Untargeted metabolomics analysis reveals the elicitation of important secondary metabolites upon treatment with various metal and metal oxide nanoparticles in Hypericum perforatum L. cell suspension cultures
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Abstract
In this study, the effects of treatment with Ag, Au, Cu, Pd, CeO2, CuO, TiO2 and ZnO nanoparticles (NPs) on plant secondary metabolism were tested using an untargeted metabolomic approach in cell suspension cultures of Hypericum perforatum L. Variations in the accumulation of secondary metabolites were observed in cultures after exposure to NPs. Overall, the number of compounds increased by metal NPs was higher than that of metal oxides. Among the metal and metal oxide NPs tested, Ag and CuO respectively induced the greatest changes in secondary metabolism. Ag NPs induced the cellular accumulation of bisxanthone (540.3 fold) gancaonin O (214.2 fold) and fusaroskyrin (98.6 fold). Compounds that were most induced by other NPs were: hyperxanthone C (Au), apigenin (Cu), emodin (Pd), emodin anthrone (CeO2), dihydroxydimethoxyxanthone I (CuO), quercetin (TiO2) and gallic acid (ZnO). The presented results show that the secondary metabolites elicited in H. perforatum vary between the types of NPs.
Nanomaterials usually have specific characteristics due to their incredibly tiny size, which also increases their surface area, providing a more interactive surface. Compared to their macro-sized counterparts, these tiny nanoparticles exhibit a multitude of size-dependent properties. Plant tissue culture (PTC) plays an important role in bioactive chemical synthesis, mass cultivation, protection, genetic control, and plant enhancement. Different nanoparticles (NPs) are utilized to improve the tissue culture responses of explants. Various nanoparticles, including cobalt, copper, silver, gold, zinc, selenium, titanium, iron, palladium, cerium, indium, manganese, aluminum, barium, silicon, nickel, zirconium, and their oxides, are used in this regard. Nowadays, it is critical to use nanosystems in conjunction with PTC for mass reproduction, conservation, genetic engineering, crop enhancement, and the synthesis of bioactive compounds. Nanostructured metal oxides play an important role in in vitro plant cultivation. The use of metal nanoparticles (MNPs) has successfully removed microbial contaminants from explants and had a favorable impact on organogenesis (increasing the growth of shoots, roots, and multiplication ratios), callus induction, metabolic changes, and the synthesis of secondary metabolites (NPs are used as elicitors or stress agents). Additionally, NPs cause somaclonal variation (modifications to DNA), improve cryopreservation (increasing the survival rate), and enhance genetic transformation (facilitating gene transformation to bypass the plant cell wall barrier and accelerating protoplast isolation). This review aims to summarize the current breakthroughs achieved by integrating nanotechnology with PTC.
Graphical abstract
... Metal and metal oxide NPs have got impact on the biosynthesis of phenylpropanoids in the Hypericum perforatum L. cells (Kruszka et al. 2022). Metal nanoparticles (Ag, Au, Cu and Pd) increased accumulation of xanthones, prenylated xanthones and beznophenones and reduced levels of flavonoids and hydroxycinnamic acid derivatives in cells. ...
... Increased quercetin content was observed in shoots and roots of Nigella arvensis L. treated with 50 mg/L NiO NPs (Modarresi et al. 2020). The level of several flavonoid aglycones like apigenin, kaempferol and quercetin was increased upon treatment with the Ag, Au, Cu and Pd NPs treatment, whereas flavonoid glucosides like quercetin 3-O-hexoside or quercetin 3-O-malonylhexoside was elicited by CuO NPs treatment in H. perforatum L. cell suspension cultures, (Kruszka et al. 2022). Anthocyanins are another subgroup of flavonoids and play an important role in the nutraceutical, pharmaceutical and food industries. ...
... Moreover, Ag NPs induced the biosynthesis of phenolic acids more strongly than AgNO 3 in the hairy root culture of Cucumis anguria L. (Chung et al. 2018b). Ag and Cu NPs stimulated the secretion of hydroxycinnamic acid and hydroxybenzoic acid derivatives from H. perforatum cells into media of cell suspension cultures (Kruszka et al. 2022). ...
Plants encounter various nanomaterials (NMs) as pesticides and fertilizers. It is also possible that nanomaterials reach plants as waste from consumer products and industry. The effects of such NMs on plants have been widely studied, and both positive and negative effects of NMs on plant growth and development have been reported. Recent metabolomics studies suggest that nanoparticles affect the concentration of secondary metabolites in plants by modulating reactive nitrogen/oxygen species, gene expression, and signaling pathways. Secondary metabolites are plant compounds that accumulate in plants through their secondary metabolism. To date, more than 200,000 defined structures of secondary metabolites have been identified, among which many of them possess antibacterial, antifungal, antiviral, anti-inflammatory, hepatoprotective, antidepressant, antioxidant, neuroprotective, and anticancer properties. The application of elicitors is a simple strategy to increase the production of secondary metabolites in plant cell and tissues. The ability of nanomaterials to induce plant secondary metabolism has recently been exploited in the elicitation of pharmaceutically important compounds from various plant species. The ability of different NMs to induce the accumulation of different classes of compounds in the same plant species has also been accomplished. The molecular mechanisms behind the effects of NMs on plant secondary metabolism revealed the putative genes involved in NM-mediated elicitation of various plant compounds in several reports. This chapter reviews the current understanding of the effects of nanoparticles on plant secondary metabolism and the elicitation of pharmacologically important compounds from plant species.
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... El-Sayed et al. (2021) studied the impact of iron oxide (FeO, < 50 nm), aluminum oxide (Al 2 O 3 , < 100 nm), zinc oxide (ZnO, < 30 nm), and titanium oxide (TiO 2 , < 50 nm) NPs at different concentrations (2.5, 5, and 10 mg L −1 ) on shoot cultures of Sequoia sempervirens and recorded the highest total phenolic and flavonoid contents in 5 mg L −1 TiO 2 NP-treated shoots when compared other treatments. Kruszka et al. (2022) studied the effects of Ag (15 nm), Au (14 nm), CuO (25-55), Pd (15 nm), CeO 2 (10 nm), TiO 2 (5-15 nm), and ZnO (30-40 nm) NPs at 0-50 mg L −1 in cell suspension cultures of Hypericum perforatum. They recorded higher concentrations of secondary compounds after treatment with metal NPs than after treatment with metal oxide NPs. ...
Specialized plant metabolites, such as phenolics, terpenes, terpenoids, nitrogen-containing compounds, and sulfur-containing compounds, are commercially valuable owing to their wide array of applications in the medical, pharmacological, cosmetic, agriculture, and food industries. Procuring valuable specialized metabolites from wild or cultivated plants is desirable; however, the concentrations and quality of secondary compounds vary between samples. Therefore, plant cells and organ cultures have been selected as viable alternatives for producing specialized metabolites. Elicitation is a strategy used to enhance the accumulation of specialized compounds in cell and organ cultures. Different biotic substances, including signaling chemicals such as salicylic acid and methyl jasmonate, elements of plant cell walls (cellulose and pectin), polysaccharides from microbes (chitin and glucan), and abiotic substances such as inorganic salts, heavy metals, UV radiation, and high salinity, have been successfully tested and used as elicitors for the hyperaccumulation of bioactive substances in cell and organ cultures. Recently, metals, metal oxide nanoparticles, and carbon-based nanomaterials have been used as unique elicitors to boost the synthesis of bioactive compounds in cell and organ cultures. The applications and usage of nanoparticles as elicitors in plant cell and organ cultures are summarized in this review. The mechanism of elicitation, toxicity, benefits, and drawbacks of using nanoparticles in plant cell and organ cultures are discussed.
Graphical abstract
... Secondary metabolites produced via plant tissue culture are of uniform quality and are available throughout the year in less time (Shasmita et al. 2018b). Several reports are available on H. perforatum where different biotechnological approaches, such as shoot culture (Gadzovska et al. 2005;Jafarirad et al. 2021), root culture (Cui et al. 2010a(Cui et al. , b, 2014Najafabadi et al. 2019), callus culture (Gadzovska et al. 2013;Ebadollahi et al. 2019), callus mediated oranogenesis (Mañero et al. 2012;Savio et al. 2012;Gaid et al. 2016) and cell suspension cultures (Gadzovska-Simic et al. 2012;Wang et al. 2015;Kruszka et al. 2022) have been used to increase the secondary metabolite content in H. perforatum. Various abiotic (e.g., amino acids, methyl jasmonate, salicylic acid, chitosan, UV rays, temperature etc.) and biotic (Aspergillus niger, A. flavus) elicitors have been used (Xu et al. 2005;Liu et al. 2007;Pavlík et al. 2007;Gadzovska-Simic et al. 2012;Gadzovska et al. 2013;Brasili et al. 2016;Tavakoli et al. 2020) which resulted in enhanced production of bioactive compounds in H. perforatum (Table 1, 2, 4, 5, 6). ...
... A combination of 2,4-D, BA and NAA are found to be optimum for the same (Table 6). In few cases, 2,4-D + KIN Walker et al. 2002), KIN + NAA + IAA (Xu et al. 2005(Xu et al. , 2008(Xu et al. , 2011, BA + Picloram (Sharafi et al. 2013) or NAA alone (Kruszka et al. 2022) have also been used. ...
Hypericum perforatum L., commonly known as St. John’s wort is an important medicinal plant, belonging to family Hypericaceae. Among all species of Hypericum, H. perforatum is most investigated and exploited. It is sold as one of the world’s topmost retailing antidepressants. This plant possesses antibacterial, antiviral, anti-inflammatory, and anticancerous properties. These medicinal properties are attributed to the presence of bioactive compounds, such as hypericins and pseudohypericins (naphthodianthrones), hyperforin and adhyperforin (prenylated acylphloroglucinols), quercetin, rutin, isoquercetin, and catechin (flavonoids), chlorogenic acid, caffeic acid, and tannic acid (phenols) and xanthones. The conventional methods of propagation of H. perforatum are time consuming and field grown plants are exposed to biotic and abiotic challenges which affect its phytochemical constituents. Moreover, these methods are also unable to meet the commercial demand of secondary metabolites. Therefore, in order to meet the growing raw material demand of pharmaceutical industries there is a need to develop effecient in vitro plant regeneration protocols for large scale production of H. perforatum plants and other biotechnological methods (cell, tissue and organ culture, cell suspension culture, hairy root culture, elicitation, etc.) for improvement of target bioactive compounds. The present review provides a comprehensive account of the available information on in vitro plant regeneration and biotechnological approaches used to enhance the secondary metabolite(s) content in H. perforatum during the past years. It also deals with the unexplored areas which might be exploited for drug discovery.
... Ag and Au NPs strongly induced the production of prenylated xanthones and HCA derivatives. Furthermore, the total content of benzoates and flavonoids showed the highest increase with the Ag and Cu NPs and Ag, Au, Cu NPs respectively (Kruszka et al., 2022). With the application of Fe 2 O 3 magnetic NPs, the total phenolic, flavonoid and lignin contents in suspension cultures of Dracocephlum polychaetum, increased compared to the control. ...
... To our knowledge, this is the first report on the impact of CNTs on flavonoid profiles in plant tissues. Previous studies have shown that metal-based nanoparticles, including Cu and Se nanoparticles, can increase the concentration of certain flavonoids and phenolic acids in plants Kruszka et al. 2022) ...
Carbon nanotubes as novel plant elicitors are intensively studied in biotechnology due to their concentration-dependent effects on plant health. This emphasizes the importance of studying nanomaterials in the field of plant nanotoxicology and enables a better understanding of their advantages and disadvantages for plant health. Researchers examined how various concentrations of multi-walled carbon nanotubes (MWCNTs) affect growth and polyphenolic accumulation in the medicinal herb, Melissa officinalis. Two-month-old plant shoots were sprayed with various concentrations (0-250 mg L −1) of COOH-functionalized MWCNTs and harvested two and three weeks after elicitation. TEM images confirmed MWCNTs uptake into the mesophyll and the vessels of leaves. Low to moderate MWCNT concentrations (50-100 mg L −1) boosted growth indices and increased total amount of phenols, flavonoids, and phenolic acids, peaking three weeks after treatment with 100 mg L −1 MWCNTs, without destroying the cells and subcellular organelles. HPLC analysis showed this treatment yielded the highest content of rosmarinic acid, salvianolic acid B, apigenin, and kaempferol. However, 250 mg L −1 MWCNTs damaged cells without boosting metabolite production. We suggest that elicitation with low to moderate MWCNT concentrations can be a useful tool for laboratory-scale production of phenolic metabolites in M. officinalis.
... Similarly, the application of AgNPs in the hairy and adventitious root cultures resulted in increased celastrol levels in the Celastrus paniculatus (Moola et al., 2022). Several bioactive secondary metabolites have been triggered in the cell suspension culture under the exposure of AgNPs, AuNPs, CuNPs, PdNPs, CeO 2 NPs, CuONPs, TiO 2 NPs, ZnONPs in the Hypericum perforatum (Kruszka et al., 2022). In another study, the 4.5 and 6 mg/L of TiO 2 NPs were suggested to be the most effective concentration ion-inducing phenolic and flavonoid compounds in the callus medium of Cicer arietinum (Al-Oubaidi and Kasid, 2015). ...
Nanotechnology has offered numerous growing opportunities in pharmaceutical, biomedical, cosmetics, textiles , electronics, automobiles, and agriculture. The broader implications on the commercial side are only recommended when a significant understanding of nano-triggered environmental toxicities is minimal or within acceptable limits. The agricultural outcomes are primarily based on the crop plantʼs performance in the growing areas. Therefore, productivity needed to be predicted by the plant responses under normal and stressed conditions. Here, the reactive oxygen species (ROS) level is a crucial and decisive factor for the overall performance of the desired genotype of particular crop plants. Since the entity is reactive and causing significant damage to the bio-machinery. Hence, regulation at the nano-dimension by the engineered nanomaterials (ENMs) is the most effective approach to mitigate the negative consequences. The preference for ENMs is strong because of having unique diversity in physical properties. Furthermore, the behavior of ROS is conditioned by hormonal concentration, secondary metabolites, and anti-oxidative systems. Therefore , a comprehensive study is needed to shed light on the ROS activity with the application of ENMs to direct research on the lesser exploited side.
... Due to the extraordinarily small dimensions, large surface areas, and high stability of Fe-NPs, it can show quite a lot of activity even at low concentration, as the solubility of iron increases (Ranjan et al. 2022). Although it is known that BIO-NPs significantly affect the secondary metabolite contents of plants (Kruszka et al. 2022), the morpho-physiological changes that occur in this process are not known. In fact, no data were found regarding physiological parameters that are coordinated with the growth and development of sweet basil seedlings of BIO-NPs. ...
Chemical fertilizers used in plant development and differentiation have become a global problem affecting the entire ecosystem, especially soil pollution. Food production demand with the increasing population has encouraged scientists to use biogenic nanoparticles in the agricultural field. Evaluation of growth, development, and differentiation processes of sweet basil (Ocimum basilicum L.) seedlings at gradually increasing concentrations of biogenic iron oxide nanoparticles (BIO-NPs) were identified by morphological and physiological parameters in this study. The results showed that growth parameters reached the maximum value at 100 mg/L but were less at other concentrations. At similar concentration, the stomatal density of the leaf was the maximum, while the stomatal area showed the lowest value. The levels of H2O2 and malondialdehyde (MDA) decreased in the treated seedlings. BIO-NPs increased the antioxidant defense and supported its growth by changing the antioxidant enzyme activities, H2O2, and MDA contents. The BIO-NP treatment provided positive improvements in phytochemical content in parallel with the growth and development of sweet basil seedlings. Different growth parameters, physiological results, supporting enzyme activities, and biochemical data revealed the contribution of the BIO-NP treatments to the growth and development of sweet basil seedlings. BIO-NPs improved higher phytochemical production of sweet basil, which may be suitable for its propagation on a commercial scale.
... In various plants, ZnONPs increased the production of bene cial compounds: essential oil ( On the other hand, some NPs, including copper hydroxide, cerium oxide, and copper oxide, actually lowered the amount of phenylpropanoids found in the plants studied (Huang et Zhao et al. 2017). Interestingly, Kruszka et al. (2022) showed that certain NPs (silver, gold, copper, and palladium) increased the amount of xanthones, prenylated xanthones, and benzophenones in St. ...
The experiment investigated the effects of different levels of zinc oxide nanoparticles (ZnONPs) (0, 10, 20, and 30 mg/L) and iron sulfate (13.9, 27.8, and 55.6 mg/L) on morphological and physiological responses of Stevia rebaudiana Bertoni plant under in vitro conditions. Results indicated that the combined application of ZnONPs at 10 mg and iron at 27.8 mg led to the highest increase in shoot number, height, and biomass, showing a respective rise of 17.37%, 39.66%, and 45.02% compared to control cultures. The highest pigment content and tissue antioxidant activity (83.48%) was observed with the combined presence of 10 mg/L ZnONPs and 27.8 mg/L iron. As ZnONP concentration increased in the culture medium, the combined effect on lipid peroxidation rate became more pronounced. The impact of ZnONPs on phenolic compound production varied depending on the specific substance. The iron content of shoots increased significantly by 41.11% under the influence of 27.8 mg/L iron and 10 mg/L ZnONP compared to control cultures. Interaction effects of treatments at various levels resulted in increased zinc content in shoots, peaking at 27.8 mg/L iron when ZnONP reached 20 mg/L, representing a 56.28% increment over control levels before slightly decreasing. The most increases in stevioside and rebaudioside were observed with the combination of 10 mg/L ZnONP and 27.8 mg/L iron, showing enhancements of 75.04% and 63.08%, respectively. These findings suggest that ZnONPs could stimulate the growth and enhance the bioactive components of stevia plants, making them a viable option as elicitors in in vitro batch cultures.
... Also, Fe 3 O 4 NPs have increased phenolic compound levels [63]. The Cu NPs and ZnO NPs minimally impacted the phenolic compound levels in infected onions, aligning with studies on Hypericum perforatum L. using various metal oxide nanoparticles [85]. The combination of salicylic acid and Fe 3 O 4 NPs yielded concentration-dependent effects on the phenolic compounds, reflecting the complex interactions between salicylic acid, nanoparticles, and the plant-pathogen system. ...
This study investigated the effectiveness of nanoparticles and chemical inducers in managing onion white rot caused by Sclerotium cepivorum. The pathogen severely threatens onion cultivation, resulting in significant yield losses and economic setbacks. Traditional fungicides, though effective, raise environmental concerns, prompting a shift toward eco-friendly alternatives. In this study, four S. cepivorum isolates were utilized, each exhibiting varying degrees of pathogenicity, with the third isolate from Abu-Hamad demonstrating the highest potency. During the in vitro studies, three nanoparticles (NPs) were investigated, including Fe3O4 NPs, Cu NPs, and ZnO NPs, which demonstrated the potential to inhibit mycelial growth, with salicylic acid and Fe3O4 NPs exhibiting synergistic effects. In vivo, these nanoparticles reduced the disease incidence and severity, with Fe3O4 NPs at 1000–1400 ppm resulting in 65.0–80.0% incidence and 80.0–90.0% severity. ZnO NPs had the most positive impact on the chlorophyll content, while Cu NPs had minimal effects. At 1000 ppm, Fe3O4 NPs had variable effects on the phenolic compounds (total: 6.28, free: 4.81, related: 2.59), while ZnO NPs caused minor fluctuations (total: 3.60, free: 1.82, related: 1.73). For the chemical inducers, salicylic acid reduced the disease (10.0% incidence, 25.0% to 10.0% severity) and promoted growth, and it elevated the chlorophyll values and enhanced the phenolic compounds in infected onions. Potassium phosphate dibasic (PDP) had mixed effects, and ascorbic acid showed limited efficacy toward disease reduction. However, PDP at 1400 ppm and ascorbic acid at 1000 ppm elevated the chlorophyll values and enhanced the phenolic compounds. Furthermore, this study extended to traditional fungicides, highlighting their inhibitory effects on S. cepivorum. This research provides a comprehensive comparative analysis of these approaches, emphasizing their potential in eco-friendly onion white rot management.
... However, Ag and CuO NPs induced greater alterations in secondary metabolism. Findings suggested that secondary metabolites elicitation in plants vary among different types of MBNPs (Kruszka et al., 2022). Moreover, Li et al. (2021) indicated that foliar application of ZnO (50 nm) and SiO 2 (20 nm) NPs at a concentration of 50 and 100 mg/L, and 20 and 60 mg/L, respectively on Cucumis sativus (cucumber) seedlings improved the metabolomic profile of plants by enhancing the TFC in the leaves, thus potentially regulating the stress resistance in the plants. ...
Nanoparticles (NPs) of metals and metal oxides have received increasing attention regarding their characteristic behavior in plant systems. The fate and transport of metal NPs and metal oxide NPs in plants is of emerging concern for researchers because they ultimately become part of the food chain. The widespread use of metal-based NPs (MBNPs) in plants has revealed their beneficial and harmful effects. This review addresses the main factors affecting the uptake, translocation, absorption, bioavailability, toxicity, and accumulation of MBNPs in different plant species. It appraises the mechanism of nanoparticle-plant interaction in detail and provides understanding of the estimation strategies for the associated pros and cons with this interplay. Critical parameters of NPs include, but are not limited to, particle size and shape, surface chemistry, surface charge, concentration , solubility, and exposure route. On exposure to MBNPs, the molecular, physiological, and biochemical reactions of plants have been assessed. We have filled knowledge gaps and answered research questions regarding the positive and negative effects of metal and metal oxide NPs on seed germination, callus induction, growth and yield of plant, nutritional content, antioxidants, and enzymes. Besides, the phytotoxicity, cytotox-icity, genotoxicity, and detoxification studies of MBNPs in plants have been outlined. Furthermore, the recent developments and future perspectives of the two-way traffic of interplay of MBNPs and plants have been provided in this comprehensive review.
... Cell culture has been used to industrially produce ginseng, comfrey, periwinkle, and Ajuga plants (Chen et al. 2016;Popova et al. 2023). In addition, suspension culture has improved the SMs of woody plants such as Ginkgo biloba and Hypericum perforatum Kruszka et al. 2022). ...
Hydrogen sulfide (H2S) as the third gasotransmitter could regulate many physiological processes such as growth and stress responses in plants. However, few studies focused on H2S affecting cell differentiation and secondary metabolism of suspension cells. In this study, we investigated the impact of sodium hydrogen sulfide (NaHS) doses (0, 0.5, 1.0, 3.0 mM) as H2S-donor at different time points on cell differentiation, antioxidant capacity, and accumulation of flavonoids (Fla) and terpene lactones (Lac) in Ginkgo biloba L. suspension cells. Results showed that distinct asymmetric division with a large and a small cell appeared in suspension cells under three NaHS treatments. Especially, the cells became enlarged, rounded and compact, and resembled embryonic cell mass in treated 3.0 mM. The 0.5 and 1.0 mM NaHS treatments efficiently promoted the growth, enhanced the enzyme activities such as SOD, POD, PAL, and 4CL (4-coumarate-Coenzyme A ligase), etc., as well as increased the levels of chlorophyll, carotenoid, AsA (Ascorbic acid), and total Fla or Lac in suspension cells. The best treatment was 1.0 mM treated for 4 or 7 days, and total Fla and Lac increased by 18.7% (4d) and 76.1% (7d) compared with the control, respectively. The high dose (3.0 mM) of NaHS treatment for 7 days restrained cells growth by decreasing photosynthetic pigments levels, and enhanced lipid peroxidation by increasing MDA content (141.5%) and the relative electrical conductivity levels (82.9%) compared to the control. High-dose NaHS also inhibited the above enzyme activities and total Flc and Lac accumulations. However, short-term 3.0 mM NaHS treatment (1d) could increase total Fla and Lac levels. This study provided new insights into the cell differentiation and the production of secondary metabolites in cell culture of G. biloba.
... Bergenin biosynthesis involves gallic acid formation as an intermediate step (Sriset et al., 2020). A similar impact of iron oxide nanoparticles as elicitors has been demonstrated in the callus cultures of Hypericum perforatum, where elicitation through iron oxide nanoparticles led to enhanced production of hyperforin and hypericin metabolites (Kruszka et al., 2022). Furthermore, the application of nano iron oxide in the callus cultures of Hyoscyamus reticulata led to the biosynthesis of the tropane alkaloids hyoscyamine and scopolamine (Lala, 2021). ...
In vitro callus cultures of Berginia ciliata were used to evaluate different ratios of iron oxide nanoparticles and methyl jasmonate for elicitation, with the goal of improving callus morphological features and bergenin bio-synthesis. Shoot explants were cultured using MS (Murashige and Skoog) media supplemented with three elicitation concentrations of iron oxide nanoparticles (30, 60, and 90 ug/l) and three elicitation concentrations of methyl jasmonate (50, 75, and 100 uM). The results were compared to the control without elicitor treatment. All the elicitation treatments significantly enhanced the phytochemical contents. In vitro callus elicitation with 75 uM MeJ and 90 ug/l iron oxide nanoparticles proved as the best among tested elicitation concentrations. The elicitation improved the water contents and fresh and dry weights of the callus. The cal-lus elicitation with 90 ug/l iron oxide nanoparticles increased the callus moisture contents by 16%. The elici-tation treatments affected callus color traits and improved the callus compactness. Callus elicitation increased the activities of bioactive antioxidants such as phenylalanine ammonia-lyase, superoxide dismu-tase, peroxidase, catalase, and ascorbate peroxidase. The contents of bergenin, a major phytochemical, were found enhanced under elicitor treatments. Furthermore, contents of total phenolics and flavonoids improved upon elicitation. The in vitro callus elicitation resulted in better antioxidant potential for B. ciliata as assessed through the DPPH free radical scavenging protocol. The callus elicitation with 90 ug/l iron oxide nanopar-ticles increased DPPH scavenging percentage by 68%. In vitro callus elicitation with iron oxide nanoparticles and methyl jasmonate is an effective measure to enhance bergenin contents and bioactive antioxidants in the callus cultures of B. ciliata.
... Celastrol, a therapeutic phytochemical 38 , was enhanced after AgNPs were applied to adventitious and hairy root cultures of Celastrus paniculatus. Recently, cell suspension cultures of Hypericum perforatum L. treated with metal (Ag, Au, Cu, Pd) and metal oxide (CeO 2 , CuO, TiO 2 , ZnO) NPs enhanced the production of a variety of bioactive secondary metabolites 39 . ...
The utilization of nanotechnology and biotechnology for enhancing the synthesis of plant bioactive chemicals is becoming increasingly common. The hairy root culture technique can be used to increase secondary metabolites such as tropane alkaloids. Agrobacterium was used to induce hairy roots from various explants of Hyoscyamus muticus. The effect of nano-silver particles (AgNPs) at concentrations of 0, 25, 50, 100, and 200 mg/L on tropane alkaloids synthesis, particularly hyoscyamine and scopolamine, was studied in transgenic hairy root cultures. Different types of explants obtained from 10-day-old seedlings of H. muticus were inoculated with two strains of Agrobacterium rhizogenes (15,834 and A4). The antimicrobial activity of an ethanolic extract of AgNPs-induced hairy root cultures of H. muticus was tested. The frequency of hairy roots was higher in hypocotyl, root, leaf, and stem explants treated with A. rhizogenes strain A4 compared to those treated with strain 15,834. In transgenic hairy root cultures, AgNPs application at a concentration of 100 mg/L resulted in the highest total tropane alkaloid production, which exhibited broad-spectrum antimicrobial activity. The study demonstrated the potential of nano-silver as an elicitor for promoting the production of target alkaloids in Hyoscyamus muticus hairy root cultures, which exhibit high biological activity.
... A recent study by Moola et al. (2022) found the treatment of adventitious and hairy root cultures of Celastrus paniculatus with silver nanoparticles (AgNPs) resulted in increased levels of celastrol, a significant phytochemical with therapeutic properties. Similarly, Kruszka et al. (2022) recently reported the elicitation of various bioactive SMs in cell suspension cultures of Hypericum perforatum L. treated with metal (Ag, Au, Cu, Pd) and metal oxide (CeO 2 , CuO, TiO 2 , ZnO) nanoparticles. This study demonstrated the potential of nanoparticles to stimulate the synthesis of biologically active compounds in plant cell cultures, which could be exploited for various medicinal and nutritional purposes. ...
... Further, Zhao et al. (2010) studied responses of different elicitor in Salvia miltiorrhiza and reported that Ag and Cd at 25 mM increased the tanshinone accumulation by about 11.5 fold. Although, our current knowledge on the mode of action of elicitors is related almost exclusively to secondary metabolism, in recent years, it has been demonstrated that primary metabolism may also be affected by elicitation (Ralph et al., 2006;Kruszka et al., 2022). Based on such observations, it has been suggested that elicitation may modulate the expression of molecules of primary metabolism involved in vacuolar transport and thereby regulates the levels of secondary metabolites (Vasconsuelo and Boland 2007). ...
... Increased bioactive compounds could be due to increased shikimate phenylpropanoid metabolism caused by ZnONPs (Zhao et al., 2020). The mechanism of action of ZnONPs in increasing charantin contents in bitter gourd may be due to better homeostasis of cytosolic Ca +2 , which may have acted as signaling molecules, boosting endogenous ROS production and subsequently increasing the phytochemical profile of bitter gourd plants (Kruszka et al., 2022). Nanoparticles mediated epigenetic regulation of modulation of Mitogen-Activated Protein Kinase (MAPK), which increases metabolite levels of flavonoids and phenolics. ...
Bitter gourd (Momordica charantia L.), grown in semi-arid and fragile soil, exhibits poor growth, yield loss, and reduced quantity of bioactive compounds which may be improved by seed priming with bio-rationale materials. A field experiment was undertaken from March to June 2022 to determine the efficacy of zinc oxide nanoparticles (ZnONPs) on bitter gourd growth, yield, and some phytochemical contents through seed priming. Plants of bitter gourd were divided into five sets. The first set served as a control and was not treated with ZnONPs. The plants of sets 2, 3, 4, and 5 were treated with ZnONPs primed seed at 0.5, 0.10, 0.15, or 0.20 mL∙L⁻¹ concentrations, and priming duration of 24 h. Seed priming with 0.20 mL∙L⁻¹ improved vine length, number of leaves, and number of branches per plant by 46, 21, and 39% compared to the control. Days to plant emergence and flowering were reduced and yield attributes improved by seed priming. Total phenolic, total flavonoids, and charantin, a steroidal saponin, contents as major secondary metabolite of fresh fruit of bitter gourd improved by 47, 36, and 52% treated with 0.20 mL∙L⁻¹ of ZnONPs primed seed. Seed priming with ZnONPs could be used to improve growth, germination, important metabolites and yield of bitter gourd.
... This indicates the signicant role of dissolved Cu ions on the toxicity of CuO NPs and MPs. Kruszka et al. 264 compared the effect of Cu and CuO NPs on the secondary metabolism of Hypericum perforatum L. cell suspension cultures and found that metal NPs induce higher metabolic changes than their counterpart metal oxide NPs. Table 7 summarizes the studies that used the metabolomics technique to assess the effect of other metal/metal oxide NPs in vitro on different cells. ...
Nowadays, nanomaterials (NMs) are widely present in daily life due to their significant benefits, as demonstrated by their application in many fields such as biomedicine, engineering, food, cosmetics, sensing, and energy. However, the increasing production of NMs multiplies the chances of their release into the surrounding environment, making human exposure to NMs inevitable. Currently, nanotoxicology is a crucial field, which focuses on studying the toxicity of NMs. The toxicity or effects of nanoparticles (NPs) on the environment and humans can be preliminary assessed in vitro using cell models. However, the conventional cytotoxicity assays, such as the MTT assay, have some drawbacks including the possibility of interference with the studied NPs. Therefore, it is necessary to employ more advanced techniques that provide high throughput analysis and avoid interferences. In this case, metabolomics is one of the most powerful bioanalytical strategies to assess the toxicity of different materials. By measuring the metabolic change upon the introduction of a stimulus, this technique can reveal the molecular information of the toxicity induced by NPs. This provides the opportunity to design novel and efficient nanodrugs and minimizes the risks of NPs used in industry and other fields. Initially, this review summarizes the ways that NPs and cells interact and the NP parameters that play a role in this interaction, and then the assessment of these interactions using conventional assays and the challenges encountered are discussed. Subsequently, in the main part, we introduce the recent studies employing metabolomics for the assessment of these interactions in vitro.
Summary
Because plants are static, they are always exposed to ambient nanoparticles (NPs) that, at sub-toxic quantities, cause abiotic stress and, at greater concentrations, cause phytotoxicity. In general, NPs cause plants to respond toxicologically by producing reactive oxygen species. Typically, plants activate genetic, biochemical, and ecological processes in response to various stresses, with both enzymatic and non-enzymatic anti-oxidant defense mechanism. The enhanced production of plants specialized natural products/metabolites, many of which have use significantly in the defensive response. Plants frequently produce or activate many kinds of specialized metabolites (such as alkaloids, terpenoids, phenolics, and flavonoids) in response to environmental stress and elicitors. These plant-specific metabolites (PSMs) play a crucial role in a plants to improve the capability to sustain the plants in a challenging environment. Many of such PSMs are utilized in variety of bioactivity and as a commercially value-added products for diverse purposes. Numerous studies on the induction of PSMs by various NPs, including metal oxide NPs, metallic NPs, and carbon-related nanomaterials, have been reported recently.
NPs have been shown to have adverse impacts on plant physiology, biochemistry, growth, and development, but still the mechanisms of specialized metabolic pathways in the plants are unknown. An improved understanding of NPs and their absorption, translocation, internalization, and elicitation mechanisms is needed in order to choose the best NP in terms of kind, size, and/or effective concentration with regard to production of PSMs in plant defense mechanisms. This review seeks to highlight the advanced research knowledge on how abiotic stressors and responses are mediated by nanoelicitors in order to establish a commercially viable and environmentally sustainable model to enhance the production of PSMs for healthcare, biotechnology, agriculture, cosmetics, pharmaceuticals, nutraceuticals, and many others purposes.
The experiment investigated the effects of different levels of zinc oxide nanoparticles (ZnONPs) (0, 10, 20, and 30 mg/L) and iron sulfate (13.9, 27.8, and 55.6 mg/L) on morphological and physiological responses of Stevia rebaudiana Bertoni plant under in vitro conditions. Results indicated that the combined application of ZnONPs at 10 mg and iron at 27.8 mg led to the highest increase in shoot number, height, and biomass, showing a respective rise of 17.37%, 39.66%, and 45.02% compared to control cultures. The highest pigment content and tissue antioxidant activity (83.48%) was observed with the combined presence of 10 mg/L ZnONPs and 27.8 mg/L iron. As ZnONP concentration increased in the culture medium, the combined effect on lipid peroxidation rate became more pronounced. The impact of ZnONPs on phenolic compound production varied depending on the specific substance. The iron content of shoots increased significantly by 41.11% under the influence of 27.8 mg/L iron and 10 mg/L ZnONP compared to control cultures. Interaction effects of treatments at various levels resulted in increased zinc content in shoots, peaking at 27.8 mg/L iron when ZnONP reached 20 mg/L, representing a 56.28% increment over control levels before slightly decreasing. The most increases in stevioside and rebaudioside were observed with the combination of 10 mg/L ZnONP and 27.8 mg/L iron, showing enhancements of 75.04% and 63.08%, respectively. These findings suggest that ZnONPs could stimulate the growth and enhance the bioactive components of stevia plants, making them a viable option as elicitors in in vitro batch cultures.
Elicitation, the process of stimulating plants to enhance the production of valuable secondary metabolites, has emerged as a significant avenue in the field of plant biotechnology. This review paper provides a comprehensive examination of elicitation, encompassing its mechanisms, the imperative for its utilization, and the diverse array of elicitors employed, including abiotic, biotic, and nanoparticle-based agents. By exploring the intricate signaling pathways and molecular responses triggered by external stimuli in this review, we gain insights into how plants tailor their metabolite production in reaction to their environment. Intricately intertwined with the mechanisms, the types of elicitors utilized are extensively discussed. Abiotic elicitors, encompassing physical factors and chemicals, biotic elicitors involving microbe-derived molecules, plant hormones and symbiotic microorganisms, and the emerging application of nanoparticles as elicitation agents are explored. The review further examines the application of these elicitors in both in-vitro and in-vivo cultures, showcasing their practical utility in controlled laboratory settings and real-world environments. By assessing the efficacy of diverse elicitors, this paper provides valuable insights into tailoring elicitation strategies to enhance secondary metabolite yields for various applications. Addressing the limitations and leveraging emerging technologies will undoubtedly steer elicitation research towards innovative breakthroughs, further enhancing our capacity to harness plants for sustainable and efficient secondary metabolite production.
Among the metals contaminants, cadmium (Cd) is one of the most toxic elements in cultivated soils, causing loss of yield and productivity in plants. Recently, nanomaterials have been shown to mitigate the negative consequences of environmental stresses in different plants. However, little is known about foliar application of titanium dioxide nanoparticles (TiO2 NPs) to alleviate Cd stress in medicinal plants, and their dual interactions on essential oil production. The objective of this study was to investigate the effects of foliar-applied TiO2 NPs on growth, Cd uptake, chlorophyll fluorescence, photosynthetic pigments, malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents, total phenols, anthocyanins, flavonoids, antioxidant enzymes (SOD, CAT and POD) activity and essential oil content of Mentha piperita L. (peppermint) under Cd stress. For this purpose, plants were grown in Cd-contaminated (0, 20, 40, and 60 mg L⁻¹) soil, and different concentrations of TiO2 NPs (0, 75, and 150 mg L⁻¹) were foliar sprayed at three times after full establishment until the beginning of flowering. Exposure to TiO2 NPs significantly (P < 0.01) increased shoot dry weight (37.8%) and the number of lateral branches (59.4%) and decreased Cd uptake in plant tissues as compared to the control. Application of TiO2 NPs increased the content of plastid pigments, and the ratio Fv/Fm (13.4%) as compared to the control. Additionally, TiO2 NPs reduced the stress markers, MDA and H2O2 contents and enhanced the activity of the phenylalanine ammonia-lyase (PAL) enzyme (60.5%), total phenols (56.1%), anthocyanins (42.6%), flavonoids (25.5%), and essential oil content (52.3%) in Cd-stressed peppermint compared to the control. The results also demonstrated that foliar spray of TiO2 NPs effectively improved the growth and chlorophyll fluorescence parameters and reduced Cd accumulation in peppermint, which was mainly attributed to the reduction of oxidative burst and enhancement of the enzymatic (SOD, CAT, and POD) antioxidant defense system due to the uptake of NPs. The findings provide insights into the regulatory mechanism of TiO2 NPs on peppermint plants growth, physiology and secondary metabolites production in Cd-contaminated soil.
Medicinal plants have been used around the world in folk medicine for generations since ancient times, and today people enjoy using these natural medicines for their healing properties, with some of their pharmacologically active phytoconstituents recently serving as a lead for new drug development. Numerous aromatic plants producing aromatic secondary metabolites (SMs), which are commonly used as culinary herbs and spices, may also exhibit medicinal properties. The final biological effects of medicinal and aromatic plants (MAPs) are determined by the amount and composition of SMs, whose biosynthesis depends on genetic, ontogenetic, morphogenetic and environmental factors. Mild abiotic stresses have been found to stimulate production of SMs in MAPs without adversely affecting plant growth and development and in certain cases may even improve MAP performance. Since MAPs are sessile organisms exposed to various environmental stresses, they have developed effective defense mechanisms allowing protection against the harmful consequences of strong abiotic stresses, especially against the adverse effect of oxidative stress, in which SMs play an important role. This chapter presents a comprehensive overview of the recent findings on the impact of abiotic stresses including drought, salinity, waterlogging, heat, cold, harmful radiation, high soil acidity, elevated levels of ozone and CO2, heavy metals and agrochemicals (fertilizers and pesticides) on MAPs. The effects of abiotic stresses on the production of SMs in MAPs, including yield and composition of essential oils are highlighted. Responses of MAPs to abiotic stresses of various intensity and corresponding defense mechanisms of MAPs against oxidative stress are discussed. Utilization of in vitro cultures and hairy roots for large-scale production of pharmacologically significant SMs of MAPs using elicitors such as metal nanoparticle is presented and advantages of MAPs cultivation in a greenhouse under controlled conditions to achieve high levels of desirable SMs is mentioned as well.
Together with toxicity, beneficial effects on plant growth have been ascribed to nanoparticles (NPs). This study aimed to survey the growth performance and metabolome adjustment of beans grown in a growth medium containing ZnONPs at different concentrations and compared to bulk ZnSO4 as a positive control. Growth parameters showed a reduction in shoot height starting from the lowest (25 mg l-1 ) concentration of ZnONPs. In comparison, growth was inhibited from 50 mg l-1 ZnSO4 , suggesting more toxic effects of nano forms of Zn. Untargeted metabolomics allowed us to unravel the biochemical processes involved in both promising and detrimental aspects. Multivariate statistics indicated that the tested Zn species substantially and distinctively altered the metabolic profile of both roots and leaves, with more metabolites altered in the former (435) compared to leaves (381). Despite having Zn forms in the growth medium, also leaf metabolome underwent a significant and extensive modulation. In general, the elicitation of secondary metabolism (N-containing compounds, phenylpropanoids, and phytoalexins) and the down-accumulation of fatty acid biosynthesis compounds were common responses to different Zn forms. However, an opposite trend could be observed for amino acids, fatty acids, carbohydrates, and cofactors being down-accumulated in ZnONPs treatment. Osmolytes, especially in ZnSO4 treatment, contributed to mitigating the effect of Zn toxicity and maintaining plant growth. Overall, the results indicated a complexity of tissue-specific and Zn-dependent response differences, resulting in distinctive metabolic perturbations. This article is protected by copyright. All rights reserved.
Due to the rapidly increasing human population and ongoing climate change, modern smart agriculture management is essential for the production of sufficient healthy food, enabling to achieve high yields of crops with improved nutritional quality at low costs and minimizing contamination of environmental matrices with compounds without adverse impact on soil degradation and nontarget organisms. One of the promising strategies to meet this challenge is the use of nanoscale agrochemicals, such as nanofertilizers and nanoherbicides enabling not only improved crops yield at using lower doses compared to conventional
agrochemicals but also ensuring improved solubility, controlled release, or targeted biodistribution of active substances, thereby reducing the adverse impact on nontarget organisms and environmental contamination. However, due to the hormetic effects of nanoscale agrochemicals, the choice of the optimal
dose for each treatment is extremely important. This chapter is focused on the beneficial and adverse impact of nanoscale agrochemicals based on metal-based nanoparticles and carbon and halloysite nanotubes on the growth and yield of plants, their beneficial impact on improved production of valuable secondary metabolites, and alleviating the adverse effects of environmental stresses on plants. The mechanisms of actions of these nanoscale materials on plants are discussed, and the benefits of using nanosized herbicidal formulations to control unwanted weeds are analyzed as well.
Dracocephlum kotschyi Boiss is a genus in Lamiaceae family and a medicinal herb native to Iran. The cell suspension cultures were treated by static magnetic field (SMF) and Fe3O4 magnetite nanoparticles (MNP) to understand the production yield of secondary metabolites. The treatment procedure was done by cultivating the cells either with 100 ppm MNP, SMFs, or simultaneous exposure to both MNP and SMFs. The SMF at 30 mT was uniformly applied to the cells either for 3 or 4 days with a 3 h per day or a 5 h per day intervals, respectively. The contents of phenolics and phytochemicals were then examined by high performance liquid chromatography and UV–Vis spectrophotometer. These treatments imposed oxidative stress and induced polyphenol oxidase and phenylalanine ammonia lyase, accompanied by enhanced production of phenolics, anthocyanins, flavonoids, and lignin. The highest membrane embrittlement and elicitation was found upon simultaneous application of the MNPs and SMFs, followed by the MNP and SMFs. The contents of naringin, rosmarinic acid, quercetin, thymol, carvacrol, apigenin, and rutin increased in the intracellular biomass of all treated cells and extracellular culture media. These findings propose the potential of these elicitors in simultaneous production and secretion of these phytochemicals into culture media.
Previous studies have reported crop toxicity of zinc oxide nanoparticles (ZnO‐NPs) by root exposure and that silica nanoparticles (SiO2‐NPs) modulate crop growth and soil physicochemical properties. In this study, we determined the effects of foliar exposure to ZnO‐NPs and SiO2‐NPs on the growth and metabolomic profile of cucumber seedlings. Foliar spraying with ZnO‐NPs and SiO2‐NPs improved cucumber plant performance, as indicated by increases in chlorophyll contents, chlorophyll fluorescence parameters, and the fresh/dry weights of leaves and roots. According to metabolomic analysis results, foliar exposure to ZnO‐NPs and SiO2‐NPs induced pronounced changes in the metabolome, especially in the leaves. Besides, ZnO‐NP or SiO2‐NP treatments either upregulated or downregulated the contents of amino acids, organic acids, sugars and glycosides, and secondary metabolites, indicating that the two NPs affected both primary metabolism and secondary metabolism. The elevated levels of several secondary metabolites involved in antioxidative defense imply that the two NPs potentially regulate stress resistance in plants. Overall, these results indicate that different from root exposure, foliar exposure to ZnO‐NPs or SiO2‐NPs is beneficial to plant growth. The present study provides a theoretical basis for the environmental assessment and application of these two NPs in agricultural production.
We report a new green route for preparing MnO2/perlite nanocomposites (NCs) by leaf extract of Hypericum perforatum. Characterization of the physicochemical properties of the MnO2/perlite-NCs was performed using XRD, FESEM, EDX, FT-IR, and DLS techniques. Furthermore, their effects on the phytochemical classification and growth parameters of H. perforatum shoot cultures were assessed. According to the FESEM image, the synthesized spherical MnO2 nanoparticles on the sheet-like structure of nano-perlite were formed, ranging about 20–50 nm. In addition, based on the EDX spectra, the elemental analysis showed the presence of Carbon, Oxygen, Silicon, Aluminum, and Manganese elements in the as-synthesized MnO2/perlite-NCs. Biological studies confirmed that nano-perlite and MnO2/perlite-NCs were non-toxic to H. perforatum shoot cultures and showed positive effects on plant growth in specific concentrations. Overall, phytochemical classification demonstrated that the terpenoids decreased in the evaluated treatments, while hypericin and pseudohypericin were increased in some treatments (25, 50 and 150 mg/L of nano-perlite) relative to control. Metabolomics results suggested that both nano-perlite and MnO2/perlite-NCs can be used as elicitors and new nanofertilizers for generating some secondary metabolites.
Purpose:
We report on the expression stability of several housekeeping/reference genes that can be used in the normalization of target gene expression in quantitative real-time PCR (qRT-PCR) analysis of plant cells challenged with metal nanoparticles (NPs).
Materials and methods:
Uniform cell suspension cultures of Hypericum perforatum were treated with 25 mg/l silver and gold NPs (14-15 nm in diameter). Cells were collected after 0.5, 4.0, and 12 h. The total RNA isolated from the cells was analyzed for the stability of ACT2, ACT3, ACT7, EF1-α, GAPDH, H2A, TUB-α, TUB-β, and 18S rRNA genes using qRT-PCR. The cycle threshold (Ct) values of the genes were analyzed using the geNorm, NormFinder, BestKeeper, and RefFinder statistical algorithms to rank gene stability. The stability of the top-ranked genes was validated by normalizing the expression of HYP1.
Results:
The expression of the tested housekeeping genes varied with treatment duration and NP types. EF1-α in gold NP treatment and TUB-α and EF1-α in silver NP treatment ranked among the top three positions. However, none of the genes retained their top ranking with time and across NP types.
Conclusion:
EF1-α can be used as a reference for treatment involving both silver and gold NPs in H. perforatum cells. TUB-α can be used only for silver NP-treated cells. The expression instability of most of the housekeeping genes highlights the importance of systematic standardization of reference genes for NP treatment conditions to draw proper conclusions on the target gene expression.
Global metabolomics based on high-resolution liquid chromatography mass spectrometry (LC-MS) has been increasingly employed in recent large-scale multi-omics studies. Processing and interpretation of these complex metabolomics datasets have become a key challenge in current computational metabolomics. Here, we introduce MetaboAnalystR 2.0 for comprehensive LC-MS data processing, statistical analysis, and functional interpretation. Compared to the previous version, this new release seamlessly integrates XCMS and CAMERA to support raw spectral processing and peak annotation, and also features high-performance implementations of mummichog and GSEA approaches for predictions of pathway activities. The application and utility of the MetaboAnalystR 2.0 workflow were demonstrated using a synthetic benchmark dataset and a clinical dataset. In summary, MetaboAnalystR 2.0 offers a unified and flexible workflow that enables end-to-end analysis of LC-MS metabolomics data within the open-source R environment.
It appears that the biologically-synthesized nanoparticles (NPs) have potential to perform as effective elicitors for the production of valuable secondary metabolites in plants. Besides, it has been reported that the toxicity of the biologically-synthesized NP is not as much as that of the chemically-synthesized NPs. Therefore, it is necessary to test their advantages aspects. In this study, the physical synthesis of perlite NPs and biologically-synthesis of TiO2/perlite nanocomposites (NCs) were conducted. Subsequently, their effects and explant source influence on the growth characteristics and secondary metabolite profiles of Hypericum perforatum callus cultures were evaluated. According to the obtained results, morphology of the synthesized perlite NPs and TiO2/perlite NCs were mesoporous and spherical with sizes ranging about 14.51–23.34 and 15.50–24.61 nm, respectively. Addition of perlite NPs and TiO2/perlite NCs to the culture medium at the concentration range of 25–200 mg/L showed no adverse impacts on the growth characteristics of H. perforatum calli. According to the GC-MS analysis, the stress caused by perlite NPs and TiO2/perlite NCs led to an increase in the variety, amount and number of volatile compounds. The calli obtained from in vitro grown plants produced more volatile compounds relative to the calli obtained from field grown plants under the nanomaterial stress conditions. The production of hypericin and pseudohypericin were also determined in the callus cultures under desired nanomaterials elicitation. Accordingly, our results suggest that perlite NPs and TiO2/perlite NCs can possibly be considered as effective elicitors for the production of volatile compounds, hypericin, and pseudohypericin in callus cultures of H. perforatum.
Among a series of xanthones identified from mangosteen, the fruit of Garcinia mangostana L. (Guttifereae), α- and γ-mangostins are known to be major constituents exhibiting diverse biological activities. However, the effects of γ-mangostin on oxidative neurotoxicity and impaired memory are yet to be elucidated. In the present study, the protective effect of γ-mangostin on oxidative stress-induced neuronal cell death and its underlying action mechanism(s) were investigated and compared to that of α-mangostin using primary cultured rat cortical cells. In addition, the effect of orally administered γ-mangostin on scopolamine-induced memory impairment was evaluated in mice. We found that γ-mangostin exhibited prominent protection against H2O2- or xanthine/xanthine oxidase-induced oxidative neuronal death and inhibited reactive oxygen species (ROS) generation triggered by these oxidative insults. In contrast, α-mangostin had no effects on the oxidative neuronal damage or associated ROS production. We also found that γ-mangostin, not α-mangostin, significantly inhibited H2O2-induced DNA fragmentation and activation of caspases 3 and 9, demonstrating its antiapoptotic action. In addition, only γ-mangostin was found to effectively inhibit lipid peroxidation and DPPH radical formation, while both mangostins inhibited β-secretase activity. Furthermore, we observed that the oral administration of γ-mangostin at dosages of 10 and 30 mg/kg markedly improved scopolamine-induced memory impairment in mice. Collectively, these results provide both in vitro and in vivo evidences for the neuroprotective and memory enhancing effects of γ-mangostin. Multiple mechanisms underlying this neuroprotective action were suggested in this study. Based on our findings, γ-mangostin could serve as a potentially preferable candidate over α-mangostin in combatting oxidative stress-associated neurodegenerative diseases including Alzheimer's disease.
Several plant bioactive compounds have exhibited functional activities that suggest they could play a remarkable role in preventing a wide range of chronic diseases. The largest group of naturally-occurring polyphenols are the flavonoids, including apigenin. The present work is an updated overview of apigenin, focusing on its health-promoting effects/therapeutic functions and, in particular, results of in vivo research. In addition to an introduction to its chemistry, nutraceutical features have also been described. The main key findings from in vivo research, including animal models and human studies, are summarized. The beneficial indications are reported and discussed in detail, including effects in diabetes, amnesia and Alzheimer's disease, depression and insomnia, cancer, etc. Finally, data on flavonoids from the main public databases are gathered to highlight the apigenin's key role in dietary assessment and in the evaluation of a formulated diet, to determine exposure and to investigate its health effects in vivo.
Nanotechnology was well developed during past decades and implemented in a broad range of industrial applications, which led to an inevitable release of nanomaterials into the environment and ecosystem. Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials in various fields, especially in the agricultural sector. Plants are the basic component of the ecosystem and the most important source of food for mankind; therefore, understanding the impacts of AgNPs on plant growth and development is crucial for the evaluation of potential environmental risks on food safety and human health imposed by AgNPs. The present review summarizes uptake, translocation, and accumulation of AgNPs in plants, and exemplifies the phytotoxicity of AgNPs on plants at morphological, physiological, cellular, and molecular levels. It also focuses on the current understanding of phytotoxicity mechanisms via which AgNPs exert their toxicity on plants. In addition, the tolerance mechanisms underlying survival strategy that plants adopt to cope with adverse effects of AgNPs are discussed.
Suspension-cultured Dracocephlum polychaetum Bornm. is a wild medicinal herb native to Iran, treated with a static magnetic field (SMF) and Fe2O3 magnetic nanoparticles (MNP). The effect of SMF (30 mT), Fe2O3 MNP (100 ppm) and the combination of these treatments on phenolic metabolism and the medicinal compounds were examined by high performance liquid chromatography and UV–Vis spectrophotometer. The activity of polyphenol oxidase and phenylalanine ammonialyase, as well as the content of total phenolics, flavonoid, anthocyanins, lignin and malondialdehyde in all treated cells, showed a significant difference with control. The intracellular content of rosmarinic acid, naringin, apigenin, thymol, carvacrol, quercetin and rutin, in all treated cells increased considerably. Their quantities also increased significantly in the treated cell culture media. These results suggest this increase is due to the increased production and secretion of the medicinal compounds from cells to the culture media upon application of SMF and Fe2O3 MNP.
Withania somnifera is an important medicinal plant due to the presence of secondary metabolites. Nanoparticles (NPs) have elicitor activity for the enhancement of secondary metabolites biosynthesis in plants. W. somnifera plants were grown in-vitro and in-vivo and treated with homologous series of Zn-Ag NPs, Ni, and CdSe. Four NPs having different molar ratio of Zn and Ag have been used for the treatment. NP1, NP2, NP3, and NP4 Exhibit 19:1, 9:1, 3:1 and 1:1 molar ratios between Zn and Ag. Among all the treated NPs NP1 showed maximum enhancement in photosynthesis rate, transpiration rate, withanolide content and increase in some carbohydrates. Genes involved in photosynthesis, Calvin cycle, carbohydrate metabolism, and withanolide biosynthesis showed up regulation in NP1 and NP3 treatments. Zn-Ag NPs showed the photocatalytic and elicitor activity which helped in the light absorption and increased oxidative stress. NP2, NP4, Ni, and CdSe treatments showed the negative impact on withanolide biosynthesis and withanolide content. NP2, NP4, Ni, CdSe treated samples showed down regulation of most of the carbohydrate and withanolide pathway genes and lower withanolide content in comparison with water treated plants. CdSe caused excess cell damage, therefore, decrease in withanolide content was recorded. Gas exchange and electron transfer rate increased in CdSe and NP1 increases due to their semiconducting properties. CdSe and NP1 can liberate significant number of electrons under the light exposure, which behaves like carriers. Cyclic electron flux was observed in all the treatments but lowest in control condition. Cyclic electron flux not only manages the electron flow around PSI under stress but also considered as essential for photosynthesis and plant growth under control condition.
The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two‐week‐old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano‐zinc oxide (ZnO) (0, 50, 100 and 200 mg/l), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine‐6‐beta‐hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO‐treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO‐treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100 mg/l of ZnO after 48 and 72 hours, respectively. Semi‐quantitative RT‐PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100 mg/l of ZnO after 24 hours. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene.
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Key message:
Water-soluble chitosan oligosaccharides (COS) affect xanthone and volatile organic compound content, as well as antifungal activity against human pathogenic fungi of extracts obtained from Hypericum perforatum root cultures. Several studies have demonstrated the elicitor power of chitosan on xanthone biosynthesis in root cultures of H. perforatum. One of the major limitations to the use of chitosan, both for basic and applied research, is the need to use acidified water for solubilization. To overcome this problem, the elicitor effect of water-soluble COS on the biosynthesis of both xanthones and volatile organic compounds (VOCs) was evaluated in the present study. The analysis of xanthones and VOCs was performed by HPLC and GC-MS headspace analysis. The obtained results showed that COS are very effective in enhancing xanthone biosynthesis. With 400 mg L-1 COS, a xanthone content of about 30 mg g-1 DW was obtained. The antifungal activity of extracts obtained with 400 mg L-1 COS was the highest, with MIC50 of 32 µg mL-1 against Candida albicans and 32-64 µg mL-1 against dermatophytes, depending on the microorganism. Histochemical investigations suggested the accumulation of isoprenoids in the secretory ducts of H. perforatum roots. The presence of monoterpenes and sesquiterpenes was confirmed by the headspace analysis. Other volatile hydrocarbons have been identified. The biosynthesis of most VOCs showed significant changes in response to COS, suggesting their involvement in plant-fungus interactions.
Due to their well-known antifungal activity, the intentional use of Ag nanoparticle (NPs) as sustainable nano-fungicides is expected to increase in agriculture. However, the impacts of AgNPs on plants must be critically evaluated to guarantee their safe use in food production. In this study, 4-week-old cucumber (Cucumis sativus) plants received a foliar application of AgNPs (4 or 40 mg per plant) or Ag+ (0.04 or 0.4 mg per plant) for seven days. Gas chromatography-mass spectrometry (GC-MS) based non-target metabolomics enabled the identification and quantification of 268 metabolites in cucumber leaves. Multivariate analysis revealed that all the treatments significantly altered the metabolite profile. Exposure to AgNPs resulted in metabolic reprogramming, including activation of antioxidant defense systems (up-regulation of phenolic compounds) and down-regulation of photosynthesis (up-regulation of phytol). Additionally, AgNPs enhanced respiration (up-regulation of TCA cycle intermediates), inhibited photorespiration (down-regulation of glycine/serine ratio), altered membrane properties (up-regulation of pentadecanoic and arachidonic acid, down-regulation of linoleic and linolenic acid), and reduced of inorganic nitrogen fixation (down-regulation of glutamine and asparagine). Although Ag ions induced some of the same metabolic changes, alterations in the levels of carbazole, indoleactate, raffinose, adenosine, lactamide, erythrose, and p-benzoquinone were AgNPs-specific. The results of this study offer new insight into the molecular mechanisms by which cucumber responds to AgNPs exposure and provide important information to support the sustainable use of AgNPs in agriculture.
The particles within the size range of 1 and 100 nm are known as nanoparticles (NPs). NP-containing wastes released from household, industrial and medical products are emerging as a new threat to the environment. Plants, being fixed to the two major environmental sinks where NPs accumulate — namely water and soil, cannot escape the impact of nanopollution. Recent studies have shown that plant growth, development and physiology are significantly affected by NPs. But, the effect of NPs on plant secondary metabolism is still obscure. The induction of reactive oxygen species (ROS) following interactions with NPs has been observed consistently across plant species. Taking into account the existing link between ROS and secondary signaling messengers that lead to transcriptional regulation of secondary metabolism, in this perspective we put forward the argument that ROS induced in plants upon their interaction with NPs will likely interfere with plant secondary metabolism. As plant secondary metabolites play vital roles in plant performance, communication, and adaptation, a comprehensive understanding of plant secondary metabolism in response to NPs is an utmost priority.
Hypericum perforatum L. is an ethnomedicine with a popular remedial legacy; especially of antidepressant and wound healing properties. Rigorous preclinical and clinical research conducted in last sesqui-decade has revealed newer facets of its therapeutic activities against psychiatric, metabolic and neoplastic disorders. Most of such curative effects are imparted synergistically by hypericin, hyperforin and flavonoids; but their action mechanisms remain ambiguous. Concomitant administration of St. John’s Wort formulation and cytochrome P450 substrate drug is limited by the episodes of herb–drug interactions; nevertheless, adverse drug reaction rate of H. perforatum remains only 2%. In present review, we aim to highlight the ‘evidence-based’ therapeutic potential of aforementioned phytopharmaceutical, which would accelerate the contemporary pharmaceutical development of this traditional phytomedicine.
Root secretion of coumarin-phenolic type compounds has been recently shown to be related to Arabidopsis thaliana tolerance to Fe deficiency at high pH. Previous studies revealed the identity of a few simple coumarins occurring in roots and exudates of Fe-deficient A. thaliana plants, and left open the possible existence of other unknown phenolics. We used HPLC-UV/VIS/ESI-MS(TOF), HPLC/ESI-MS(ion trap) and HPLC/ESI-MS(Q-TOF) to characterize (identify and quantify) phenolic-type compounds accumulated in roots or secreted into the nutrient solution of A. thaliana plants in response to Fe deficiency. Plants grown with or without Fe and using nutrient solutions buffered at pH 5.5 or 7.5 enabled to identify an array of phenolics. These include several coumarinolignans not previously reported in A. thaliana (cleomiscosins A, B, C, and D and the 5′-hydroxycleomiscosins A and/or B), as well as some coumarin precursors (ferulic acid and coniferyl and sinapyl aldehydes), and previously reported cathecol (fraxetin) and non-cathecol coumarins (scopoletin, isofraxidin and fraxinol), some of them in hexoside forms not previously characterized. The production and secretion of phenolics were more intense when the plant accessibility to Fe was diminished and the plant Fe status deteriorated, as it occurs when plants are grown in the absence of Fe at pH 7.5. Aglycones and hexosides of the four coumarins were abundant in roots, whereas only the aglycone forms could be quantified in the nutrient solution. A comprehensive quantification of coumarins, first carried out in this study, revealed that the catechol coumarin fraxetin was predominant in exudates (but not in roots) of Fe-deficient A. thaliana plants grown at pH 7.5. Also, fraxetin was able to mobilize efficiently Fe from a Fe(III)-oxide at pH 5.5 and pH 7.5. On the other hand, non-catechol coumarins were much less efficient in mobilizing Fe and were present in much lower concentrations, making unlikely that they could play a role in Fe mobilization. The structural features of the array of coumarin type-compounds produced suggest some can mobilize Fe from the soil and others can be more efficient as allelochemicals.
There has been an increasing influx of nanopesticides into agriculture in recent years. Understanding the interaction between nanopesticides and edible plants is crucial in evaluating the potential impact of nanotechnology on the environment and agriculture. Here we exposed lettuce plants to Cu(OH)2 nanopesticides (1050-2100 mg/L) through foliar spray for one month. Inductively coupled plasma-mass spectrometry (ICP-MS) results indicate that 97-99% (1353-2501 mg/kg) of copper was sequestered in the leaves and only a small percentage (1-3%) (17.5-56.9 mg/kg) was translocated to root tissues through phloem loading. Gas Chromatography-Time-of-Flight Mass Spectrometry (GC-TOF-MS) based metabolomics combined with Partial Least Squares-Discriminant Analysis (PLS-DA) multivariate analysis revealed that Cu(OH)2 nanopesticides altered metabolite levels of lettuce leaves. Tricarboxylic (TCA) cycle and a number of amino acid-related biological pathways were disturbed. Some antioxidant levels (cis-caffeic acid, chlorogenic acid, 3,4-dihydroxycinnamic acid, dehydroascorbic acid) were significantly decreased compared to the control, indicating that oxidative stress and a defense response occurred. Nicotianamine, a copper chelator, increased by 12-27 fold compared to the control, which may represent a detoxification mechanism. The up-regulation of polyamines (spermidine and putrescine) and potassium may mitigate oxidative stress and enhance tolerance. The data presented here provide a molecular-scale perspective on the response of plants to copper nanopesticides.
Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3′,4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3′-hydroxylation of 2,4,6-trihydroxybenzophenone and C–O phenol coupling of the resulting 2,3′,4,6-tetrahydroxybenzophenone. Relative to the inserted 3′-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5-trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C–O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs.
Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM) production (the "Plant Cell Factory" concept). Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.
Since copper nanoparticles are being increasingly used in agriculture as pesticides, it is important to assess their potential implications for agriculture. Concerns have been raised about the bioaccumulation of nano-Cu and their toxicity to crop plants. Here, the response of cucumber plants in hydroponic culture at early development stages to two concentrations of nano-Cu (10 and 20 mg/L) was evaluated by proton nuclear magnetic resonance spectroscopy (1H NMR) and Gas Chromatography-Mass Spectrometry (GC-MS) based metabolomics. Changes in mineral nutrient metabolism induced by nano-Cu were determined by ICP-OES. Results showed that nano-Cu at both concentrations interfere with the uptake of a number of micro- and macro-nutrients, such as Na, P, S, Mo, Zn, and Fe. Metabolomics data revealed that nano-Cu at both levels triggered significant metabolic changes in cucumber leaves and root exudates. The root exudate metabolic changes revealed an active defense mechanism against nano-Cu stress: up-regulation of amino acids to sequester/exclude Cu/nano-Cu; down-regulation of citric acids to reduce the mobilization of Cu ions; ascorbic acid up-regulation to combat reactive oxygen species; up-regulation of phenolic compounds to improve antioxidant system. Thus, we demonstrate that non-targeted 1H NMR and GC-MS based metabolomics can successfully identify physiological responses induced by nanoparticles. Root exudates metabolomics revealed important detoxification mechanisms.
Plant cell culture systems have become an attractive and sustainable approach to produce high-value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohor-mones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed-, stimulus-responsive-, and hormone-carrying-NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP-mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP-associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy.
Heavy metals (HMs) are affected by natural and human activities and contaminate the soil environment, endanger plant growth and pose a serious threat to the ecosystem. HMs interfere with plant metabolism and some protein activities and cause oxygen stress. Plants combat excess HMs in the environment through a complex array of biological activities (such as antioxidant mechanisms, chelation, and regionalization of HM ions). Metabolomics is an emerging technique utilized for the qualitative and quantitative analysis of all low-molecular-weight metabolites that is now widely used in toxicology, disease diagnosis, plant research and other fields and has become an important component of systems biology. This paper reviews the progress and current status of metabolomics research on plants under HM stress and introduces plant detoxification and phytoremediation mechanisms.
Nanoscale zero-valent iron (nZVI) has been widely applied in the environmental field to degrade organic pollutants. The potential risk posed from nZVI on crop species is not well understood and is critical for sustainable application in the future. In this study, maize (Zea mays L.) plants were cultivated in field soils mixed with nZVI at 0, 50, and 500 mg/kg soil for four weeks. Upon exposure to 500 mg/kg nZVI, ICP-MS results showed that Fe accumulated by roots and translocated to leaves was increased by 36% relative to untreated controls. At 50 mg/kg, root elongation was enhanced by 150–200%; at 500 mg/kg, pigments, lipid peroxidation, and polyphenolic levels in leaves were increased by 12, 87 and 23%, respectively, whereas the accumulation of Al, Ca, and P were decreased by 62.2%, 19.7%, and 13.3%, respectively. A gas chromatography–mass spectrometry (GC–MS) based metabolomics analysis of maize roots revealed that antioxidants and stress signaling-associated metabolites were downregulated at 50 mg/kg, but were upregulated at 500 mg/kg. At 50 mg/kg, the content of glutamate was increased by 11-fold, whereas glutamine was decreased by 99% with respect to controls. Interestingly, eight metabolic pathways were disturbed at 50 mg/kg, but none at 500 mg/kg. This metabolic reprogramming at the lower dose represented potential risks to the health of exposed plants, which could be particularly important although no phenotypic impacts were noted. Overall, metabolites analysis provides a deeper understanding at the molecular level of plant response to nZVI and is a powerful tool for full characterization of risk posed to crop species as part of food safety assessment.
The aim of the study was to determine the effects of CeO2, Fe2O3, and SiO2 nanoparticles on the metabolism of phenols and flavonoids and the antioxidant status of butterhead lettuce and sweet pepper seedlings. Nanoparticles were used as a 1.5% suspension on the leaves. Phenolic and flavonoid contents increased as well as the antioxidant capacity of sweet pepper after exposure to Fe2O3-NPs. Phenolic and flavonoid concentrations in lettuce were the highest when CeO2-NPs were applied, while glutathione content increased due to Fe2O3-NPs and CeO2-NPs treatment. The highest ascorbic acid concentration was found in sweet pepper exposed to CeO2-NPs. The levels of dehydroascorbic acid, monodehydroascorbate, and L-galactono-1,4-lactone were the highest as a result of foliar spraying of sweet pepper with SiO2-NPs, but the ascorbic acid content in that plant was the lowest. Carotenoids increased after spraying of lettuce with SiO2-NPs. Individual metabolites from phenolic and flavonoid metabolism were determined. In lettuce seedlings, five phenolic compounds were decreased (3,4-diOH-benzaldehyde, ferulic acid, p-coumaric acid, salicylic acid, and vanillin) and two compounds (gallic acid and vanillic acid) were increased in comparison to control plants, while for sweet pepper an increase was observed for four compounds (chlorogenic acid, neochlorogenic acid, ferulic acid, and protocatechuic acid). To the best of our knowledge, this is the first study on the phenolic profile of edible juvenile plants treated with such metal/metalloid nanoparticles. We may also conclude that various nanoparticles may interact differently with phenolic phytochemicals depending also on plant species that have varying levels of stress tolerance.
With the increasing use of zinc oxide nanoparticles (ZnO NPs) in industry, there is an increased release of these NPs into ecosystem, with potential impact on the ecological environment. Herein, we investigated the physiological and molecular mechanisms underlying ZnO NP-mediated plant growth in tomato plants. Foliar spraying with ZnO NPs (20 and 100 mg L⁻¹) improved tomato growth by increasing the chlorophyll content and photosystem II activity. Comparative transcriptomic analysis revealed that ZnO NPs upregulated the expression of a set of genes involved in nutrient element transport, carbon/nitrogen metabolism, and the secondary metabolism in tomato, with the metabolome analysis further supporting this result. Foliar spraying with ZnO NPs increased iron (Fe) accumulation by 12.2% in tomato leaves; we thus examined the effects of ZnO NPs in tomato plants in response to Fe deficiency. Interestingly, foliar spraying with ZnO NPs markedly improved Fe deficiency tolerance in tomato. Physiological analysis indicated that ZnO NPs reduced Fe deficiency-induced oxidative damage and improved the metal nutrient element contents in tomato. Further, transcriptomic and metabolomic analyses indicated that foliar spraying with ZnO NPs increased the expression of genes encoding antioxidative enzymes, transporters, and the enzymes or regulators involved in carbon/nitrogen metabolism and secondary metabolism, thereby improving the levels of antioxidation, sugars, and amino acids in Fe-deficient tomato plants. Taken together, these results contribute to our understanding of the ecological effects of ZnO NPs.
Biotechnological strategies are needed to produce larger quantities of biomass and phytochemicals. In this study, callus cultures of Fagonia indica were elicited with different concentrations of chemically and biologically synthesized silver nanoparticles (chem- and bioAgNPs) to compare their effects on biomass, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of the extracts from callus. The results revealed that bioAgNPs being more biocompatible produced the highest biomass initially on day 10 (FW = 4.2152 ± 0.13 g; DW = 0.18527 ± 0.01 g) and day 20 (FW = 7.6558 ± 0.10 g; DW = 0.3489 ± 0.01 g) when supplemented in media as 62.5 µg/mL and 250 µg/mL, respectively. Initially, the highest TPC (319.32 ± 8.28 µg GAE/g of DW) was recorded on day 20 in chemAgNPs (31.25 µg/mL) induced callus as compared to TPC = 302.85 ± 3.002 µg GAE/g of DW in bioAgNPs-induced callus. Compared to the highest values of TFC (108.15 ± 2.10 µg QE/g of DW) produced in 15.6 µg/mL chemAgNPs-induced callus on day 20, TFC produced in bioAgNPs (62.5 µg/mL) was 168.61 ± 3.17 µg GAE/g of DW on day 10. Similarly, chemAgNPs-induced callus (62.5 µg/mL) showed the highest free radical scavenging activity (FRSA) i.e. 87.18% on day 20 while bioAgNPs (125 µg/mL) showed 81.69% FRSA on day 20 compared to highest among control callus (63.98% on day 40). The highest total antioxidant capacity of chemAgNPs-(125 µg/mL) induced callus was 330.42 ± 13.65 µg AAE/g of DW on day 20 compared to bioAgNPs-(62.5 µg/mL) induced callus (312.96 ± 1.73 µg AAE/g of DW) on day 10. Conclusively, bioAgNPs are potent elicitors of callus cultures of F. indica.
The plant kingdom produces hundreds of thousands of low molecular weight organic compounds. Based on the assumed functions of these compounds, the research community has classified them into three overarching groups: primary metabolites, which are directly required for plant growth; secondary (or specialized) metabolites, which mediate plant-environment interactions; and hormones, which regulate organismal processes and metabolism. For decades, this functional trichotomy of plant metabolism has shaped theory and experimentation in plant biology. However, exact biochemical boundaries between these different metabolite classes were never fully established. A new wave of genetic and chemical studies now further blurs these boundaries by demonstrating that secondary metabolites are multifunctional; they can function as potent regulators of plant growth and defense as well as primary metabolites sensu lato. Several adaptive scenarios may have favored this functional diversity for secondary metabolites, including signaling robustness and cost-effective storage and recycling. Secondary metabolite multifunctionality can provide new explanations for ontogenetic patterns of defense production and can refine our understanding of plant-herbivore interactions, in particular by accounting for the discovery that adapted herbivores misuse plant secondary metabolites for multiple purposes, some of which mirror their functions in plants. In conclusion, recent work unveils the limits of our current functional classification system for plant metabolites. Viewing secondary metabolites as integrated components of metabolic networks that are dynamically shaped by environmental selection pressures and transcend multiple trophic levels can improve our understanding of plant metabolism and plant-environment interactions.
Background
Tuberculosis (TB) is one of the infectious diseases associated with high rate of morbidity and mortality and still remains one of the top-ten leading causes of human death in the world. Development of new anti-TB drugs is mandatory due to existence of latent infection as well as the expansion of the resistant Mycobacterium tuberculosis (MBT) strains. Xanthones encompass a wide range of structurally diverse bioactive compounds, obtained either naturally or through chemical synthesis. There is a growing body of literature that recognizes the antitubercular activity of xanthone derivatives.
Objective
The objective of this review is to highlight the main natural sources along with the critical design elements, structure-activity relationships (SARs), modes of action and pharmacokinetic profiles of xanthone-based anti-TB compounds.
Method
In the present review, the anti-TB activity of xanthones reported in the literature from 1972 to date is presented and discussed.
Results
Exploration of xanthone scaffold led to identification of several members of this class having superior activity against both sensitive and resistant MBT strains with distinctive mycobacterial membrane disrupting properties. However, studies regarding their modes of action, pharmacokinetic properties and safety are so limited.
Conclusion
Comprehendible data and information are afforded by this review and it would certainly provide scientists with new thoughts and means which will be conducive to design and develop new drugs with excellent anti-TB activity through exploration of xanthone scaffold.
The rapid development of nanotechnology has raised concern regarding the environmental toxicity of nanoparticles (NPs). However, little is known about the molecular mechanisms underlying NP toxicity in plants. Broad bean (Vicia faba L.) plants were cultivated in soil amended with 0, 10, and 100 mg cadmium sulfide (CdS)-NPs kg soil⁻¹ for 4 weeks and then the phenotypic, biochemical, and metabolic responses of the plants to CdS-NPs stress were evaluated. Metabolomics analysis revealed the significant up-regulation (1.2- to 39.2-fold) of several antioxidative metabolites, including N-acetyl-5-hydroxytryptamine, 2-hydroxybutanoic acid, putrescine, and flavone, upon CdS-NPs exposure, but no negative phenotypic effects were visible (plant biomass, photosynthetic pigment contents, and lipid peroxidation). This observation was in accordance with the observed regulation of antioxidative-defense-related metabolic pathways (tyrosine pathway and phenylpropanoid biosynthesis) that were identified by biological pathway analysis. Importantly, twice as many metabolites were modulated in leaves than in roots, including three nitrogen-related (purine metabolism; alanine, aspartate, and glutamate metabolism; β-alanine metabolism) and two carbon-related (pantothenate and CoA biosynthesis and carbon fixation) metabolic pathways. These results indicate that to alleviate the toxicity of CdS-NPs exposure in soil, plants significantly reprogram the metabolic profiles of leaves rather than of roots, which might subsequently impact both harvest and crop quality.
Given the wide applications of engineered nanomaterials (ENMs) in various fields, the ecotoxicology of ENMs has attracted much attention. The traditional plant physiological activity (e.g., reactive oxygen species and antioxidant enzymes) are limited in that they probe one specific process of nanotoxicity, which may result in the loss of understanding of other important biological reactions. Metabolites, which are downstream of gene and protein expression, are directly related to biological phenomena. Metabolomics is an easily performed and efficient tool for solving the aforementioned problems because it involves the comprehensive exploration of metabolic profiles. To understand the roles of metabolomics in phytotoxicity, the analytical methods for metabolomics should be organized and discussed. Moreover, the dominant metabolites and metabolic pathways are similar in different plants, which determines the universal applicability of metabolomics analysis. The analysis of regulated metabolism will globally and scientifically help determine the ecotoxicology that is induced by ENMs. In the past several years, great developments in nanotoxicology have been achieved using metabolomics. However, many knowledge gaps remain, such as the relationships between biological responses that are induced by ENMs and the regulation of metabolism (e.g., carbohydrate, energy, amino acid, lipid and secondary metabolism). The phytotoxicity that is induced by ENMs has been explored by metabolomics, which is still in its infancy. The detrimental and defence mechanisms of plants in their response to ENMs at the level of metabolomics also deserve much attention. In addition, owing to the regulation of metabolism in plants by ENMs affected by multiple factors, it is meaningful to uniformly identify the key influencing factor.
Shoot cultures of eight Hypericum species belonging to the sections Hypericum, Oligostema, Ascyreia and Webbia were evaluated for their phytochemical profiles by high-performance liquid chromatography. In total, 17 secondary metabolites assigned to the groups of anthraquinones, phloroglucinols, hydroxycinnamic acids and flavonoids were detected. Furthermore, the elicitation potential of 18 biotic factors derived from saccharides, endophytic fungi and Agrobacterium rhizogenes was examined and statistically analysed with the paired two-sample t-test and principal component analysis. The production of naphthodianthrones and emodin was predominantly stimulated by elicitors derived from Fusarium oxysporum and Trichoderma crassum, while Piriformospora indica promoted the phloroglucinols production. Among flavonoids, the aglycone amentoflavone was readily increased by several elicitors up to 15.7-fold in H. humifusum treated by potato-dextrose broth. However, the chlorogenic acid proved to be the most susceptible metabolite to elicitation, when 31.7-times increase was detected in H. maculatum shoots upon D-glucose treatment. In spite of several biotic factors have been tested, no metabolite was commonly induced in all Hypericum spp. as a response to elicitor treatments.
This study describes the influence of bio-synthesized silver nanoparticles (AgNPs) on phytochemicals and their pharmacological activities in the cell suspension cultures (CSC) of bitter gourd. To standardize the effect of sucrose, plant growth regulators, medium, AgNPs and growth kinetics for the biomass and bioactive compounds accumulation in CSC of bitter gourd. The medium comprising MS salts, sucrose (30 g/L) with 2,4-D (1.0 mg/L) and TDZ (0.1 mg/L) at 28 days of CSC was appropriate for biomass and bioactive compound accumulation. The contents of silver, malondialdehyde and hydrogen peroxide were highly elevated in AgNPs (10 mg/L)-elicited CSC when compared with non-elicited CSC. AgNPs (5 mg/L) elicited CSC extracts had significantly enhanced the production of total phenolic (3.5 ± 0.2 mg/g), and flavonoid (2.5 ± 0.06 mg/g) contents than in the control CSC extracts (2.5 ± 0.1 and 1.6 ± 0.05 mg/g). AgNPs (5 mg/L) elicited CSC showed a higher amount of flavonols (1822.37 µg/g), hydroxybenzoic (1713.40 µg/g) and hydroxycinnamic (1080.10 µg/g) acids than the control CSC (1199, 1394.42 and 944.52 µg/g, respectively). Because of these metabolic changes, the pharmacological activities (antioxidant, antidiabetic, antibacterial, antifungal and anticancer) were high in the AgNPs (5 mg/L)-elicited CSC extracts in bitter gourd. The study suggested the effectiveness of elicitation process in enhancing the accumulation of phenolic compounds and pharmacological activities. AgNPs-elicited CSC offered an effective and favorable in vitro method to improve the production of bioactive compounds for potential uses in pharmaceutical industries.
Targeted metabolomics aims to provide a new approach to investigate metabolites and gather both qualitative and quantitative information. We describe a protocol for extraction and analysis of plant metabolites, specifically 13 secondary metabolites (antioxidants) using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), with high linearity (R2 > 0.99) and reproducibility (0.23-6.23 R%) with low limits of detection (>0.001 ng/mL) and quantification (>0.2 ng/mL). The protocol was applied to study the antioxidant response of cucumber plants exposed to nanocopper pesticide. Dose-dependent changes in antioxidant concentrations were found, and 10 antioxidants were significantly consumed to scavenge reactive oxygen species, protecting plants from damage. Levels of three antioxidants were up-regulated, as a response to the depletion of the other antioxidants, signaling activation of the defense system. We demonstrated that the reported LC-MS/MS method provides a quantitative analysis of antioxidants in plant tissues, for example to investigate interactions between plants and nanomaterials.
This study investigated the toxicity of CuO nanoparticles (NPs) to Nicotiana tabacum L. cv. Bright Yellow-2 (BY-2) cells. CuO NPs higher than 2 mg/L began to exhibit significant inhibition on the growth of plant cells, and the 24-h median effective concentration was calculated as 12 mg/L. CuO NPs (12 mg/L) showed much higher toxicity than the released Cu2+ (0.8 mg/L) and bulk particles (BPs, 12 mg/L) during all the exposure times (0-24 h). CuO NPs were internalized by plant cells through endocytosis, and then located in cytoplasm, mitochondria and vacuoles. Under CuO NPs exposure, the generation of total reactive oxygen species increased by 38.3%, and 40.9% compared with CuO BPs, and Cu2+ treatments, respectively. The main radical species were identified as H2O2 and OH·. CuO NPs significantly reduced the activities of complexes I and III on mitochondrial electron transport chain, blocked the electron transfer from NADH to ubiquinone, and from ubisemiquinone to ubiquinol, induced oxidative stress, and finally led to membrane damage as indicated by an increase in the contents of malondialdehyde and lactate dehydrogenase. Global gene expression analysis from RNA-sequencing showed that CuO NPs (12 mg/L) significantly induced 2692 differentially expressed genes (P-value < 0.05, fold-change > 2) including 1132 up-regulated and 1560 down-regulated genes in BY-2 cells. The differentially expressed genes related to oxidative stress and mitochondria were identified according to Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis. This work provides insights into the molecular mechanism of nanotoxicity toward plants.
Plants of the genus Hypericum (Hypericaceae) are used in folk medicine all over the world, H. perforatum being the most well-known species. Standardized extracts of this plant are commercially-available to treat mild to moderate depression cases. The present review summarizes the literature published up to 2016 concerning the phloroglucinol derivatives isolated from Hypericum species, together with their structural features and biological activities. These phytochemical studies led to the isolation of 101 prenylated phloroglucinols, chromanes and chromenes, 35 dimeric acylphloroglucinols, 235 polycyclic polyprenylated acylphloroglucinols, 25 simple benzophenones and 33 phloroglucinol-terpene adducts. These compounds show a diverse range of biological activities, such as antimicrobial, cytotoxic, antinociceptive and antidepressant-like effects.
Excess copper may disturb plant photosynthesis and induce leaf senescence. The underlying toxicity mechanism is not well understood. Here, 3-week-old cucumber plants were foliar exposed to different copper concentrations (10, 100 and 500 mg/L) for a final dose of 0.21, 2.1 and 10 mg/plant, using CuSO4 as the Cu ion source for seven days, three times per day. Metabolomics quantified 149 primary and 79 secondary metabolites. A number of intermediates of the tricarboxylic acid (TCA) cycle were significantly down-regulated 1.4-2.4 fold, indicating a perturbed carbohydrate metabolism. Ascorbate and aldarate metabolism and shikimate-phenylpropanoid biosynthesis (antioxidant and defense related pathways) were perturbed by excess copper. These metabolic responses occur even at the lowest copper dose considered although no phenotype changes were observed at this dose. High copper dose resulted in a 2-fold increase in phytol, a degradation product of chlorophyll. Polyphenol metabolomics revealed that some flavonoids were down-regulated, while the nonflavonoid 4-hydroxycinnamic acid and trans-2-hydroxycinnamic acid were significantly up-regulated 4- and 26-fold compared to the control. This study enhances current understanding of copper toxicity to plants, and demonstrates that metabolomics profiling provides a more comprehensive view of plant responses to stressors, which can be applied to other plant species and contaminants.
Chemically synthesized nanoparticles (NPs) are widely used in industry and concern over their impact on the environment is rising. In this study, greenhouse grown bean (Phaseolus vulgaris L.) plants were treated with CeO2 NPs suspensions at 0, 250, 500, 1000, and 2000mgL(-1) either aerially by spraying or via soil application. At 15days after treatment, plants were analyzed for Ce uptake, morphological and biochemical assays, as well as high-resolution mass spectrometry based metabolomics and proteomics. The results from ICP-MS assays showed a dose dependent absorption, uptake and translocation of Ce through both roots and leaves; Ce content increased from 0.68 up to 1894mgkg(-1) following spray application, while concentrations were three orders lower following soil application (0.59 to 2.19mgkg(-1)). Electrolyte leakage increased with NPs rate, from 25.2% to 70.3% and from 24.8% to 32.9% following spray and soil application, respectively. Spraying lowered stomatal density (from 337 to 113 per mm(2)) and increased stomatal length (from 12.8 to 19.4μm), and altered photosynthesis and electron transport chain biochemical machinery. The increase in Ce content induced accumulation of osmolites (proline increased from 0.54 to 0.65mg/g under spray application), phytosiderophores (muconate and mugineate compounds showed increase fold-changes >16) and proteins involved in folding or turnover. NPs application induced membrane damage, as evidenced by the increase in membrane lipids degradates and by the increase in electrolyte leakage, and caused oxidative stress. Most of the responses were not linear but dose-dependent, whereas metabolic disruption is expected at the highest NPs dosage. Both proteomics and metabolomics highlighted a stronger effect of CeO2 NPs spraying, as compared to soil application. High concentrations of NPs in the environment have been confirmed to pose toxicity concern towards plants, although important differences could be highlighted between aerial deposition and soil contamination.
This study investigates the phytotoxicity of chronic exposure (up to 20 d) of different TiO2 nanoparticles (TiO2-NP) concentrations (5, 50, 150 mg L(-1)) in Triticum aestivum. Germination was not affected by TiO2-NP exposure and seedling shoot length (3 d) was enhanced. Contrarily, plants' shoot growth (20 d) was impaired. Effects on membrane permeability and total antioxidant capacity in TiO2-NP chronic exposure were organ dependent: increased in leaves and decreased in roots. Roots also showed lower levels of lipid peroxidation. Flow cytometry revealed no changes in ploidy levels as well as in the cell cycle dynamics for both organs. However, TiO2-NP induced clastogenic effects in roots with increases in micronucleated cells in root tips in a dose dependent manner. Also, increases of DNA single/double strand breaks were found in leaves, and effects were similar to all doses. Ti uptake and translocation to leaves were confirmed by ICP-MS, which was dependent on NP concentration. Overall, these data indicate that TiO2-NP phytotoxicity is more severe after longer exposure periods, higher doses and more severe for shoots than roots. The observed effects are a result of both direct and indirect (oxidative stress and/or water imbalances) action of TiO2-NP. Additionally, results highlight the negative impact that TiO2-NP may have on crop growth and production and to the risk of trophic transfer.
Mass spectrometry reveals the biochemical changes in Arabidopsis thaliana resulting from exposure to sub-lethal concentrations of silver nanoparticles.
False positive and false negative peaks detected from extracted ion chromatograms (EIC) are an urgent problem with existing software packages that preprocess untargeted liquid or gas chromatography-mass spectrometry metabolomics data because they can translate downstream into spurious or missing compound identifications. We have developed new algorithms that carry out the sequential construction of EICs and detection of EIC peaks. We compare the new algorithms to two popular software packages XCMS and MZmine 2, and present evidence that these new algorithms detect significantly fewer false positives. Regarding the detection of compounds known to be present in the data, the new algorithms perform at least as well as XCMS and MZmine 2. Furthermore, we present evidence that mass tolerance in m/z should be favored rather than mass tolerance in ppm in the process of constructing EICs. The mass tolerance parameter plays a critical role in the EIC construction process and can have immense impact on the detection of EIC peaks.
While the use of nanopesticides in modern agriculture continues to increase, their effects on crop plants are still poorly understood. Here, 4-week-old spinach plants grown in an artificial medium were exposed via foliar spray to Cu(OH)2 nanopesticide (0.18 and 18 mg/plant) or Cu ions (0.15 and 15 mg/plant) for 7 days. A gas chromatography-time of flight-mass spectrometry metabolomics approach was applied to assess metabolic alterations induced by Cu(OH)2 nanopesticide in spinach leaves. Exposure to Cu(OH)2 nanopesticide and copper ions induced alterations in the metabolite profiles of spinach leaves. Compared to the control, exposure to 18 mg Cu(OH)2 nanopesticide induced significant reduction (29-85%) in antioxidant or defense-associated metabolites including ascorbic acid, alfa-tocopherol, threonic acid, β-sitosterol, 4-hydroxybutyric acid, ferulic acid and total phenolics. The metabolic pathway for ascorbate and aldarate was disturbed in all exposed spinach plants (nanopesticide and Cu2+). Cu2+ is responsible for the reduction in antioxidants and perturbation of the ascorbate and aldarate metabolism. However, nitrogen metabolism perturbation was nanopesticide specific. Spinach biomass and photosynthetic pigments were not altered, indicating metabolomics can be a rapid and sensitive tool to detect earlier nanopesticide effects. Consumption of antioxidants during the antioxidant defense process resulted in reduction of nutritional value of exposed spinach.
The wide occurrence and high environmental concentration of titanium dioxide nanoparticles (nano-TiO2) have raised concerns about their potential toxic effects on crops. In this study, we employed a GC-MS-based metabolomic approach to investigate the potential toxicity of nano-TiO2 on hydroponically-cultured rice (Oryza sativa L.) after exposed to 0, 100, 250 or 500 mg/L of nano-TiO2 for fourteen days. Results showed that the biomass of rice was significantly decreased and the antioxidant defense system was significantly disturbed after exposure to nano-TiO2. One hundred and five identified metabolites showed significant difference compared to the control, among which the concentrations of glucose-6-phosphate, glucose-1-phosphate, succinic and isocitric acid were increased most, while the concentrations of sucrose, isomaltulose, and glyoxylic acid were decreased most. Basic energy-generating ways including tricarboxylic acid cycle and the pentose phosphate pathway, were elevated significantly while the carbohydrate synthesis metabolism including starch and sucrose metabolism, and glyoxylate and dicarboxylate metabolism were inhibited. However, the biosynthetic formation of most of the identified fatty acids, amino acids and secondary metabolites which correlated to crop quality, were increased. The results suggest that the metabolism of rice plants is distinctly disturbed after exposure to nano-TiO2, and nano-TiO2 would have a mixed effect on the yield and quality of rice.
Objectives
In this review, we aim at updating the available information on the improvement of the Hypericum perforatum L. (Hypericaceae) phytochemical pro- file and pharmacological properties via elicitation.
Key findings
Hypericum perforatum seedlings, shoots, roots, calli and cell sus- pension cultures were treated with diverse elicitors to induce the formation of secondary metabolites. The extracts of the elicitor-treated plant material contain- ing naphthodianthrones, phloroglucinols, xanthones, flavonoids and other new compounds were quantitatively analysed and tested for their bioactivities. While hypericins were mainly produced in H. perforatum cultures containing dark nod- ules, namely shoots and seedlings, other classes of compounds such as xanthones, phloroglucinols and flavonoids were formed in all types of cultures. The extracts obtained from elicitor-treated samples generally possessed better bioactivities compared to the extract of control biomass.
Summary
Although elicitation is an excellent tool for the production of valuable secondary metabolites in H. perforatum cell and tissue cultures, its exploitation is still in its infancy mainly due to the lack of reproducibility and difficulties in scal- ing up biomass production.
Objectives:
The most widely applied qualitative and quantitative analytical methods in the quality control of Hypericum perforatum extracts will be reviewed, including routine analytical tools and most modern approaches.
Key findings:
Biologically active components of H. perforatum are chemically diverse; therefore, different chromatographic and detection methods are required for the comprehensive analysis of St. John's wort extracts. Naphthodianthrones, phloroglucinols and flavonoids are the most widely analysed metabolites of this plant. For routine quality control, detection of major compounds belonging to these groups seems to be sufficient; however, closer characterization requires the detection of minor compounds as well.
Conclusions:
TLC and HPTLC are basic methods in the routine analysis, whereas HPLC-DAD is the most widely applied method for quantitative analysis due to its versatility. LC-MS is gaining importance in pharmacokinetic studies due to its sensitivity. Modern approaches, such as DNA barcoding, NIRS and NMR metabolomics, may offer new possibilities for the more detailed characterization of secondary metabolite profile of H. perforatum extracts.