Nanoparticles Alter Secondary Metabolism in Plants via ROS Burst

1The National Science Foundation (NSF) projects that the global market for products incorporating nanotechnology could amount to three trillion USD by 2020. Currently, more than 1000 commercial products containing nanoparticles (NPs) are available in the market. The NPs commonly found in household, industrial and healthcare products are Au (Gold), Ag (silver), ZnO (zinc oxide), CuO (copper oxide), TiO2 (titanium dioxide), Fe3O4/Fe2O3 (iron oxides), and CeO2 (cerium oxide). Similarly, incorporation of Ag, ZnO, TiO2, and SiO2 (silicon dioxide) NPs into agrochemicals (pesticides, fungicides, herbicides, fertilizers, etc.) is expected to have great potential in nanotechnology-driven smart agriculture. The expanding applications of nanotechnology in domestic, industrial and agricultural sectors are also increasing the possibilities of NPs reaching the environment as nanomaterial-containing wastes. As the consequences of NP pollutants reaching the environment in significant quantities are unknown, understanding the plant’s response to NPs is an intensive area of research.

2Most studies with NPs indicated a certain degree of phytotoxicity, especially at high concentrations. Depending on their size, NPs can enter plant cells from the apoplast, crossing the plasma membrane via endocytosis; subsequently they can be translocated from one part to another through symplastic flow. There is also evidence for the transport of NPs into subcellular organelles such as the nucleus, plastids, and vacuoles.

3Arabidopsis thaliana (L.) Heynh seedlings grown on soil treated with ZnONPs were observed to have reduced growth, chlorophyll content and rates of photosynthesis. These effects were concentration dependent with growth compromised 20 and 80%, respectively, with 200 and 300 mg/L treatments. At 300 mg/L, the chlorophyll content, net rate of photosynthesis, leaf stomatal conductance, intercellular CO2 concentration and transpiration rate were all reduced more than 50%. Similarly, an increasing concentration (0, 2.5, 10, 50, 100, and 1,000 mg/L) of CuONPs negatively affected Oryza sativa L. seedling growth in a hydroponic system. Photosynthetic rate, transpiration rate, stomatal conductance, maximal quantum yield of PSII photochemistry, and photosynthetic pigment contents declined, with a complete loss of PSII photochemical quenching at 1,000 mg/L. ZnONPs inhibited the expression of genes involved in chlorophyll synthesis and photosystem structure. Accumulation of CuONPs in the chloroplasts was accompanied by a lower number of thylakoids per granum. AgNPs inhibited Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and the photo-protective capacity of PSII in the model aquatic higher plant Spirodela polyrhiza (L.) Schleid.

4In addition to reduced photosynthetic rates the growth inhibition caused by NPs has also been associated with increased oxidative stress. However, whether the arrest of photosynthesis or the induction of oxidative stress is the dominant impact of NPs is a subject of debate, since both of them go hand in hand. Although the accumulation of NPs in chloroplasts and damage to the photosynthetic apparatus supports the former, the fact that to reach the chloroplast NPs must cross the plasma membrane, where they can induce reactive oxygen species (ROS) via NADPH oxidases argues the reverse. ROS production, damage to the membrane structure and function, and fluctuation in antioxidant enzymatic activities are documented across plant species as common responses to NPs. A few studies have also demonstrated that treatment of plants and photosynthetic microorganisms with NPs resulted in increased production of phenolics, which might act as antioxidants to scavenge the ROS.

5The possibility of NP-induced disturbance in ROS homeostasis and associated signaling pathways as a major factor underlying the changes in plant secondary metabolism is explored in this perspective.

6Oxidative burst has been consistently reported in plants exposed to toxic levels of NPs. Exposure to various NPs, for example Ag, ZnO, and Al2O3 (aluminum oxide), also induced reactive nitrogen species (∗NO, nitric oxide) and H2O2 in duckweed, corn and tobacco bright yellow (BY2) cells. In tobacco BY2 cells, Al2O3NPs also induced the production of superoxide anion (O⋅2-), one of the highly reactive forms of ROS. Although it is debated whether ROS activation stems, actually, from intact particles or, rather, from ions released from NPs, recent studies supports the latter. In S. polyrhiza, internalized Ag, regardless of whether the exposure was Ag+ ions or AgNPs, had the same capacity to generate ROS supporting the hypothesis that intracellular AgNPs dissociate into highly toxic Ag+ ions. Similarly, dissolution of ZnO, CuO, and CeO2 (cerium oxide) into their respective ions (Zn2+, Cu2+, or Ce4+) has been established in other studies.

7The mechanisms through which NPs induce ROS production and trigger oxidative stress at the cellular level have also been investigated. AgNPs triggered Ca2+ and ROS signaling through the induction of Ca2+-permeable pores and direct oxidation of apoplastic L-ascorbic acid. A. thaliana root hair defective 2 (rhd2) mutant lacking NADPH oxidase RBOHC showed a significantly lower level of ROS generation in response to AgNPs compared with wild type plants, indicating that the accumulation of ROS in cells is mediated by plasma membrane-bound NADPH oxidases (RBOH) enzymes that produce ROS at the apoplast. On the other hand, chloroplastic ROS generation was observed in S. polyrhiza, based on the ability of AgNPs to inhibit Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and the photo-protective capacity of PSII.

8A common consequence of harmful levels of ROS is the damage to cellular macromolecules including membrane lipids that leads to cell death. Growth inhibition coupled with lipid peroxidation has been reported in O. sativa seedlings treated with 0.5, 1.0, and 1.5 mM CuONPs and in 5 mg/L TiO2NPs treated Nitzschia closterium. NPs could also damage other macromolecules like DNA. AgNPs and AuNPs affected cell division in Allium cepa L. root tip cells, the former causing chromatin bridge, chromosomal stickiness, disturbed metaphase, multiple chromosomal breaks, and cell disintegration. DNA damage, mitochondrial dysfunction, and cell apoptosis were also observed in eggplant, as a consequence of oxidative stress induced by Co3O4.

9In order to mitigate the effects of oxidative stress plants activate both enzymatic and non-enzymatic antioxidant defense machinery to scavenge excess ROS. Correspondingly, NP-mediated stress also activates plant’s antioxidant machinery/enzymes. Briefly, superoxide dismutase (SOD) that catalyzes detoxification of O⋅2- into either ordinary molecular oxygen (O2) or H2O2 and ascorbate peroxidase (APX), which detoxifies peroxides such as H2O2 using ascorbic acid (Asc) as a substrate, were up-regulated in plants upon treatment with NPs. Whereas, dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDAR) enzymes that regulate the cellular Asc redox state were downregulated. Proteomic analysis of AgNPs treated O. sativa roots revealed an increased abundance of SOD, APX, and glutathione-S-transferase (GST). These NPs also stimulated the activities of SOD and APX significantly, while inhibiting glutathione reductase (GR) and DHAR in Pisum sativum L. seedlings. Catalase (CAT), another enzyme that protects the cells from oxidative damage, was significantly elevated upon treatment of wheat roots with 500 mg/kg CuONPs. Maize plants germinated and grown on soil amended with 0, 400, and 800 mg/kg CeO2NPs showed a concentration dependent increase in the accumulation of H2O2 when tested after 10 days, but on day 20 did not show any difference. A similar pattern in the increase of CAT and APX activities protected CeO2NP treated maize seedlings from lipid peroxidation.

10As disruption of ROS homeostasis impairs plant growth and development, whereas maintenance of ROS levels within appropriate parameters promotes plant health, it is emerging that the induction of antioxidant machinery by NPs might promote plant growth as reported in a few studies as long as a harmful level of ROS is not reached in the cells, whereas, once breached, this may lead to impaired organelle function, membrane damage, and eventually phytotoxicity.

11So far, a handful of studies have showed that NPs could affect microbial and plant secondary metabolism. For example, the concentration of phenolic compounds secreted to an extracellular medium was increased 127.5 and 22.1%, respectively, in Arthrospira platensis Gomont (cyanobacterium) and Haematococcus pluvialis Flotow (microalga) after treating with 100 mg/L TiO2NPs. Artemisinin content was increased 3.9-fold in Artemisia annua L. hairy root cultures after 900 mg/L AgNPs treatment for 20 days. This increase was associated with oxidative stress (H2O2 production), lipid peroxidation and CAT activity. A substantial increase in plant growth and diosgenin concentration was observed in fenugreek after 2 μg/kg AgNP treatment. Ferulic acid and isovitexin were increased in barley plants exposed to CdO (cadmium oxide) NPs in air for 3 weeks at a concentration of 2.03 ± 0.45 × 105 particles cm-3. In A. thaliana, anthocyanin and flavonoid biosynthetic genes were upregulated in response to AgNPs.

12Although all the studies discussed above provide evidence for NP-mediated modulation of plant secondary metabolism, the following studies provide an indirect link between ROS and secondary metabolism. Satureja khuzestanica Jamzad calli growth improved significantly with increasing concentrations of carbon nanotubes (CNTs) in culture medium up to 50 mg/L, and then began to decrease at 500 mg/L. At this toxic concentration (500 mg/L), the highest level of H2O2 was observed together with significantly higher polyphenol oxidase (PPO), peroxidase (POD), and secondary metabolic activities. Similarly, when A. thaliana was exposed to 250 and 1000 mg/L CeO2 and indium oxide (In2O3) NPs, in addition to excessive ROS production, the activities of phenylalanine ammonia lyase (PAL) and PPO were greatly induced revealing a possible role of secondary metabolism in protection against oxidative stress. Furthermore, PAL is the first enzyme of the general phenylpropanoid pathway that catalyses the deamination of phenylalanine to cinnamic acid and play a key role in diverting aromatic amino acids from primary metabolism to phenylpropanoid pathway.

13There are several lines of evidence available in the literature implicating ROS-mediated signaling events as inductive cues for plant secondary metabolism. ROS themselves are signaling molecules, capable of inducing plant secondary metabolism. This could be observed during the wound-induced activation of secondary metabolism where ROS plays a key role as signaling molecule. In addition, ROS can also serve as signals for other messengers like jasmonic acid (JA), salicylic acid (SA), ethylene (ET), NO, brassinosteroids (BRs), etc., which are capable of modulating secondary metabolisms directly or indirectly.

14To support the notion that ROS induced by NPs acts as signals for secondary metabolism, many indirect lines of evidence are available. ZnONP treatment induced SA, whereas it suppressed JA in A. thaliana. Moreover, SA-mediated systemic acquired resistance (SAR) against microbial pathogens was compromised in A. thaliana after treatment with Ag, TiO2NPs, and CNTs, resulting in an increased colonization by Pseudomonas syringae pv. tomato, Pst. These authors further suggested that SA pathway repression is a common feature of NP exposure, as an inducible kinase in the pathway that activates basal immune response upon perception of bacterial flagellin namely FLG22-induced receptor-like kinase 1 (FRK1) was downregulated in response to NPs. In addition to SA-mediated SAR, other signaling pathways such as ET, BRs, and NO were also affected by NPs. In A. thaliana plants treated with AgNPs expression of ET biosynthetic components 1-aminocyclopropane-1-carboxylate synthase ACC and ACC oxidase 2 was reduced, suggesting that these NPs could inhibit ET perception and affect its biosynthesis. ET is an important signaling molecule mediating sesquiterpenoid biosynthesis in the Atractylodes lancea (Thunb.) endophytic fungi Gilmaniella sp. AL12 interaction. BRs, the steroidal phytohormones that play important role in plant growth, secondary metabolite accumulation, stress responses and adaptation could ameliorate ZnONP-induced oxidative stress by improving antioxidant potential and redox homeostasis in tomato seedlings. NO, another universal signaling molecule that plays a central role in secondary metabolite production in plant cells, is also involved in plant–NP interactions. For instance, AgNP-induced phytotoxicity could be alleviated by NO in P. sativum seedlings. Correspondingly, O. sativa NO excess mutant (noe1) plants were tolerant to ZnONP treatment, whereas OsNOA1-silenced (noa1) plants were susceptible to ZnONP-induced phytotoxicity.

15Although the aforementioned reports suggest that NPs are interfering with various signaling pathways and capable of modulating plant secondary metabolism, the exact mechanism through which this modulation could occur is not understood. We believe that the initial responses of plants to NPs might include elevated levels of ROS, cytoplasmic Ca2+ and upregulation of mitogen-activated protein kinase (MAPK) cascades similar to other abiotic stresses (Figure 1) because of the following reasons. Recognition of AgNPs by plasma membrane bound receptors triggered a Ca2+ burst and ROS induction in A. thaliana. Ca2+ levels and associated signaling pathway proteins were found to be upregulated in the proteomic analysis of AgNP treated O. sativa roots. These authors hypothesized that AgNPs, or ions released thereof, impede cell metabolism by binding to Ca2+ receptors, Ca2+ channels, and Ca2+/Na+ ATPases. As sensed by calcium binding proteins (CaBPs) or other NP-specific proteins, NPs either mimic Ca2+ or signaling molecules in the cytosol. MAPK phosphorylation, and activation of downstream transcription factors generally lead to the transcriptional reprogramming of secondary metabolism in plants. Although no direct evidence for the involvement of MAPK pathways in plant-NP interactions is available, animal and human cell line studies revealed that analogous pathways are involved in AgNP-induced signaling, and it has been postulated that plants may also utilize MAPK cascade upon exposure to Ag NPs.

16As discussed in this article, exposure to NPs has the potential to alter plant secondary metabolism. Secondary metabolites can act as phytoalexins/phytoanticipins to protect plants from herbivores and pathogenic microbes, as signals for plant symbiotic interactions with beneficial microbes and as allelopathic agents to protect plants from rhizosphere competitors. In addition, they also serve as physical and chemical barriers to abiotic stressors and as antioxidants to scavenge ROS. Although NP-mediated changes in plant secondary metabolism would affect the optimal interaction of plants with their surrounding environment and possibly growth and productivity, substantial research is needed to understand the exact impact.

17The presence of NPs in the environment might affect the pharmacological properties of medicinal plants, as many phytomedicines exert their beneficial effects through additive or synergistic actions of several compounds acting on single or multiple target sites associated with a physiological process. While it is necessary to tackle these adverse effects, NP-mediated changes in secondary metabolism could also be beneficial if harnessed in such a way that NPs are used as elicitors in molecular pharming to enhance the production of desired secondary metabolites. For example, the content of important drugs like artemisinin and diosgenin were enhanced in plants treated with NPs. The ability of NPs to adsorb secondary metabolites could be exploited for purification of precious compounds from plants via nanotrapping, if harnessed properly. Similarly, in vitro green synthesis of NPs using plant extracts can be further extended to develop high throughput tools to purify specific classes of compounds, as green synthesized NPs are often found as conjugates of secondary metabolites.

18Paucity of knowledge on the exact consequences of NP accumulation in the environment on plant metabolism is exacerbated by the fact that most of the studies have been conducted under controlled laboratory conditions and typically at much higher concentrations than what could be expected in the environment. For instance, to induce statistically significant changes in the growth characteristics of A. thaliana plants, the minimum concentration of AgNPs was 300 mg/L under laboratory conditions, a value much higher compared to the predicted environmental concentration of AgNPs in different environmental compartments: e.g., 1.3–4.4 mg/kg in sewage sludge. Moreover, the ecologically relevant concentration of NPs largely depends on their environmental fate, plant species, characteristics of NPs, the medium through which it reaches the plant, etc., in addition to other, yet unknown, parameters. Although a recent study showed that ecologically relevant size and concentration of CdONPs could activate secondary metabolism in barley plants, it is difficult to generalize the impact of NPs on plant secondary metabolism in the environmental perspective. However, it is necessary to improve our understanding on the environmental fate of NPs and their hazards/risks, testing ecologically relevant conditions and concentrations in the context of plant secondary metabolism. Considering that plant secondary metabolism includes a vast array of compounds that are tightly controlled by signaling events and environmental cues, a case-by-case analysis might be necessary to have a deeper understanding.

19GM collected information on NPs and secondary metabolism. CS prepared the possible mechanisms of plant secondary metabolism induction by NPs. GF conceived the idea of this perspective and collected all other information on ROS, singling pathways, and phytotoxicity in response to NPs. All the authors participated in writing and approved the manuscript for publication.

20The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.