Nanoparticles: Synthesis, Morphophysiological Effects, and Proteomic Responses of Crop Plants

Plant cells are frequently challenged with a wide range of adverse environmental conditions that restrict plant growth and limit the productivity of agricultural crops. Rapid development of nanotechnology and unsystematic discharge of metal containing nanoparticles (NPs) into the environment pose a serious threat to the ecological receptors including plants. Engineered nanoparticles are synthesized by physical, chemical, biological, or hybrid methods. In addition, volcanic eruption, mechanical grinding of earthquake-generating faults in Earth’s crust, ocean spray, and ultrafine cosmic dust are the natural source of NPs in the atmosphere. Untying the nature of plant interactions with NPs is fundamental for assessing their uptake and distribution, as well as evaluating phytotoxicity. Modern mass spectrometry-based proteomic techniques allow precise identification of low abundant proteins, protein–protein interactions, and in-depth analyses of cellular signaling networks. The present review highlights current understanding of plant responses to NPs exploiting high-throughput proteomics techniques. Synthesis of NPs, their morphophysiological effects on crops, and applications of proteomic techniques, are discussed in details to comprehend the underlying mechanism of NPs stress acclimation.


Introduction
Rapid advancement in nanotechnology has taken the food industry to a new height [1]. Nanoparticles (NPs) are ultrafine particles with a size of less than 100 nm in at least one dimension [2]. Owing to having unique physical and chemical properties, such as high surface area and nanoscale size, these microscopic particles have the potential to improve the quality of food processing, packaging, storage, transportation, functionality, and other safety aspects of food [2]. Moreover, in recent years, nanotechnology has gained tremendous attention in agriculture sector as promising agents for plant growth, fertilizers, and pesticides, ensuring sustainable crop production [3]. The engineered nanomaterials have a wide range of applications in the healthcare industry, including drug delivery [4], cellular imaging and diagnosis [5], cancer therapy [6], antimicrobials [7], biosensors [8], anti-diabetic agents [9], and cosmetics [10]. Nevertheless, unsystematic release of nano-containing biosolids and agrochemicals is a serious threat to the environment, including plants [11].
Among metal based NPs, iron NPs are widely used in environmental remediation, biomedical, diagnostic field, and drug delivery because of their unique properties, such as excellent biodegradability, low cytotoxicity, and ability to attach with multiple targeted ligands or antibodies [12,13]. Few studies have been conducted to assess the impact of iron NPs on plants [14,15]. Kim et al. [14] reported that exposure of iron NPs triggered root elongation in Arabidopsis thaliana by nZVI-mediated OH radical-induced cell wall loosening. Conversely, iron-ion/NPs did not affect physiological parameters in lettuce plant [15]. Similar to iron, copper NPs have diverse applications, such as electro metallic agent, wood preservative, bioactive, and lubricant [16]. However, unmanaged discharge of copper NPs into the environment poses an increasing threat to plants [17]. Hence, there is urgent need of in-depth research for understanding the various pathways involved in NPs stress response mechanisms in plants.
Most of the phytotoxicity research so far conducted is focused on effects of NPs on seed germination and, at very early growth stages, of the plants [18]. Techniques, including cytotoxicity study [19], transcriptomics [20], and proteomics [21] have been widely used for analyzing uptake, bioaccumulation, biotransformation, and risks of NPs for food crops. Moreover, NP-mediated phytotoxicity as well as their ecotoxicity was conducted on mammalian cells [22]. These high-throughput genome-based omics techniques have been used extensively to dissect plant responses to NPs [23]. Although transcriptional analysis was performed in a variety of organisms including microbes, humans, mammalian cell lines, and other model organisms [24], information about plant-NPs interactions and NP-mediated phytotoxicity is still limited.
The high-throughput techniques used in proteomics focus on revealing structure and conformation of proteins, protein−protein, and protein−ligand interactions. Proteomics offer several advantages over the genome or transcriptome-based technologies as it directly deals with the functional molecules rather than DNA or mRNA [25]. Gel-based or gel-free proteomic techniques, protein chips/microarrays, and protein biomarkers have been widely used for reliable identification and accurate quantitation of stress responsive proteins for dissecting plant stress signaling pathways [26]. Improved protein extraction protocol and advancement in mass spectrometry have made proteomics a rapid, sensitive, and reliable technique for identification and characterization of differentially modulated proteins to assess the possible impact of NPs on crops. Alternative to single omics approach, multi-omics techniques, such as combination of transcriptomics, proteomics, and metabolomics offer more advantages in identifying the underlying response mechanisms of plants towards the environmental contaminants, including NPs [27]. This review highlights the various methods used for synthesis of NPs, their morphophysiological impact on crop plants, and applications of proteomic techniques to comprehend the underlying mechanism of NPs stress acclimation.

Methods for NPs Synthesis
The size, concentration, and stability of NPs primarily determine their effects on plants [23]. The characteristics of NPs largely depend on their mode of synthesis. There are various physical, chemical, and biological methods for the synthesis of economically important NPs [28]. Although the methods of NPs synthesis are diverse, there is a bare necessity to develop some ecofriendly processes so that they may be less hazardous to the environment (Table 1).

Physical Methods for NPs Synthesis
These methods are being used for the synthesis of various economically important NPs, such as silver, copper, iron, titanium, and others. The method of tube furnace was used for the synthesis of spherical silver NPs [29]; while laser ablation resulted in the formation of triangular bipyramidal nanocrystals of silver [30]. NPs synthesized by Ytterbium fiber laser ablation were spherical in shape and polycrystalline in nature [31]. Iron NPs with the globular shape were produced using the thermal dehydration method [32]; whereas irregular shape was attained with thermal decomposition approach [33]. Furthermore, copper NPs with spherical shaped and uniform diameters were synthesized using the thermal decomposition approach [34]. The topographic map indicated that NPs synthesized through sodium borohydrate as the reducing agent produced the NPs with irregular surfaces [35], while the polyol method synthesized pure crystalline copper NPs with cubic surface [36]. When tween 80 was added as modification in the polyol method, it resulted in the formation of crystalline copper NPs [37]. The physical approaches mainly synthesized the NPs with uniform morphological characteristics, which ultimately affected their response towards the environment as well as to the living ecosystem.

Chemical Methods for NPs Synthesis
The chemical reduction using a variety of organic/inorganic reducing agents, electrochemical techniques, physicochemical reduction, and radiolysis is a well-accepted approach for the synthesis of NPs [38]. The process of reduction through various chemicals led to the synthesis of the diverse shape of properties of NPs, such as silver nitrate reduction with sodium borohydrate resulted in the mixture of spherical and rod shaped silver NPs [39]; however, iron NPs were spherical when iron salt was reduced with sodium borohydrate [40]. The reduction of copper salts with sodium borohydrate produced spherical [41] and irregular NPs [35]. Sonochemical and thermal reduction of copper hydrazine carboxylate produced a network of irregular shaped copper NPs [42]. Wet chemical synthesis involving stoichiometric reaction also produced spherical copper NPs [43]. Moreover, wet chemical method produced nanowires of silver [44]; while spherical silver NPs were produced on ascorbic acid as a reducing agent [45]. Mesoporous silica resulted in the formation of iron NPs having uniform pore size and large surface area [46]. The zinc NPs with crystalline shaped morphology were obtained using ammonium carbamate as a precipitating agent [47]; while refluxing zinc acetate precursor in diethylene and triethylene glycol synthesized oval to rod shaped NPs [48]. Due to the usage of various chemicals for NPs synthesis, there is growing concern about the possible release and effect of NPs in the surrounding environment.

Biological and Green Methods for NPs Synthesis
In biological and green methods, living organisms, such as bacteria, viruses, and plants, are used as capping and reducing agents. . Copper NPs were produced using extracts of Ocimum sanctum leaf [59], Cassia alata flower [60], Capparis zelynica leaf [61], and Syzygium aromaticum solution [62]. Studies have shown that green synthesis methods exploiting plants or microorganisms are relatively safe, inexpensive, and environment-friendly.

Morphological and Physiological Effects of NPs on Crops
The most advanced interdisciplinary tool with the larger potential in agriculture for increased crop productivity is the nanotechnology in which NPs with varying size, concentration, and surface charge influenced the growth and development of diverse plant species [67]. A variety of NPs have been tested against germination of seeds, growth of shoot/root, and crop production [68]. NPs exert species-specific toxicity, plant organ specificity, as well as stress dependency (Table 2).

Plant Species Specificity of NPs
The impact of NPs depends on the type of plant species used. The aqueous suspension of aluminum oxide NPs improved the root growth of radish [69] but reduced in cucumber [70]. The aqueous suspension of titanium oxide NPs increased root length of wheat [71] but inhibited in cucumber [72]. The iron NPs aqueous suspension increased root length of Arabidopsis thaliana [14] and restricted in lettuce [15]. The aqueous suspension of titanium oxide NPs inhibited root elongation in cucumber [69] and carrot [70], but enhanced the growth of maize [1], wheat [73], and spinach [74][75][76][77]. The carbon-nanotubes suspension increased germination rate, fresh biomass, and seedling length in Solanum lycopersicum [78], Allium cepa [79], and wheat [80], while reduced in Cucurbita pepo [81], rice [72], and lettuce [79]. These studies have increased our understanding of phytotoxicity and plant responses towards NPs.

Plant Organ Specific Effects of NPs
The carbon nanotubes, copper-oxide NPs, and titanium-dioxide NPs increased resistance to fungal infection by altering the level of endogenous hormones [82]. The direct application of silver NPs reduced seedling biomass of wheat [83], zucchini [81], mung bean [83], and cabbage [84]; while it regulated the seedling growth in maize [84] and Vigna radiata [83]. The hydroponic applications of silver NPs enhanced root elongation in rice [85]; while it reduced in zucchini [81]. Changes in the morphological characteristics of treated plants depend on the types of NPs used. Silver NPs and aluminum-oxide NPs reduced [86] and improved [87], respectively, growth of wheat. The iron NPs enhanced germination ratio and plant growth [88]; while copper NPs inhibited the growth of wheat [89]. The flowering and yield of rice reduced on carbon nanotubes exposure [73]; while enhanced under cerium-oxide NPs treatment [90]. Silver NPs [84] and cerium-oxide NPs [91] improved the growth of maize; while aluminum-oxide NPs [70], titanium-oxide NPs [1], and copper NPs [80] treatments led to growth reduction. Keeping in view these studies, NPs might be involved in the alteration of growth in plants.

Stress Dependency of NPs
Various modes of applications determine the effects of NPs on growth and productivity of plants. Direct application of aluminum oxide NPs improved root length of wheat [87]; while reduced in maize in hydroponic condition [70]. Exposure of aluminum oxide NPs improved survival percentage and weight/length of root including hypocotyl of soybean under flooding stress [92,93]. There are some NPs with the capability to keep the same effects on the plant, though, applied through various ways, e.g., titanium-oxide NPs improved the growth of spinach when applied through seed treatment [94] and foliar spray [95]. Similarly, soil or direct application of iron NPs increased the growth [96] and yield [97] of wheat. The alteration in the morphology of plants is dependent on the mode of application and the type of NPs exposure is dependent on the mode of application.

Applications of Proteomic Techniques to Assess the Impact of NPs on Crops
With the advancements in mass spectrometry, proteomics has become a powerful technology for the identification and characterization of stress-induced proteins. Detailed proteome analysis of plant organelles generates comprehensive information about the intrinsic mechanisms of plant stress responses towards NPs. Proteomic analyses of various crops exposed to different NPs are summarized in Table 3.

Proteomic Analysis of Silver NPs Challenged Crops
Silver NPs are considered as a promising antibacterial agent due to their strong biocidal effect against microorganisms [115]. These NPs are synthesized through different physical, chemical, and biological methods and well-defined parameters of size and shape [28]. The effects of silver NPs were initially analyzed using proteomic techniques in Chlamydomonas [116], Escherichia coli [117], and Bacillus thuringiensis [118]. Currently, various crop plants were exposed to silver NPs and their effects were analyzed using gel-based or gel-free proteomic techniques. Our gel-free proteomic study revealed restricted growth of soybean seedlings under silver NPs treatment [119]. Proteins related to secondary metabolism, cell organization, and hormone metabolism were mostly influenced by silver NPs exposure. In contrast, silver NPs of 15 nm in size significantly improved the soybean growth under flooding stress by enhancing proteins linked to amino acid synthesis [120]. In wheat, the accumulation of different cellular compartmental proteins on silver NPs exposure in shoot and root was mainly involved in metabolism and cell defense [86]. Silver NPs with chemical exposure increased the proteins related to photosynthesis and protein synthesis, while decreased the glycolysis, signaling, and cell wall related proteins in wheat [121]. Large numbers of proteins involved in the primary metabolism were increased in soybean [119]. Silver NPs treatment increased the proteins related to protein degradation, while decreased protein synthesis related proteins in soybean; indicating that it might improve the growth of soybean under flooding stress through protein quality control [122]. Proteins related to the oxidative stress, signaling, transcription, protein degradation, cell wall synthesis, cell division, and apoptosis were found to be increased in silver NPs exposed rice [118]. In Eruca sativa, proteins associated with the endoplasmic reticulum and vacuole were differentially modulated under silver NPs exposure [86]. These findings indicate that silver NPs primarily influence various metabolic processes in wheat and protein quality control in soybeans; thus, improving plant growth.

Proteomic Analysis of Aluminum Oxide NPs Stressed Crops
Aluminum oxide NPs are mostly used in military and commercial products [123]. Extensive usage of aluminum oxide NPs leads towards their possible leakage into environment, which ultimately interacts with living organisms including plants [124]. Proteomic analysis of soybean root treated with aluminum oxide NPs revealed an increase in the number of proteins related to protein synthesis, transport, and development during the recovery from flooding [92]. A study by Mustafa et al. [120] revealed that proteins associated with the ascorbate-glutathione cycle, as well as ribosomal proteins, were differentially influenced by aluminum oxide NPs. Moreover, high abundance of proteins involved in oxidation-reduction, stress signaling, hormonal pathways related to growth and development, were evident in aluminum oxide NPs challenged soybean [119]. A separate study has shown growth promoting effects of aluminum oxide NPs in the soybean under flooding stress by regulating energy metabolism and cell death [125].

Proteomic Analysis of Crops Exposed to Copper NPs and Iron NPs
Among the various metal-based NPs, copper NPs are by far the most well studied NPs whose toxicity has been tested in wide range of crops. They have wide applications in electronics, air/liquid filtration, ceramics, wood preservation, bioactive coatings, and films/textiles [16]. At the cellular level, copper acts as structural and catalytic component of many proteins involved in various metabolic processes. In wheat seedlings, abundance of proteins associated with glycolysis and tricarboxylic acid cycle was found to be increased; while, photosynthesis and tetrapyrrole synthesis related proteins were decreased on exposure to copper nanoparticles [97]. Wheat grains obtained after NPs exposure were analyzed through gel-free proteomic technique, which indicated an increase in proteins involved in starch degradation and glycolysis [96].
Similar to copper NPs, iron NPs have extensive industrial, commercial, and biomedical applications [12]. Because of their high reactivity and magnetic property, iron NPs have been used as remediation agents for environmental applications [13]. Iron NPs have known stimulatory effects on the seed germination and plant growth of wheat [96]. Authors exploited gel-free/label-free proteomic technique to elucidate the impact of iron NPs on shoot growth of drought tolerant and salt tolerant wheat varieties. A study revealed that differentially expressed proteins in both varieties were mainly associated with photosynthesis. Notably, proteins related to light reaction were enhanced in the salt tolerant variety compared to drought tolerant wheat on iron NPs exposure. A separate study on grain analysis of wheat indicated an increase in the number of proteins related to starch degradation, glycolysis, and the tricarboxylic acid cycle [103].

Proteomic Analysis of Other NPs Challenged Crops
One of the most commonly used nanomaterials in agriculture and the energy sector is titanium dioxide NPs [126]. They have diverse applications in personal skincare products, water-treatment agents, and bactericidal agents owing to their high stability and anticorrosive/photocatalytic properties [127,128]. The toxicological effects of nanometer titanium dioxide on a unicellular green alga Chlamydomonas reinhardtii were accessed by monitoring the changes in the physiology and cyto-ultrastructure [129]. Authors reported nano titanium dioxide mediated inhibition in photosynthetic efficiency and cell growth, with increased contents of carotenoids and chlorophyll b.
In addition, various NPs are being extensively utilized to improve the growth and productivity of crop plants. However, application of zinc oxide NPs had marked effects on soybean seedling growth, rigidity of roots, and root cell viability [119]. Gel-free proteomic analysis revealed down regulation oxidation-reduction cascade associated proteins, including GDSL motif lipase 5, SKU5 similar 4, galactose oxidase, and quinone reductase in zinc oxide NPs exposed roots. A separate study on cerium oxide NPs treatment in maize indicated enhanced accumulation of heat shock proteins (HSP70) and increased activity of ascorbate peroxidase and catalase [130]. This up regulated antioxidant defense system might help maize plants to overcome NPs-induced oxidative stress damages.
All of these studies indicate that NPs have the potential to modulate plant metabolic processes, and impact of NPs could be either positive or negative, depending on the plant species and type of nanoparticles used, their size, composition, concentration, and physical/chemical properties.

NPs Uptake and Mode of Action
The phytotoxicity of NPs largely depends on the particle size, concentration and chemistry of NPs, in addition to the chemical milieu of the subcellular sites at which the NPs are deposited [23].
Plants, being an indispensable component of terrestrial ecosystems, serve as a potential route for the factory discharged-NPs to enter the plant root system and their transportation to other parts of the plants, resulting bioaccumulation in the food chain [132]. The physico-chemical properties of soil matrix (viz. mineral composition, pH, ionic strength, dissolved organic matter, etc.) as well as the of metal based NPs (viz. size, surface charge, surface coating, etc.) are the determining factors for NPs mobility [133]. Primary-lateral root junctions are the prime sites through which NPs could enter xylem via cortex and finally reach the central cylinder [23]. Study on the uptake pathways of zinc oxide NPs by maize roots reveals that majority of the total zinc oxide NPs undergo dissolution in the exposure medium, and the released Zn 2+ ions are only taken up by the roots [134]. Only a small fraction of zinc oxide NPs adsorbed on the root surface can cross the root cortex as a result of speedy cell division and root tip elongation, apart from their entry to vascular system through the gap of the Casparian strip at the sites of the primary-lateral root junction.
Once NPs enter the root cells, these ultrafine particles upon dissolution discharge metal ions that interact with the functional groups of proteins (carboxyl and sulfhydryl groups) causing altered protein activity. The released redox-active metal ions could trigger reactive oxygen species (ROS) generation through the Fenton and Haber-Weiss reactions [135]. In these reactions, the hydrogen peroxide (H 2 O 2 ) is decayed by the metal ions leading to the formation of more toxic ROS, namely hydroxyl radical ( • OH) and hydroxyl anion (OH − ). Elevated ROS generation was documented in leaves of soybean exposed to zinc oxide NPs and silver NPs [119] as well as in copper oxide NPs challenged rice [105]. These NPs mediated excess ROS formation disturbs the cellular redox system in favor of oxidized forms, causing oxidative damage to vital cellular components including nucleic acids, lipids, and proteins [135].
Cellular compartments with extremely high oxidizing metabolic activity or with an intense rate of electron flow, such as mitochondria, chloroplasts, and peroxisomes, constitute a major source of ROS production in plants [136]. Investigations have revealed that zinc oxide NPs mediated deregulation of photosynthetic efficiency in plants is due to the down regulation of chlorophyll synthesis genes and structural genes of photosystem I [137,138]. To protect cells against such oxidative damages, plants have developed robust multi-component antioxidant defense system comprising of both enzymatic and non-enzymatic machineries [119,139]. The enzymatic antioxidant defense system chiefly includes ROS scavenging enzymes of the ascorbate-glutathione cycle, which operates in nearly all plant cell organelles [140]. The orchestrated action of key antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) is an adaptive strategy of plant to cope with the NPs induced oxidative stress damages.
Moreover, NPs exposure often leads to disruption of cellular redox homeostasis and cause cell membrane damage through lipid peroxidation [105,106,108]. Among the ROS, hydroxyl radical ( • OH) is known to be the most reactive, capable of stealing hydrogen atom from a methylene (-CH 2 -) group present in polyunsaturated fatty acid side chain of membrane lipids and, thus, initiates lipid peroxidation [141]. Since, • OH is derived from H 2 O 2 as a consequence of one electron reduction, H 2 O 2 scavenging peroxides play essential roles in protecting lipid membranes from NPs mediated oxidative stress. Among ROS, a recent study revealed down regulation of ascorbate peroxidase (APX1) in zinc oxide NPs challenged maize leaves with concomitant increased malondialdehyde (MDA) level, an indicative of oxidative stress induced damage to the lipid membrane [108]. The NPs-induced higher membrane damage is in accordance with the previous reports in rice [105] and Syrian barley [106].
Apart from enzymatic component of ascorbate-glutathione cycle, plants have evolved a second line of defense to cope with the NPs induced oxidative stress. The thioredoxin (Trx) family protein is one of them, engage in mitigating oxidative damages by providing reducing power to reductases, detoxifying lipid hydroperoxides or repairing oxidized proteins. They also act as regulators of scavenging mechanisms and key components of signaling pathways in the plant antioxidant network [142]. In addition, these proteins are necessary for their potential roles as facilitators and regulators of protein folding and chaperone activity [143]. Furthermore, plant quinone reductases (QRs) are involved redox reactions and act as detoxification enzymes of free radicals. Soybean seedlings exposed to zinc oxide NPs and silver NPs treatments exhibited significantly declined abundance of Trx and QR proteins [119]. Severe oxidative burst evident in zinc oxide NPs and silver NPs challenged soybean might be the result of such declined protein abundance affecting optimum growth of seedlings. Enzymes of shikimate pathway involved in the synthesis of amino acids (phenylalanine, tryptophan, and tyrosine) were also found to be affected under NPs exposure. These aromatic amino acids not only act as substrates for the protein synthesis, but are also linked with formation of secondary products, including lignin, suberin, and phytoalexins. The abundance of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway, was reported to be decreased in soybean under silver NPs treatment [119]. The reduced shoot length of silver NPs exposed soybean seedlings might be the result of such marked decline in DAHP synthase level. In a nutshell, low abundance of proteins involved in oxidation-reduction, shikimate pathway might limit the growth of the silver NPs challenged soybean seedlings up to a certain level. Summarizing all these findings, a comprehensive model of cellular responses to NPs is presented in Figure 1.

Conclusions
Nanotechnology has gained tremendous momentum in recent times because of the wide applications of NPs in agriculture, cosmetic industry, cellular imaging, medical diagnosis, biosensing, drug delivery, and cancer therapy. Nevertheless, unintended release of such commercially manufactured nanomaterials into the environment has raised global concern. Hence, considerable attention is now being paid to the methods and strategies of NPs synthesis, plant-nanomaterials interactions, and their environmental fate. As compared to traditional physical Figure 1. Schematic illustration of diverse cellular responses to nanoparticles (NPs). Exposure to metal based-NPs triggers oxidative stress through enhanced reactive oxygen species (ROS) generation, disruption of redox homeostasis, impaired photosynthetic activity, mitochondrial dysfunction, lipid peroxidation, and membrane damage. Upward arrows indicate increased and downward arrows indicate decreased protein abundance in response to NPs stress, respectively. Abbreviations: APX, ascorbate peroxidase; AsA, reduced ascorbate; CAT, catalase; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHAR, dehydroascorbate reductase; ETC, electron transport chain; H 2 O 2 , hydrogen peroxide; MDA, malondialdehyde; MDA-radical, monodehydroascorbate radical; MDAR, monodehydroascorbate reductase; • OH, hydroxyl radical; OEE, oxygen-evolving enhancer; PS, photosystem; QR, quinone reductase; ROS, reactive oxygen species; SOD, superoxide dismutase; Trx, thioredoxin.

Conclusions
Nanotechnology has gained tremendous momentum in recent times because of the wide applications of NPs in agriculture, cosmetic industry, cellular imaging, medical diagnosis, biosensing, drug delivery, and cancer therapy. Nevertheless, unintended release of such commercially manufactured nanomaterials into the environment has raised global concern. Hence, considerable attention is now being paid to the methods and strategies of NPs synthesis, plant-nanomaterials interactions, and their environmental fate. As compared to traditional physical and chemical processes, green synthesis of NPs using microorganisms and plants is an environment-friendly, cost effective, safe, biocompatible, green alternative approach for large scale production of NPs. Morphophysiological as well as proteomic studies on NPs-induced phytotoxicity reveal that particle size, concentration, and chemistry of NPs, as well as the type of plant species used, are the key factors determining the type and magnitude of the cellular responses. However, more initiatives must be taken to find out whether the metal-based NPs exert phytotoxicity exclusively due to their high surface area and nanoscale size or due to the released metal ions. Moreover, there is a need for more comprehensive omics approach integrating genomics, transcriptomics, proteomics, and metabolomics, so that the impact of the applied NPs on plants can be assessed well in time.