were respectively identified by treatment with KAR 1 at 24 and 48 h. Among these DEGs, 865 DEGs (24 h) and 596 DEGs (48 h) were respectively classified into KEGG pathways. The result indicated that a significant number of these DEGs were enriched in phenylpropanoid biosynthesis. The expression of most glycosylsis-, glycosyltransferase-related genes were largely induced by KAR 1 . Furthermore, the genes that participated in biosynthesis of salvianolic acids were up-regulated after KAR 1 treatment, including 4-coumaric acid CoA-ligase (4CL, 2 genes), phenylalanine ammonia-lyase (PAL, 3 genes), tyrosine aminotransferase (TAT, 5 genes), 4-hydroxyphenylpyruvate reductase (HPPR, 7 genes), rosmarinic acid synthase (RAS, 18 genes) and cinnamic acid 4-hydroxylase (C4H, 24 genes). The results revealed that genes related to the salvianolic acid biosynthesis were regulated by KAR 1 in S. miltiorrhiza hairy roots. The findings provide insights into the molecular mechanism of salvianolic acid biosynthesis and a reference for the genetics research of S. miltiorrhiza next.

Alleviation of the Byproducts Formation Enables Highly Efficient Biosynthesis of Rosmarinic Acid in Saccharomyces cerevisiae.

Rosmarinic acid as a polyphenolic compound has great values in the pharmaceutical, cosmetic, and food industries. To achieve efficient biosynthesis of rosmarinic acid, the major obstacles such as imbalanced metabolic flux among branching pathways and substrate promiscuity of pathway enzymes should be eliminated. Here, a rosmarinic acid producing Saccharomyces cerevisiae strain was constructed by introducing codon optimized d-lactate dehydrogenase gene mutant (OD-LDHY52A), 4-coumarate CoA ligase gene (OPc4CL2), and rosmarinic acid synthase gene (OMoRAS) into a previously constructed caffeic acid hyper-producer. To identify the metabolic bottleneck, the substrate specificity of OPc4CL2 and OMoRAS was figured out by bioconversion experiments and HPLC-MS/MS analysis. Subsequently, the byproducts formation was alleviated by removing prephenate dehydratase and tuning down the expression level of OPc4CL2. The final strain YRA113-15B produced 208 mg/L rosmarinic acid in a shake-flask culture (a 63-fold improvement over the initial strain), which was the highest rosmarinic acid titer by engineered microbial cells reported to date. This work provides a promising platform for fermentative production of rosmarinic acid and offers a strategy to overcome the intrapathway competition.

Hydroxycinnamoyltransferase and CYP98 in phenolic metabolism in the rosmarinic acid-producing hornwort Anthoceros agrestis

Main conclusionAnthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered.Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.

Integrative Omics Analyses Reveal the Effects of Copper Ions on Salvianolic Acid Biosynthesis.

Salvianolic acids, a group of secondary metabolites produced by Salvia miltiorrhiza, are widely used for treating cerebrovascular diseases. Copper is recognized as a necessary microelement and plays an essential role in plant growth. At present, the effect of copper on the biosynthesis of SalAs is unknown. Here, an integrated metabolomic and transcriptomic approach, coupled with biochemical analyses, was employed to dissect the mechanisms by which copper ions induced the biosynthesis of SalAs. In this study, we identified that a low concentration (5 μM) of copper ions could promote growth of S. miltiorrhiza and the biosynthesis of SalAs. Results of the metabolomics analysis showed that 160 metabolites (90 increased and 70 decreased) were significantly changed in S. miltiorrhiza treated with low concentration of copper ions. The differential metabolites were mainly involved in amino acid metabolism, the pentose phosphate pathway, and carbon fixation in photosynthetic organisms. The contents of chlorophyll a, chlorophyll b, and total chlorophyll were significantly increased in leaves of low concentration of copper-treated S. miltiorrhiza plants. Importantly, core SalA biosynthetic genes (laccases and rosmarinic acid synthase), SalA biosynthesis-related transcription factors (MYBs and zinc finger CCCH domain-containing protein 33), and chloroplast proteins-encoding genes (blue copper protein and chlorophyll-binding protein) were upregulated in the treated samples as indicated by a comprehensive transcriptomic analysis. Bioinformatics and enzyme activity analyses showed that laccase 20 contained copper-binding motifs, and its activity in low concentration of copper ions-treated S. miltiorrhiza was much higher than that in the control. Our results demonstrate that enhancement of copper ions of the accumulation of SalAs might be through regulating laccase 20, MYBs, and zinc finger transcription factors, and photosynthetic genes.

Inductive effect of titanium dioxide nanoparticles on the anticancer compounds production and expression of rosmarinic acid biosynthesis genes in Dracocephalum kotschyi transformed roots

Methoxylated flavonoids, mainly xanthomicrol and cirsimaritin that can be extracted from Dracocephalum kotschyi Boiss, have anticancer, antispasmodic and antiplatelet effects. The production of these valuable pharmaceutical compounds is one of the major goals of biotechnology studies. In this work, induced transformed roots were influenced by various concentrations of titanium dioxide nanoparticles (TiO2 NPs) at 24 or 48 h exposure time. The effects of TiO2 NPs were assessed on growth rate, activity of antioxidant enzymes, total phenol and flavonoid content (TPC and TFC) and rosmarinic acid (RA) and some flavonoids accumulation. The gene expression level of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) genes were assessed by real time PCR analysis. The transformed roots biomass was substantially increased in elicited roots in comparison with the control. The TPC, TFC and antioxidant enzymes activitywere affected by TiO2 NPs concentration and exposure time. Valuable flavonoids with anticancer characteristics along with xanthomicrol, cirsimaritin and isokaempferide exhibited an increase (70, 34.28 and 7.81-fold, respectively) versus the control. The maximum content of RA (530.5 μg g−1 FW), which was 4.30 times as great as that of control was detected in samples treated with TiO2 NPs (50 mg L−1) 24 h after elicitation. Real-time PCR analysis revealed a considerable increase in pal and ras expression rate engaged by TiO2 NPs levels and exposure time. Overall D. kotschyi transformed roots elicitation by TiO2 NPs led to a massive increment in the production of valuable anticancer flavonoids such as xanthomicrol, cirsimaritin and RA as polyphenol.

Increased phenylpropanoids production in UV-B irradiated Salvia verticillata as a consequence of altered genes expression in young leaves

Ultraviolet-B (UV–B) radiation as an environmental potential elicitor induces the synthesis of plant secondary metabolites. The effects of UV-B radiation on photosynthetic pigments and dry weight, biochemical and molecular features of old and young leaves of Salvia verticillata were investigated. Plants were exposed to 10.97 kJ m−2 day−1 of biologically effective UV-B radiation for up to 10 days. The sampling process was performed in four steps: 1, 5, 10, and 13 days (recovery time) after the start of irradiation. As a result of plant investment in primary and secondary metabolism, the production of phenolic compounds increased, while chlorophyll levels and leaf dry weight (%) declined. Under long-term UV-B exposure, young leaves exhibited the most significant reduction in chlorophyll a and b content and leaf dry weight. The highest level of total phenol (1.34-fold) and flavonoid concentration (2-fold) relative to the control was observed on the 5th day and recovery time, respectively. Young leaves demonstrated the highest amount of phenolic acids in recovery time. Young leaves on the 5th day of the experiment exerted the highest level of antioxidant activity when compared to the control. A positive correlation was observed between antioxidant activity and the amount of phenolic compounds. Regarding the expression of phenylpropanoid pathway genes, UV-B enhanced the expression of phenylalanine ammonia-lyase, tyrosine aminotransferase, and rosmarinic acid synthase with the highest level in young leaves on the 10th day. Overall, young leaves of S. verticillata indicated higher sensitivity to UV-B radiation and developed more tangible reactions to such radiation.

Effect of graphene / metal nanocomposites on the key genes involved in rosmarinic acid biosynthesis pathway and its accumulation in Melissa officinalis

BackgroundRecently, numerous investigations have been done to study graphene and silver nanoparticle in the fields of agriculture and medicine. In the present study, the green synthesis of nanoparticles with two concentrations (0, 40, 60 mM) and their effect on the molecular and biochemical biosynthesis pathway of rosmarinic acid in a new method, low cost, and safe for the environment has been investigated. The transcript levels of key genes in the rosmarinic acid biosynthesis pathway (Tyrosine aminotransferase, rosmarinic acid synthase, and phenylalanine-ammonia lyase) were studied using real-time quantitative polymerase chain reaction. Then, the rosmarinic acid content was evaluated using HPLC.ResultsThe results showed that a concentration-dependent manner was observed in treated plants. At the biochemical level, the use of nanocomposites at concentration of 40 mM showed higher soluble carbohydrate (37%), flavonoids (21%), total phenol (35%) as well as total protein (47%) compared to the control plants. HPLC results showed that rosmarinic acid content in the treated plants with a low concentration of nanocomposite (40 mM) was more affected than plants treated with a high concentration of nanocomposite (60 mM) (26%) and also compared to other treatments. At the molecular level, the result showed that Tyrosine aminotransferase and rosmarinic acid synthase gene expression was positively correlated with both silver nanoparticle concentrations and nanocomposite treatments, but phenylalanine-ammonia lyase gene expression was positively correlated only with nanocomposite at 40 mM concentration.ConcludeIt can conclude that the nanocomposite at low concentration is more likely to induce molecular and biochemical parameters. And also, in the rosmarinic acid biosynthesis pathway, the Tyrosine aminotransferase -derived pathway is more efficient than the phenylalanine-ammonia lyase -derived pathway by causing a nano-elicitor. Therefore, it was concluded that studied elicitor at low concentration, can create plants with higher production capacity.

SmJRB1 positively regulates the accumulation of phenolic acid in Salvia miltiorrhiza

Phenolic acids rank high among the major active metabolites produced in the Chinese herb, Salvia miltiorrhiza, which has a rapidly escalating medicinal demand. Phenolic acid biosynthesis can be induced by methyl jasmonate (MeJA), although the underlying molecular mechanism remains obscure. A jasmonic acid-responsive basic helix-loop-helix (bHLH) transcriptional factor, SmJRB1, was identified in this study from the MeJA-induced transcriptome library of S. miltiorrhiza. The SmJRB1 is localized in the nuclei illustrated by the transient-transformation of epidermal cells in tobacco leaves. Overexpression of the SmJRB1 significantly promoted the accumulation of phenolic acids and upregulated the genes that encode the principal enzymes such as rosmarinic acid synthase I (RAS1) and cytochrome P450-dependent monooxygenase (CYP98A14), which are known to promote the phenolic acid biosynthetic pathway. Further investigation revealed that SmJRB1 triggered the activation of RAS1 transcription, according to the Dual-Luciferase (Dual-LUC) and yeast one-hybrid (Y1H) assays. Furthermore, yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays demonstrated that SmJRB1 interacted with the Jasmonate ZIM-domain proteins (JAZ) SmJAZ3 and SmJAZ9, and was most likely responsible for the co-regulation of the biosynthesis of phenolic acids. Overall, these results demonstrate that the SmJRB1 is an activator that enhances the phenolic acid accumulation in S. miltiorrhiza, and the study provides insights into the phenolic acid accumulation-related regulatory mechanisms in S. miltiorrhiza, as well as on the metabolic manipulation of the bioactive substances present.

The Biosynthesis of Phenolic Compounds Is an Integrated Defence Mechanism to Prevent Ozone Injury in Salvia officinalis

Specialized metabolites constitute a major antioxidant system involved in plant defence against environmental constraints, such as tropospheric ozone (O3). The objective of this experiment was to give a thorough description of the effects of an O3 pulse (120 ppb, 5 h) on the phenylpropanoid metabolism of sage, at both biochemical and molecular levels. Variable O3-induced changes were observed over time among the detected phenylpropanoid compounds (mostly identified as phenolic acids and flavonoids), likely because of their extraordinary functional diversity. Furthermore, decreases in the phenylalanine ammonia-lyase (PAL), phenol oxidase (PPO), and rosmarinic acid synthase (RAS) activities were reported during the first hours of treatment, probably due to an O3-induced oxidative damage to proteins. Both PAL and PPO activities were also suppressed at 24 h from the beginning of exposure, whereas enhanced RAS activity occurred at the end of treatment and at the recovery time, suggesting that specific branches of the phenolic pathways were activated. The increased RAS activity was accompanied by the up-regulation of the transcript levels of genes like RAS, tyrosine aminotransferase, and cinnamic acid 4-hydroxylase. In conclusion, sage faced the O3 pulse by regulating the activation of the phenolic biosynthetic route as an integrated defence mechanism.

Silver nanoparticle pollutants activate oxidative stress responses and rosmarinic acid accumulation in sage.

In this study, physiological and molecular responses of sage (Salvia officinalis) to silver nanoparticles (SNPs) were studied. It is supposed that sage oxidative responses can be activated to overcome the negative effects of SNPs. Results showed the penetration of SNPs via leaf epidermis into the parenchyma cells after foliar application. A significant decrease of photosynthetic pigments and increase of cell injury indicators, the activity of enzymatic antioxidants and also the content of non-enzymatic antioxidants were observed after exposure of sage plants to 50 and 1000 mg l-1 SNPs compared to control plants. Phenolic compounds generally increased, but not in linear response to the dose level. The most abundant phenolic acid, rosmarinic acid (RA), increased more than eightfold at 100 mg l-1 SNPs. Furthermore, the content of RA, salvianolic acid A and B was positively correlated with the activity of phenylalanine ammonia-lyase and RA synthase, but not with tyrosine aminotransferase. It could be concluded that the content of phenolic compounds increased in response to lower SNPs concentrations (50 and 100 mg l-1 ). However, the oxidative stress responses continued above these concentrations.