Epigenetic Modifications and Gene Expression Alterations in Plants Exposed to Nanomaterials and Nanoplastics: The Role of MicroRNAs, lncRNAs and DNA Methylation

Abstract

Nanomaterials (NMs) are currently widely used in a wide range of industrial production and scientific applications, starting from molecular and medical diagnostics to agriculture. In the agricultural and food systems, NMs are now used in various ways, to improve the nutritional value of crops, detect microbial activity and inhibit biofilms, encapsulate and deliver pesticides, protect plants from chemical spoilage, as nanosensors and more. Despite these applications, NMs are described as “dual-face technologies”: they can also act as environmental contaminants. For instance, nanoplastics (NPs) dispersed in the environment can damage plants at different levels and undermine their viability. Epigenetic modifications induced by NMs have potentially wider and longer-term impacts on gene expression and plant functions. Therefore, it is important to verify whether plants are also affected by NMs on the molecular level, including epigenetic mechanisms and any induced variation on the epigenome. This review focusses on gene expression modulation and epigenetic alterations such as DNA methylation and the role of microRNAs and long non-coding RNAs (lncRNAs) induced in plants and crops by NMs and NPs.

1. Introduction

1.1. Nanomaterials

Nanomaterials (NMs) are substances with dimensions between 1 and 100 nanometers (nm) in at least one of three dimensions [1]. Their smaller dimensions compared to micromaterials give them particular chemical–physical properties that make them interesting for new applications. For instance, dispersed NMs have a higher number of particles per unit and a larger surface area, making them more reactive [1,2]. They can be classified in many ways based on their properties (Figure 1), including their chemical composition [3]. NMs can be synthesized starting with organic compounds and give rise to a very large class of nanomaterials [4]. They can be carbon-based or also formed by biological molecules like micelles [5,6]. Additionally, NMs can be synthesized with inorganic elements. An immediate schematization consists of grouping them into metal or metal-oxide nanoparticles. They lack carbon atoms and have a very wide range of applications in the medical and manufacturing fields. They can be ambivalent in the sense that studying their toxicity is very important because they can induce adverse outcomes, such as titanium NMs [7], or are non-reactive, like gold-based NMs [8]. NMs are currently widely used in various production contexts and in a wide range of scientific fields, from molecular and medical diagnostics to agriculture [9]. In the agricultural food field, NMs have potential benefits in favoring sustainability, such as in the precision dosing of fertilizers and pesticides and potential uses in soil remediation [10]. In the first case, NMs not only proved to be the optimal carrier of pesticides but also could be directly used as pesticides themselves. Silica NMs at 0.5 and 1 g/kg showed a biocide ability against weevils with Orzya sativa through a mechanism still not clarified [11]. Famous additional examples are gold (Au) and silver (Ag) NMs, which are also used as antimicrobial agents in cosmetics [12]. Au NMs were demonstrated to be active against bacterial genera such as Fusarium, Aspergillus and Staphylococcus while Ag NMs proved their efficacy against insects as Hippobosca maculata [13,14]. Moreover, encapsulating pesticides in NMs may increase their effects as biocides and reduce toxicity in animals. When compared to free imidacloprid, the pesticide imidacloprid encapsulated in calcium alginate nanoparticles was less cytotoxic against Vero cells at 5 and 10 mg/L. However, following 11 to 15 days of application as a spray, it proved more effective against leafhoppers (pesticide concentration 0.145 mg/L) [15,16].

In addition to these applications, the fact that different plants may respond in very different ways should be underlined. NMs and nanoplastics (NPs) are now used in various contexts such as improving the nutritional value of crops, detecting microbial activity and inhibiting biofilms, the encapsulation and delivery of pesticides, protecting plants from chemical spoilage, nanosensors and more; it is important to find the perfect balance between the benefits for plant growth and the toxic effects [17]. In fact, it must also be considered that NMs can act as environmental contaminants: for example, NPs present in the environment can damage plants at different levels and undermine their viability [18,19]. NPs can be classified as a specific type of environmental incidental NM, and scientific investigations should take into account the existing profound knowledge on nanotechnology/nanomaterials when discussing issues around NPs. Today, they are the most significant environmental pollutants by quantity, so investigating their impact on plants is crucial. Moreover, the composition of micro- and nanoplastics is heterogeneous as they degrade from a vast number of different plastics containing a wide number of additives and other substances. This issue makes it more complex to generalize their hazards to living beings. NPs absorbed and accumulated by crops contaminate the food chain and can pose unforeseen and unknown risks to human and animal health. In general, further studies are needed to understand which NMs and NPs are useful and how they should be used for crops of agri-food interest. An important first step is to identify NMs that are potentially useful for plant viability and above all are non-toxic. In addition to the classic toxicity studies, it is important to verify whether plants are also affected by NM effects at the molecular level, also with reference to epigenetic mechanisms and any induced variations on the epigenome. The need to understand epigenetic impacts is driven by a combination of deliberate applications in agri-food sectors and unintentional impacts, i.e., pollution from other sources, considering that their modifications can be inherited by the following generations [20]. The main goal of this review is to highlight the epigenetic modification that plants go through in response to NMs exposure. In addition to the standard epigenetic modifications, such as methylation and histone modifications, we support the concept of and the need for including RNA-mediated regulations in these kinds of responses. In some cases, we also reported the effects of microplastics and bigger particles due to the lack of studies at the “nanoscale” to solicitate the urgency of further studies.

Nanomaterials are mainly classified by their chemical composition. The two most important categories are organic NMs and inorganic NMs. Then, their three-dimensional structure determines their properties and applications. The figure illustrates the most used NMs in agriculture and clinical practice. Organic NMs are composed mainly of carbon atoms variably bonded with other molecules. For instance, when composed of lipids, they are called micelles or liposomes. Based on spatial geometry and carbon number, they are differently classified. Fullerenes are spherical, composed of repeated pentagonal or hexagonal units; nanotubes have a tubular form; and nanofibers are characterized by a long structure. Inorganic NMs, because of their high chemical variability, can be summarized in a lot of different groups. In the figure, we focus on the main ones. Metal NMs can be considered both for clinical therapies and as pollutants. For the first case, gold NMs are considered the most useful ones because of their inert properties and for the absence of toxic effects. All the other NMs composed of metals are instead more or less toxic based on the reactive potential of the elements they are composed of with biological systems. To increase the reactivity of the metal NMs, they are often used as “oxide NMs”. Unluckily, the increased reactivity is also translated to living organisms when exposed to them. Therefore, if NMs are considered pollutants, their increased reactivity, useful for industrial and biomedical applications, can pose a higher risk to plants and other living beings. Indeed, oxide NMs are more toxic than the respective non-oxides [4,8]. For instance, zinc oxide NMs have a higher antimicrobial effect when compared to zinc NMs. Quantum dots exploit quantum confinement to generate fluorescence. This property makes them suitable for diagnostics and imaging. They are semiconductors composed principally of zinc. Sometimes they can be coated with carbon sheets. Only recently a new typology made by two graphene sheets has been developed, rendering the classic inorganic quantum dots a technology bouncing between organic and inorganic NMs [4,8].

1.2. Plants Epigenetics

The term epigenetics refers to alterations in gene expression that are not caused by mutations in the DNA sequence and that can be inherited (schematized in Figure 2) [21]. DNA methylation is historically the most studied epigenetic mechanism [22]. It consists of the addition of a methyl group (-CH₃) to the fifth carbon of cytosine, forming 5-methylcytosine [22]. This is a widely conserved mechanism from plants (in Arabidopsis thaliana, almost 14% of its genome is methylated) to Drosophila melanogaster (only 0.03%) [23,24]. In plants, methylation occurs particularly on three sites: CG, CXG and CXX (where x is one base between adenine, cytosine and thymine) [24]. It is an enzyme-mediated process that is driven by three factors: methyltransferase 1 (MET1), chromomethylase 3 (CMT3) and domain-rearranged methylase 1 and 2 (DRM1 and DRM2) [25]. The inhibition of MET1 can lead to defects in seed development and reduced methylation at sequences such as transposons. It is directed at CGs [25]. CMT3 is specific to CXGs. Both of them are essential for guaranteeing symmetric methylation. DRM1 and DRM2 are responsible for ex novo methylation [26].

Histone modifications are the second most-studied type of epigenetic mechanisms. They consist of adding chemical groups such as methyl and acetyl to aminoacidic residues like arginine or lysine. These additions may lead to chromatin modifications inducing or repressing gene expression. Usually, methylation is associated with reduced gene expression, while acetylation is associated with increased gene expression. Histone acetyltransferases and histone deacetylase drive the addition or removal of the acetyl group, while lysine and arginine methyltransferases are responsible for methylation [24]. Histone modifications are relevant biological processes contributing to the regulation of flower development and photosynthesis [27,28]. Histone ubiquitination is still an unclear process. It probably contributes to histone trimethylation and to mediating transcriptional changes [29].

The figure summarizes the most frequently found types of epigenetic modifications occurring in plants. Methylation is mainly represented by the addition of a methyl group to cytosine. Histone modifications are usually characterized by the presence of one or more methyl groups or by the addition or removal of acetyls from lysine (K) or arginine residues. Ubiquitination is a lesser-known process whose functions need to be further investigated.

Non-coding RNAs (ncRNAs) have a decisive role in regulating cell functionalities. They can be classified as housekeeping if they are RNAs needed for basic cellular processes such as protein synthesis and ribosome functions. In this case, the main components are messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). NcRNAs were discovered in the 1960s. Till then, RNAs were considered only as molecules involved in gene expression. Therefore, their revelation was a groundbreaking discovery [30]. Additionally, all of the ncRNAs that were found throughout the course of the following two decades were detected by chance, before any theories regarding their potential roles were even hypothesized. Many of the ncRNAs were thought to be residual fragments that represented the introns that had been cut out of pre-mRNAs until the transcription and processing of mRNAs were clarified. Meanwhile, it was shown that several of the ncRNAs were implicated in exon splicing and intron elimination. When systematic searches for non-coding RNAs (ncRNAs) were finally conducted in the late 1990s, they turned up a veritable universe of ncRNAs ranging in length [31]. Based on the number of base pairs, they are classified in small non-coding RNA (sncRNAs, <50 nucleotides) and long non-coding RNA (lncRNAs, >50 nucleotides). MicroRNAs (miRNAs) are a subtype of sncRNAs. They are able to repress the expression of a gene interacting with the 3′ region of the target RNA blocking its translation [32]. NcRNAs are composed of a wide range of subtypes. They were discovered in 1991, clarifying the mechanism of X chromosome inactivation through XIST silencing in animals [33]. They can be classified as (1) long intergenic ncRNA (lincRNA); (2) intron ncRNA (IncRNA); (3) antisense RNA and natural antisense transcripts (NATs); (4) divergent lncRNA; and (5) enhancer RNA (eRNA), based on the genetic locus they originated from [34]. LncRNAs can interact with proteins, guide RNA complexes, act as scaffold molecules and even interact with miRNAs [34,35,36,37]. Considering all of these functions, it appears clear that their role in controlling gene expression without altering the DNA sequence is fundamental. They also proved to act on levels of DNA methylation. Wassenegger discovered so-called RNA-directed DNA methylation (RdDM) in plants [38]. The interaction with AGO4 (ARGONAUTE4) lead to the methylation of target genes by DRM2 (de novo methyltransferase) [38].

1.3. Epigenetics and Environmental Stress

Epigenetic mechanisms lead to responses to environmental stresses, allowing faster reactions to different stimuli rather than positive mutations. It has been argued that, under some circumstances, epigenetic memory in the genome helps remember the stress tolerance strategy adopted by the plant [39].

Plants, like all eukaryotes, regulate gene expression primarily through transcription factors, which respond to many other mechanisms, including epigenetic ones such as molecular, genetic and metabolic changes, in order to react to external stimuli. In plant ecology, recent studies have investigated the role and contribution of epigenetics to plant phenotypes, stress responses and adaptation to habitats. While there has been some progress in revealing the role of epigenetic in ecology, the studies with non-model species have so far been limited to describing broad patterns based on markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contributed to a more detailed understanding but have limited ecological realism [40].

Studies on the epigenetic effects of NMs and NPs could reveal whether plants change their metabolic activity and respond at the systemic and/or cellular and tissue levels. In addition, it is important to understand whether subsequent generations inherit an epigenetic fingerprint that makes them more resistant to NMs or even more sensitive. This review focuses attention on the gene expression modulation and epigenetic alterations induced by microRNAs, lncRNAs and DNA methylation by NPs and NMs in plants.

2. Epigenetic and Gene Expression Modulation by Environmental Factors in Plants: NPs and NMs

Considering the challenges that climate change represents, research on plants may offer improved insights into the molecular bases of adaptability and epigenetic stress memory, enabling (epi) genome editing of crops for climate resilience and sustainable agriculture [41]. The field of environmental epigenetics studies how environmental variables might modify an individual’s epigenetic profile [42]. Numerous investigations have demonstrated the ability of environmental contaminants to cause epigenetic changes. Transgenerational epigenetic stress memory should be meiotically stable and pass down to at least two generations without stress [43]. One well-known example of pollutants inducing epigenetic effects is arsenic, which, through the arsenic detoxification route, causes global DNA hypomethylation, cancer and cardiovascular disorders in animals [44,45,46]. In plants, it has been observed that exposing rice to drought stress repeatedly over several generations improves adaptation through epimutations and changes in DNA methylation that are passed to the progenies [47]. The MorpheusMolecule1 (MOM1) and DDM1 genes have been related to the absence of stress-induced epigenetic markers in Arabidopsis, but the descendants of a double mutant for ddm1–mom1 were observed to inherit stress-induced epigenetic markers. In heterozygosis—either mom1 or ddm1—plants were unable to inherit the epigenetic mark caused by stress. These results suggest that DDM1 and MOM1 prevent stress-induced epimarks from being inherited [48]. Concluding, plants adjust to changes in their environment by remembering stressful events and becoming more resilient to similar stressors in the future thanks to a multitude of mechanisms that can regulate epigenetic responses and memory (Figure 3).

Plants are exposed both to abiotic and biotic stressors. They react by activating genes related to self-defense. This response can be controlled by epigenetic mechanisms, or in an opposite way, it can lead to epigenetic modifications. In both cases the epigenetic mechanisms leave a footprint on the DNA that can be inherited by the following generations, generating more resistant individuals (A). The main plant epigenetic mechanisms are shown in (B), with a focus on the alterations induced by NMs in (C).

The study of epigenetics associated with NMs and NPs is still not popular enough despite the relevance that the topic is acquiring, and as a result, we have analyzed the few articles that are currently accessible on model organisms including plants and crops, to our knowledge. Multi-Walled Carbon Nanotubes (MWCNTs, 5–10 μg/mL) in Allium cepa showed the ability to alter methylation patterns following experimental exposure [49]. The authors referred to the sequence 5′-CCGG-3′ as an epigenetic marker. In particular, methylation at this locus stopped cleavage with the HpaII restriction enzyme but not with MspI. The authors did not observe any change with MspI, but the amount of cleavage increased with HpaII at 5 μg/mL. The higher concentration led to the block of HpaII activity. These results indicated the presence of hypermethylation following MWCNT exposure. In the same study, the cytotoxicity and genotoxicity of nanomaterials were also proved. Zea mays roots were shown to be sensitive to Single-Walled Carbon Nanotubes (SWCNTs, 30 nm, 20 mg/L) [50]. Following SWCNT exposure, the authors detected a decrease in HDA108, HD1b and HD2 genes in parallel to an overexpression of HDA101, HDA102 and HDA106. In addition, a global increase in H3K9 acetylation was also observed. More studies exist today about NP-induced modifications. The effects of polyethylene terephthalate nanoplastics (PET-NPs) were tested on the model Spirodela polyrhiza (L.) focusing on particle-induced epigenetic effects, like alterations in the DNA methylation pattern [51]. The methylation-sensitive amplification polymorphism (MSAP) technique was applied. They employed MspI and HpaII as restriction enzymes as described above. Results showed hypermethylation in 5′CCGG sites implicated in DNA defense from dangerous factors like reactive oxygen species (ROS) or in the presence of new epialleles. This work, to our knowledge, is the first demonstration of PET nanoplastic-induced epigenetic modifications in plant organisms, but also the first, in general, that considered epigenetic outcomes in plants. We also included papers on gene expression regulation as a result of exposure to NPs, considering the relationship between epigenetic modifications and changes in gene expression. It is well known that exposure to NMs generally alters the expression of genes in a variety of exposed organisms, including microorganisms and plants, particularly when the particles are charged or comprise metals, and we briefly describe a few examples. NPs are easily absorbed by plant roots, and reports of NPs being transported to the upper portions of plants through xylem have been made [52]. It has been demonstrated that NPs can cause changes in gene expression in a variety of plant organisms, in addition to the previously mentioned toxicity pathways. Lagarde et al. [53] demonstrated that changes could occur in genes unrelated to the detoxification of particles in the microalga Chlamydomonas reinhardtii when it was exposed to 400 and 1000 μm polypropylene and high-density polyethylene. A decrease in UDP-glucose-4-dehydratase was noted, along with an increase in UDP-glucuronate decarboxylase and UDP-glucose-4-epimerase, which are involved in the production of xylose. When 100 nm nanopolystyrene was applied to Allium cepa roots, a dose-dependent trend in decreased CDC2 expression was seen, resulting in a reduction in the plant’s ability to undergo cell division [54]. Sun et al. [55] found that exposure to differently charged nanopolystyrene caused a rise in flavonoids and a decrease in peroxidases, which heightened the oxidative stress-induced genotoxic impact in roots. In cucumber (Cucumis sativus L.), the impacts of polystyrene NPs on photosynthesis, carbon and nitrogen metabolism were discovered by a combination of transcriptomics, metabolomics and physiological characteristics. Despite extensive research, it is still unknown how microplastic and nanoplastic contamination affects plant photosynthesis and the metabolism of carbon and nitrogen. Indeed, Zhuang et al. [56] utilized 5 μm and 0.1 μm polystyrene microplastics (PS-MPs) and nanoplastics (PS-NPs), respectively, to investigate if they affected cucumber photosynthesis, as well as carbon and nitrogen metabolism. Cucumber mesophyll cell integrity and photosynthetic metrics were dramatically decreased by the PS-MP treatments, whereas the amount of soluble sugar in cucumber leaves was increased. Cucumber leaf photosynthetic ability was reduced as a result of the 5 μm PS alteration in the expression of genes necessary for the generation of ATP and NADPH. Attenuating the detrimental effects on photosynthetic efficiency, 0.1 μm PS changed the expression of the genes phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK), which in turn modified the intercellular CO₂ concentration. Additionally, PS decreased the expression levels of nitrate/nitrite transporter (NRT) and nitrate reductase (NR), which decreased the efficiency of nitrogen usage in cucumber leaves and caused damage to mesophyll cells by increasing the accumulation of γ-glutamylcysteine (γ-GC) and citrulline and reduced glutathione (GSH).

Jang. M. et al. [57] demonstrated that polystyrene nanoparticles can penetrate grains, albeit it is still unclear how they would affect crops cultivated in contaminated soil. The authors focused on the translocation of PS-NPs in crops, such as rice (Oryza sativa L.) and peanuts (Arachis hypogaea L.). They found that PS-NPs may move from the root to the grain during the maturity stage. This is interesting as it suggests that NPs transfer into the reproductive meristem, both via xylem and into gametes. Kim et al. [58] recently examined the parental transfer of nanoplastics by exposing Pisum sativum (pea) plants to NPs over an extended period of time via a soil medium. Using confocal laser scanning microscopy, they detected the presence of NPs in harvested fruits and a later generation of plants transplanted in uncontaminated soil. The fluorescence was found in the vascular bundles’ cell walls rather than in the epidermis, which suggests that NPs were transferred from their parents. They also measured the amount of each nutrient in peas and estimated the reproductive parameters to find out how NPs affected the health of the next plant generations. Changes in the amount and composition of nutrients, as well as reductions in crop production and fruit biomass, were observed. Kim et al. [58] investigated the effects of the chronic exposure of pea plants to MPs in the soil and observed the transfer of MPs in the subsequent generations of plants, from F0 to F1. First, the presence of MPs in the harvested fruits and subsequent plants was confirmed based on significant fluorescence in the roots and stems. The fluorescence was detected in the vascular bundles rather than in the epidermis, confirming that it was not due to any external exposure but rather due to parental transfer.

Significant safety concerns were raised by the potential dramatic impacts of NPs on plants throughout generations, affecting plant species as well as their predators in terrestrial ecosystems. The long-term impacts of NPs on plants may be dangerous to the availability of food, as this study demonstrates evidence of the parental transfer of NPs in the soil through plants.

3. The Role of MicroRNAs (miRNAs) and Long Non-Coding RNAs (lncRNAs) as Epigenetic Factors in Response to NPs, MPs and NMs Stress

Plant growth and development are highly precisely regulated, and this regulation is impacted by both internal genetic information and external environmental influences. Plant growth and development depend on the normal expression of miRNAs. Previous research has demonstrated that miRNA controls plant growth and development extensively [59]. Research on Arabidopsis thaliana and other plants has demonstrated the involvement of miRNAs in a wide range of biological functions. In contrast to hormones’ widespread effects on plants, miRNAs play a critical role in the accurate regulation of gene expression. Further research is needed to understand how plants modify tissue formation by integrating the fine regulation of miRNAs into the hormone system pathways. Understanding the function of miRNA requires an understanding of the role and mechanism of miRNA migration between cells and tissues. Crop breeding depends on our ability to comprehend the complex regulation of plant development by miRNAs. Plant mortality is frequently caused by dominant gene knockout; however, miRNAs can safely alter gene expression to some extent and enhance plant development. Grain yield in rice is determined by the number of branches, including tiller and inflorescence branches [59]. Researchers studying plants have recently placed a great deal of emphasis on the mobility of short RNA inside and between cells, tissues and organisms. Determining the function and process of small RNA migration is a laborious task. Thus far, research suggests that miRNA has the ability to migrate and generate gradient distributions across many tissues. Following biogenesis, miRNA is transferred to target cells while being shielded from deterioration. Further research is needed to understand how intermediate steps affect miRNA mobility and its non-cellular autonomous function.

Additionally, plants can be induced to synthesize siRNA by biotic and abiotic stressors. For instance, in response to stressors like nitrogen shortage, A. thaliana is able to express a significant amount of 22 nt siRNAs that are dependent on DCL2 and RDR6. The reason why just a limited subset of A. thaliana gene loci is able to make 22 nt siRNAs is still a mystery. More miRNAs, their action mechanisms and their regulatory pathways need to be found in model plants with the aid of various single-cell omics and nanopore sequencing techniques. This will provide a solid theoretical foundation for comprehending how miRNA controls plant growth and development and can then be applied to agriculturally significant plants [59].

Long non-coding RNAs (lncRNAs) have been extensively studied in the literature, revealing their diverse regulatory roles in plant’s responses to abiotic stressors and their crucial roles in environmental adaptation. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with a length of more than 200 nucleotides that are known to affect multiple biological processes. These processes include plant drought resistance, temperature regulation, salt tolerance and heavy metal stress. Future research showing the true scope and intricacy of the plant non-coding transcriptome may lead to further changes in the definition and categorization of lncRNAs [60,61]. As such, our understanding of plant lncRNAs is very limited and needs further exploration. Growing amounts of data from several plant species demonstrate the vital functions that lncRNAs play in abiotic stress responses. On the other hand, fewer lncRNAs have been investigated in bryophytes and ferns, whereas the majority of functional plant lncRNAs were discovered in angiosperms. The majority of research on stress-responsive lncRNAs has been concentrated on a single stress type or single stress. Consequently, further work is required to identify and clarify the molecular processes and mechanisms of plant lncRNAs. Recently, according to [62], lncRNAs have been found to play a crucial role in regulating many phytohormone pathways in plants in response to various abiotic and biotic stimuli.

Comprehensive screening and cross-referencing of lncRNAs would offer fresh perspectives on identifying and functionally characterizing putative important lncRNAs that are critical for plant adaptation and memory of diverse and recurring abiotic stressors. Furthermore, it is still not entirely obvious how distinct plant groups—like Eudicotyledoneae and Monocotyledoneae, for example—conserve their biological roles and metabolisms. Consequently, extensive evaluations of plant lncRNA activity and comparative studies of lncRNA conservation among plant species will be effective methods for lncRNA identification.

While many species’ responses to abiotic stressors are mediated by lncRNAs, it is still unclear whether or not these transcripts could serve as useful markers or genomic resources in plant molecular breeding. Furthermore, it is necessary to continue developing breeding techniques based on lncRNAs in order to optimize the balance between abiotic stress conditions and plant growth.

Plant-based foods have the potential to expose humans to micro- and nanoplastics. Numerous cellular processes in humans can be impacted by NPs, including necrotic, inflammatory and apoptotic processes. Low concentrations of polystyrene NPs have been shown to cause dysregulation of miRNAs in human and animal cells at the molecular level. NPs may have the ability to impact biological processes at the nuclear level, such as changing DNA methylation and controlling the expression of these molecules [63].

There are few studies on how plastics affect higher terrestrial plants, particularly long-lived trees. A thorough investigation of the physiological and biochemical reactions of seedlings of the conifer species Torreya grandis cv. Merrillii (family Taxacea) to 100 nm-diameter polystyrene nanoplastics (PSNPs) was conducted by [64], offering new information on how forest plants react to NP treatment. One-year-old T. grandis plants were used in this study, and the length of the experiment was 14 days. The findings demonstrated that the activities of catalase and peroxidase, as well as the buildup of the reactive material thiobarbituric acid, were increased by nanoplastics. Moreover, a number of genetic markers of T. grandis, including short RNA, gene transcription, protein expressions and metabolite accumulation, were significantly impacted by PSNP treatment. According to a multiomic study, PSNPs control protein expression and short RNA transcription to influence the pathways involved in the manufacture of terpenoid and flavonoid compounds. Analyses of small RNA, the microRNAome, the transcriptome and the proteome revealed that nanoplastics might have an impact on T. grandis shoot photosynthesis. By modifying small RNA expression and protein levels, PSNPs can also alter the pathways involved in the manufacture of terpenoid and flavonoid compounds, according to multiomic research.

4. Discussion and Conclusions

The increase in pollutants in the environment has introduced the need to understand how living organisms and humans are affected by them. Plants represent the basal constituents of ecosystems, determining their main features. In addition, they constitute one of the main sources of food for humans. The understanding of how they are impacted by pollutants represents a fundamental step both for environmental studies and for the assessment of human hazard. NPs are now the most significant environmental pollutant by quantity; therefore, investigating their impact on plants is crucial. In addition to classical toxicity studies, it is important to see if plants are also affected by the presence of NMs at the molecular level, with reference to epigenetic mechanisms and gene expression. These could tell us whether plants change their metabolic activity or respond systemically. In addition, it is important to understand whether subsequent generations inherit an epigenetic fingerprint that makes them more resistant to pollution or NMs. As for the specific epigenetic mechanisms involved, the current literature has demonstrated, by both in vitro and in vivo experiments, the ability of miRNAs and lncRNAs to dysregulate gene expression due to exposure to environmental factors, including NPs in plants/crops and animals (Figure 3). Banikazemi et al. [63] reviewed the role of microRNAs and long non-coding RNAs and their biological effects on epigenetic alterations in response to NP exposure. The evolution, roles, processe, and applications of plant lncRNAs under abiotic challenges, like NMs and NPs exposure, remain unclear despite the pertinent results that have been reported recently [65]. Actually, as shown in Figure 4, a major part of the papers focused on NMs as pollutants, especially investigating their amounts in different environments with descriptive studies. In addition, papers on plants also focused on physiological and phenotypical aspects, with low interest on the molecular level. Microplastics remain the most-studied followed by NPs. Animals are more considered than plants. In conclusion, we underline that there are not lots of scientific papers on plants/epigenetics/nanoparticles and nanomaterial arguments, but this research field is in fast starting development and needs more comprehensive data on the epigenetics/epigenomics affecting plants via NPs/NMs.

The figure illustrates the results of a bibliometric analysis highlighting connections between the main topics published to date using the keywords “nanoplastics, microplastics” AND “plants” on PubMed. A total of 10,000 papers were collected as a list, downloaded from the database and then processed with Vosviewer. The analysis was conducted with all the possible results till January 2025. Even if the main keywords were “nanoplastics and microplastics”, MPs remain the principal object of study, with polystyrene being the most used polymer type.