Recent Advances in Metal-Based Nanoparticle-Mediated Biological Effects in Arabidopsis thaliana: A Mini Review

The widespread application of metal-based nanoparticles (MNPs) has prompted great interest in nano-biosafety. Consequently, as more and more MNPs are released into the environment and eventually sink into the soil, plants, as an essential component of the ecosystem, are at greater risk of exposure and response to these MNPs. Therefore, to understand the potential impact of nanoparticles on the environment, their effects should be thoroughly investigated. Arabidopsis (Arabidopsis thaliana L.) is an ideal model plant for studying the impact of environmental stress on plants’ growth and development because the ways in which Arabidopsis adapt to these stresses resemble those of many plants, and therefore, conclusions obtained from these scientific studies have often been used as the universal reference for other plants. This study reviewed the main findings of present-day interactions between MNPs and Arabidopsis thaliana from plant internalization to phytotoxic effects to reveal the mechanisms by which nanomaterials affect plant growth and development. We also analyzed the remaining unsolved problems in this field and provide a perspective for future research directions.


Introduction
Metal nanoparticles (MNPs) are widely used in biosensors, medical imaging, diagnostic and therapeutic materials, antimicrobial agents and drugs, chemical catalysis, optoelectronics and other areas due to their unique physical and excellent chemical properties [1][2][3][4][5][6]. However, during their production and recycling process, MNPs are inevitably leaked into the environment and may become exogenous stimulation for plants.
MNPs have been extensively studied for their effects on plant growth and development. For instance, TiO 2 NPs were found to promote seed germination in tomato, onion and radish, Cu NPs concentration-dependently inhibit seedling growth and root growth in both mung bean and wheat, and Al 2 O 3 NPs were observed to have no pharmacological effect on the root elongation of Arabidopsis, radish, rape, ryegrass, lettuce or cucumber [7][8][9].
Soil has gradually become a significant reservoir for MNP deposits in the environment. Several previous studies have shown that the number of residual nanoparticles in the soil of certain regions could reach up to 1.9-865 mg/kg [10]. These MNPs were absorbed by plant roots and leaves and transported to other tissues via the plant's vascular system [11][12][13]. However, most studies found that MNPs have a low rate of internalization [14][15][16]. For example, the translocation of Ce from the roots to the stems was shown to be around 1.44% and 1.79% [17]. Nevertheless, these internalized MNPs still pose a risk due to their ability to translocate, accumulate and even transform within the plants to interact with biomolecules, leading to changes in the morphological characteristics, physiological

Absorption and Transport of MNPs in Arabidopsis thaliana
Based on studies performed to assess the ecological safety of nanomaterials, it is generally believed that nanoparticles exposed to the surface of plants can be attached to tissue surfaces and hinder the transmission of water, nutrients and ion exchange. Some hydrophilic nanoparticles have been found to cross plants' cell walls and accumulate between cell walls and cell membranes or between cell walls of adjacent cells, indicating a potential plasmatic exosomal transport mode for nanoparticles in plant tissues. Despite limited evidence showing that intact plant roots can absorb and translocate nanoparticles [18][19][20][21], there are still controversies surrounding this issue [22,23].

Absorption and Transport of Monometallic Nanoparticles in Arabidopsis thaliana
Geisler-Lee et al. tested different sizes of Ag NPs (20, 40, 80 nm) in a hydroponic growth media using different microscopy methods to study the effects of Ag NPs' toxicity in Arabidopsis root tips. They found that Ag NPs were absorbed and gradually accumulated in the root tips, from the marginal cells to the root cap, epidermis and columella, and then penetrated the initial part of the root meristem (Figure 1a). At low concentrations, smaller Ag NPs accumulated more than larger ones, while at high concentrations, the opposite occurred [24,25]. Ag NPs were first absorbed by underground tissues (primary root and lateral roots) and then transferred to aboveground parts (stems, leaves, flowers, etc.) where they tended to influence the growth and development of Arabidopsis thaliana. They appeared to accumulate in the plastid exosomes of root tissues while only a tiny fraction was transported to aboveground tissues. In the places they accumulated, i.e., on the surface of bare plant roots and leaves, they demonstrated a low internalization rate. It was found that the particle size of Ag NPs in plant tissues was larger than their initial diameter, suggesting that the internalized Ag NPs no longer existed as intact individual particles but rather appeared to aggregate and biotransform in the plants [26]. In contrast to observations made for Ag NPs, Au NPs (60 nm) tended to remain unaggregated after being absorbed by Arabidopsis roots. Yeonjong Koo et al. compared leaf acoustic signal distributions from Arabidopsis leaves exposed to media with high (2.4 × 10 10 NP mL −2 ) or low (4.8 × 10 8 NP mL −2 ) GNP concentrations. The high GNP concentration increased the percentage of the leaf surface area, but regardless of concentration, nearly all the signals remained in the 90-200 mV amplitude range. A lack of high-amplitude signals suggests that GNPs did not aggregate in plants (Figure 1b) [27]. Thus, it seems that in addition to the changes in morphology and concentrations of monometallic nanomaterials that occur in Arabidopsis, other factors also affect the state of MNPs in plants. (b1,b2) The frequency of leaf signal amplitudes is compared between (b1) high-and low-GNP-concentration exposure to detached leaf petioles and (b2) high-and low-GNP-concentration exposure to whole plants for two different durations. Signal amplitudes below 200 mV and above 200 mV are indicated on upper side of each graph. (b3) Percentage of leaf surface that emitted detectable signal (% surface with signal, x axis) and acoustic signal amplitude (average signal amplitude over 90 mV-average signal amplitude below 90 mV, y axis) from (b1) and (b2) are plotted. Detached leaf data are shown in green; whole-plant exposure data are shown in orange. Reprinted with permission from Refs. [27,28].
The surface charge of nanoparticles is generally assumed to be a key factor affecting their uptake and translocation. Using DF-HSI and nano-CT, Astrid et al. observed that negatively charged nanoparticles were transported along plastid exosome in Arabidopsis while positively charged nanoparticles uptake occurred to a small extent, possibly through other processes, such as clathrin-mediated endocytosis, in the phytoplankton ( Figure 2) [29]. However, Milewska-Hendel et al. modified the surface of AuNPs using polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) and citrate to achieve neutral, positive and negative charges, as demonstrated by HRTEM analysis, which demonstrated that, regardless of the surface charge of Au NPs, they did not traverse the cell wall barrier of Arabidopsis root bark cells or root cap cells but were internalized by the protoplasm [30]. Although there seems to be some strong co-localization of Au NPs in root tips, it has not yet been possible to determine whether Au NPs are adsorbed on or accumulated in the roots. Percentage of leaf surface that emitted detectable signal (% surface with signal, x axis) and acoustic signal amplitude (average signal amplitude over 90 mV-average signal amplitude below 90 mV, y axis) from (b 1 ) and (b 2 ) are plotted. Detached leaf data are shown in green; whole-plant exposure data are shown in orange. Reprinted with permission from Refs. [27,28].
The surface charge of nanoparticles is generally assumed to be a key factor affecting their uptake and translocation. Using DF-HSI and nano-CT, Astrid et al. observed that negatively charged nanoparticles were transported along plastid exosome in Arabidopsis while positively charged nanoparticles uptake occurred to a small extent, possibly through other processes, such as clathrin-mediated endocytosis, in the phytoplankton ( Figure 2) [29]. However, Milewska-Hendel et al. modified the surface of AuNPs using polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) and citrate to achieve neutral, positive and negative charges, as demonstrated by HRTEM analysis, which demonstrated that, regardless of the surface charge of Au NPs, they did not traverse the cell wall barrier of Arabidopsis root bark cells or root cap cells but were internalized by the protoplasm [30]. Although there seems to be some strong co-localization of Au NPs in root tips, it has not yet been possible to determine whether Au NPs are adsorbed on or accumulated in the roots.

Absorption and Transport of Metal Oxide Nanoparticles in Arabidopsis thaliana
The use of zinc oxide nanoparticles (ZnO NPs) as Zn fertilizer has been shown to be effective for correcting Zn deficiency in soils [31]. However, it has also been shown that ZnO NPs may dissolve rapidly once they are released into the soil, releasing Zn ions, and may lead to a far higher concentration of Zn than expected [32]. In plants, Zn homeostasis is mediated through transporter proteins involved in the intracellular acquisition of Zn, mobilization and sequestration [33]. The Arabidopsis transporter proteins AtZIP4, AtZIP9 and AtZIP12 are involved in the acquisition of Zn from roots and subsequent mobilization to aerial tissues, while AtHMA3 and AtHMA4 mediate root-to-crown Zn transport [34,35]. Prakash et al. observed Arabidopsis seedlings after treatment with ZnO NPs under fluorescent labeling. They detected an intense green fluorescence in the primordial root tip region, primordial lateral root junctions and aboveground root junctions, but ZnO NPs treatment resulted in Zn accumulation only in the root apex and root-shoot junctions, whereas Zn ion treatment caused a root-to-shoot uptake and translocation of the element (Figure 3) [36].

Absorption and Transport of Metal Oxide Nanoparticles in Arabidopsis thalia
The use of zinc oxide nanoparticles (ZnO NPs) as Zn fertilizer has bee effective for correcting Zn deficiency in soils [31]. However, it has also be ZnO NPs may dissolve rapidly once they are released into the soil, releasin may lead to a far higher concentration of Zn than expected [32]. In plants, Z is mediated through transporter proteins involved in the intracellular acq mobilization and sequestration [33]. The Arabidopsis transporter proteins A In experiments where Arabidopsis was exposed to 5-40 mg/L of CuO NPs, the Cu content in Arabidopsis roots was significantly increased compared to the Cu content in Arabidopsis stems and leaves. Additionally, while the transfer rate of CuO NPs from root to shoot was found to be low (1.1-2.8%), under the same conditions, that of Cu 2+ occurred at a higher rate (10.8%), indicating a weak transport capacity of CuO NPs (Figure 4) [37]. Wang et al. exposed Arabidopsis to 50 mg/L of CuO NPs and found that the Cu contents in the roots were significantly higher than those in leaves, flowers and harvested seeds in the investigated ecotypes of Arabidopsis. In all the tissues tested, the Cu contents were significantly higher after exposure to 50 mg/L of CuO NPs than exposure to 0.15 mg/L of Cu 2+ , indicating that a large number of CuO NPs were transformed and transported as Cu 2+ in Arabidopsis [38]. Thus, based on metal oxide nanoparticles' solubility, comparing the effect of the nanoparticles themselves with that of a single metal ion is important to determine the extent of their internalization in plants. In experiments where Arabidopsis was exposed to 5-40 mg/L o content in Arabidopsis roots was significantly increased compared to th abidopsis stems and leaves. Additionally, while the transfer rate of Cu shoot was found to be low (1.1-2.8%), under the same conditions, tha a higher rate (10.8%), indicating a weak transport capacity of CuO N Wang et al. exposed Arabidopsis to 50 mg/L of CuO NPs and found tha the roots were significantly higher than those in leaves, flowers and the investigated ecotypes of Arabidopsis. In all the tissues tested, the C nificantly higher after exposure to 50 mg/L of CuO NPs than expos Cu 2+ , indicating that a large number of CuO NPs were transformed Cu 2+ in Arabidopsis [38]. Thus, based on metal oxide nanoparticles' so the effect of the nanoparticles themselves with that of a single metal determine the extent of their internalization in plants.  In experiments where Arabidopsis was exposed to 5-40 mg/L of CuO NPs, the content in Arabidopsis roots was significantly increased compared to the Cu content in A abidopsis stems and leaves. Additionally, while the transfer rate of CuO NPs from root shoot was found to be low (1.1-2.8%), under the same conditions, that of Cu 2+ occurred a higher rate (10.8%), indicating a weak transport capacity of CuO NPs ( Figure 4) [3 Wang et al. exposed Arabidopsis to 50 mg/L of CuO NPs and found that the Cu contents the roots were significantly higher than those in leaves, flowers and harvested seeds the investigated ecotypes of Arabidopsis. In all the tissues tested, the Cu contents were s nificantly higher after exposure to 50 mg/L of CuO NPs than exposure to 0.15 mg/L Cu 2+ , indicating that a large number of CuO NPs were transformed and transported Cu 2+ in Arabidopsis [38]. Thus, based on metal oxide nanoparticles' solubility, compari the effect of the nanoparticles themselves with that of a single metal ion is important determine the extent of their internalization in plants. Unlike highly soluble MNPs, TiO2 NPs are difficult for plant roots to absorb due their low solubility. In addition, titanium also plays a key role in plants as it stimula the production of more carbohydrates and helps in encouraging growth and the rate Unlike highly soluble MNPs, TiO 2 NPs are difficult for plant roots to absorb due to their low solubility. In addition, titanium also plays a key role in plants as it stimulates the production of more carbohydrates and helps in encouraging growth and the rate of photosynthesis. Ti/TiO 2 , widely used in the agricultural sector, exhibited both phytotoxic and positive effects on the size, concentration and plant species tested [39].
Although Ti elements are non-essential elements for Arabidopsis thaliana because their cell membranes lack corresponding transport receptors, Kurepa et al. found that TiO 2 NPs (<5 nm) could be absorbed, translocated and distributed among the tissues and cells of Arabidopsis seedlings [12]. Via morphological and histological assessment of ultrasmall TiO 2 NPs, García-Sánchez et al. observed that TiO 2 NPs could enter Arabidopsis cells, accumulate in subcellular (including vesicular) locations such as the cytosol and root cell nuclei and further disrupt Arabidopsis microtubule dynamics [12,40,41]. This suggests that there are still other unknown ways and pathways for MNPs to enter Arabidopsis, and it would be helpful to further assess TiO 2 NPs using traceable signals.
CeO 2 NPs are a class of MNPs that tend to aggregate and precipitate in aqueous solutions due to their size and surface properties. In a study by Yang et al., the investigators introduced an agar curing medium to prevent the aggregation of CeO 2 NPs, allowing them to be uniformly dispersed. It was found that the transport of Ce compounds by Arabidopsis grown in the agar medium behaved similarly to internalized CuO NPs in plants [42]. Ma et al. digested and analyzed Arabidopsis exposed at 0-1000 ppm CeO 2 NPs by ICP-MS and observed measurable amounts of the elements in the root and stem tissues of Arabidopsis. However, the underlying mechanism of this transport is yet to be uncovered. Despite these observations, the accumulation and translocation of CeO 2 NPs in plants seem to vary depending on the plant species. Birbaum et al. found that CeO 2 NPs did not undergo translocation in maize, while Ce elements were found to be accumulated in plants such as alfalfa, cucumber and tomato [43][44][45][46].

Absorption and Transport of Other Metal-Based Nanoparticles in Arabidopsis thaliana
Given their promising water solubility and small size, quantum dots (QDs) were believed to be easily absorbed by plants; this was also confirmed in the recent study of pumpkin's physiological responses to zinc oxide quantum dots and nanoparticles [47]. The experimental results for water-dispersible CdSe/ZnSe QDs showed no significant results [48]. Using confocal fluorescence microscopy, Navarro et al. found that watersoluble CdSe/ZnS QDs with carboxyl groups were strongly adsorbed to polar/charged root surfaces but could not enter the roots. Moreover, despite a 7-day exposure period, the plant cells remained impermeable to QDs, and therefore, QDs could neither be endocytosed nor passively or actively transported through the plant root system ( Figure 5), suggesting the significant effect of the surface charge of nanoparticles on their uptake by Arabidopsis. In addition to the barrier created by the plant's cell wall, when QDs are electrostatically adsorbed on the root surface, they form bulky agglomerates, which further impedes their entry as endocytosis cannot occur [49].
Taken together, the current body of literature suggests that although the uptake of most MNPs is associated with ion transporters on Arabidopsis root cell membranes, they have a low rate of internalization [50]. Small numbers of MNPs that are ingested or able to enter root cells via other routes are biotransformed into an ionic state and transported to other parts of Arabidopsis. Besides this, the importance of nanomaterials' entry through stomata has also been extensively studied [51,52]. Moreover, the size, charge and growth media of nanoparticles affect the extent to which they are absorbed and transported. tosed nor passively or actively transported through the plant root system ( gesting the significant effect of the surface charge of nanoparticles on their abidopsis. In addition to the barrier created by the plant's cell wall, when Q statically adsorbed on the root surface, they form bulky agglomerates, wh pedes their entry as endocytosis cannot occur [49].

Phytotoxic Effects of MNPs on Arabidopsis thaliana
The large surface area and small size of NPs are some of their desirable attributes that allow them to substantially ameliorate plants' physiological processes. Nevertheless, the results derived from such research have not always been positive as NPs have been shown, in some cases, to negatively affect plants due to their potentially toxic nature [53]. Despite the low internalization and transfer rate of MNPs in Arabidopsis, most studies confirmed that MNPs have predominantly harmful effects on the plant. Table 1 summarizes the phytotoxic effects of different MNPs on Arabidopsis. Separated border-like cell sheets (isolated from the root) and associated mucus accumulated and trapped NPs independent of particle charge, in contrast to the marginal cells on the root crown that exhibited charge specificity. [29] Au NPs 10-18 nm 100 mg/mL Au NPs had significant effects on the lateral roots of Arabidopsis. At the highest concentration, the minimal Au NPs inhibited the length of primary roots but contrarily also promoted the growth of hairy roots. [61] Au NPs 24 nm 10, 80 µg/mL Exposure to Au NPs at 24 nm at concentrations of 10 and 80 µg/mL significantly increased seed germination, nutritional growth and free-radical scavenging activity. [62] CdSe/ZnS QDs 6.3 ± 0.7 nm 5 µg/mL The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was reduced in the plants. [ [68] CuO NPs 20-40 nm 20, 50 mg/mL The growth of Arabidopsis seedlings of different ecotypes (Col-0, Bay-0 and Ws-2) and the germination of their pollen and harvested seeds were inhibited. [38] CuO NPs -5, 10 µg/mL Elevated endogenous H 2 S and Cys content inhibited Arabidopsis root elongation in a dose-dependent manner. [69] CuO NPs -10, 20 µg/mL Strongly inhibited the growth of Arabidopsis.
[67] The 3 mg/L treatment had no significant effect on seedling and root length, and the 25 mg/L treatment resulted in a reduction in seedling and root length. [73]

Toxic Effects at the Morphological Level
Growth potential, seed germination, biomass and leaf surface area are the commonly used parameters to assess morphological changes in plants and phytotoxicity [74][75][76][77][78]. Kaveh et al. found that exposure to Ag NPs (from 5 to 20 mg/L) resulted in reduced biomass of Arabidopsis, while Ag NPs at concentrations of 50 mg/L and 60 mg/L had a positive effect on root growth. In addition, the shapes of Ag NPs were also found to be an important factor influencing root growth [79]. Ag NPs with triangular and decahedral shapes promoted root growth, while spherical Ag NPs had no effects on Arabidopsis seedlings [80].
The number and length of Arabidopsis lateral roots have been found to be significantly reduced after treatment with different sizes and concentrations of Au NPs solutions, whereby smaller (10 nm) Au NPs negatively affected primary root growth but significantly promoted root hair growth ( Figure 6) [61].
Although some studies have shown that ZnO NPs could significantly inhibit Arabidopsis growth and biomass accumulation, low concentrations of less than 20 mg/L are considered non-effective. Treatment at concentrations greater than 50 mg/L was found to significantly reduce the fresh weight and primary root length of Arabidopsis seedlings, decrease leaf size and yellowing and alter root structure [36,65]. Although some studies have shown that ZnO NPs could significant bidopsis growth and biomass accumulation, low concentrations of less tha considered non-effective. Treatment at concentrations greater than 50 mg/L significantly reduce the fresh weight and primary root length of Arabidopsis crease leaf size and yellowing and alter root structure [36,65].
Unlike other metal oxide nanoparticles, iron oxide nanoparticles (IO lieved to interact with Arabidopsis in a charge-dependent manner as charge be detected in Arabidopsis's root, leaf, flower and hornbeam tissues. Furth charged IONP had a significant inhibitory effect on Arabidopsis seedlin length, while positively and negatively charged IONP inhibited pollen vi tube growth and seed yield (Figure 7). These results indicate the detrim IONPs on the whole plant's reproduction cycle. This phytotoxicity has also be related to the concentration of IONPs, and the number of IONPs expose [73]. Unlike other metal oxide nanoparticles, iron oxide nanoparticles (IONPs) are believed to interact with Arabidopsis in a charge-dependent manner as charged IONP could be detected in Arabidopsis's root, leaf, flower and hornbeam tissues. Further, negatively charged IONP had a significant inhibitory effect on Arabidopsis seedlings and roots' length, while positively and negatively charged IONP inhibited pollen viability, pollen tube growth and seed yield (Figure 7). These results indicate the detrimental effect of IONPs on the whole plant's reproduction cycle. This phytotoxicity has also been found to be related to the concentration of IONPs, and the number of IONPs exposed to the plant [73].
The morphotropic effects of MNPs on Arabidopsis may also be related to the alteration of the porosity of the plant's cell walls when stimulated under different concentrations of MNPs. While larger pores tend to promote the uptake of nutrients and MNPs from the soil, MNPs tend to simultaneously cause root clogging and impede the entry of nutrients.

Toxic Effects at the Physiological Level
Oxidative-stress-induced by CuO NPs or their released Cu 2+ is the primary mechanism by which CuO NPs induce their phytotoxic effects [81]. In 2017, Ke et al. demonstrated that the phytotoxic effects of CuO NPs mainly originated from the nanoparticles themselves; dissolved Cu 2+ contributed only a tiny fraction to the toxicity induced by CuO NPs. Upon 2 h of exposure to CuO NPs (10 mg/L and 20 mg/L), severe damage of Arabidopsis root cells was observed, whereas corresponding exposure with dissolved Cu 2+ (0.80 mg/L and 1.35 mg/L) did not damage the roots and only exhibited partial phytotoxic effects after 12 days of exposure [67]. Moreover, studies have shown that Arabidopsis roots, leaves, flowers and harvested seeds treated with CuO NPs contained significantly higher Cu elements than CuO bulk particles (BPs) and Cu 2+ treatment, demonstrating that CuO BPs cannot be readily accumulated and distributed in the plant (Figure 8), indicating that CuO NPs themselves have high mobility in Arabidopsis. In combined ethylene experiments, CuO NPs were observed to induce oxidative stress and inhibit growth by affecting the rosette size, biomass, chlorophyll content, lipid peroxidation, accumulation of oxygen species and cellular ultrastructure of Arabidopsis [38,68]. Interestingly, Jia et al. found that the Cys cycle affected the uptake and intracellular transport of CuO NPs. The activity and toxicity of CuO NPs were reduced by promoting the production of chelators while stabilizing the level of CuO NP-induced reactive oxygen species [69].
Unlike other metal oxide nanoparticles, iron oxide nanoparticles (IO lieved to interact with Arabidopsis in a charge-dependent manner as charge be detected in Arabidopsis's root, leaf, flower and hornbeam tissues. Furth charged IONP had a significant inhibitory effect on Arabidopsis seedlin length, while positively and negatively charged IONP inhibited pollen vi tube growth and seed yield (Figure 7). These results indicate the detrime IONPs on the whole plant's reproduction cycle. This phytotoxicity has also be related to the concentration of IONPs, and the number of IONPs expose [73]. Single asterisks represent treatments that were significantly different from the control with a p < 0.05; two asterisks indicate treatments that were significantly different with a p < 0.01. Reprinted with permission from Ref. [73]. erials 2022, 15, x FOR PEER REVIEW 11 of tube growth in A. thaliana. Treatments with significant differences from the control are marked w an asterisk (p < 0.05). (d) Treatment of A. thaliana with IONPs resulted in reduced seed producti Single asterisks represent treatments that were significantly different from the control with a 0.05; two asterisks indicate treatments that were significantly different with a p < 0.01. Reprin with permission from Ref. [73].
The morphotropic effects of MNPs on Arabidopsis may also be related to the alterati of the porosity of the plant's cell walls when stimulated under different concentrations MNPs. While larger pores tend to promote the uptake of nutrients and MNPs from t soil, MNPs tend to simultaneously cause root clogging and impede the entry of nutrien

Toxic Effects at the Physiological Level
Oxidative-stress-induced by CuO NPs or their released Cu 2+ is the primary mech nism by which CuO NPs induce their phytotoxic effects [81]. In 2017, Ke et al. demo strated that the phytotoxic effects of CuO NPs mainly originated from the nanopartic themselves; dissolved Cu 2+ contributed only a tiny fraction to the toxicity induced by Cu NPs. Upon 2 h of exposure to CuO NPs (10 mg/L and 20 mg/L), severe damage of A bidopsis root cells was observed, whereas corresponding exposure with dissolved C (0.80 mg/L and 1.35 mg/L) did not damage the roots and only exhibited partial phytoto effects after 12 days of exposure [67]. Moreover, studies have shown that Arabidopsis roo leaves, flowers and harvested seeds treated with CuO NPs contained significantly high Cu elements than CuO bulk particles (BPs) and Cu 2+ treatment, demonstrating that Cu BPs cannot be readily accumulated and distributed in the plant (Figure 8), indicating th CuO NPs themselves have high mobility in Arabidopsis. In combined ethylene expe ments, CuO NPs were observed to induce oxidative stress and inhibit growth by affecti the rosette size, biomass, chlorophyll content, lipid peroxidation, accumulation of oxyg species and cellular ultrastructure of Arabidopsis [38,68]. Interestingly, Jia et al. found th the Cys cycle affected the uptake and intracellular transport of CuO NPs. The activity a toxicity of CuO NPs were reduced by promoting the production of chelators while sta lizing the level of CuO NP-induced reactive oxygen species [69]. At low concentrations, CeO2 NPs can promote growth, but at high concentrations g/L), they induce glutathione metabolism (oxidative stress response) and inhibit chlo phyll production and plant growth [42,82]. These changes in physiological levels are lieved to be caused by the nanoparticles themselves, rather than by dissolved Ce 4+ , a are particle-specific [72].
Although it is generally accepted that oxidative stress (ROS) could be one of the rect sources of nanomaterials' toxicity, relevant experiments showed that changes in R At low concentrations, CeO 2 NPs can promote growth, but at high concentrations (1 g/L), they induce glutathione metabolism (oxidative stress response) and inhibit chlorophyll production and plant growth [42,82]. These changes in physiological levels are believed to be caused by the nanoparticles themselves, rather than by dissolved Ce 4+ , and are particle-specific [72].
Although it is generally accepted that oxidative stress (ROS) could be one of the direct sources of nanomaterials' toxicity, relevant experiments showed that changes in ROS may not be the direct cause of the toxic effects of TiO 2 NPs. TiO 2 NPs are extremely easy to agglomerate in aqueous solutions due to their large specific surface area, high surface energy and severe lack of coordination, making it necessary to ensure the stability of TiO 2 NP dispersions in all relevant plant studies. Interestingly, it was found that the presence of tetracycline (TC) could significantly reduce the accumulation of Ti 4+ released by TiO 2 NPs in branches and roots [71]. Further, Arabidopsis could also mitigate chloroplast oxidative damage caused by low doses of TiO 2 NPs through autophagy [70]. These findings provide essential information needed to understand the interaction between metal-based nanoparticles and contaminants, such as antibiotics, in the plant systems.
Additionally, MNPs have been found to affect the photosynthetic efficiency of Arabidopsis in several other ways. For example, CuO NPs, CeO 2 NPs and ZnO NPs can disrupt the energy transfer or oxidation from the photosystem to the Calvin cycle and reduce the gas exchange dynamics [83]. Further, ZnO NPs can also alter the photosynthetic core by releasing Zn 2+ instead of Mg 2+ in the chlorophyll center, and exhibit tissue specificity and concentration dependence. These observations corroborate experiments from phytohormone analysis [64,84]. Ag NPs, on the other hand, can accumulate in Arabidopsis leaves through the particles themselves to further disrupt the cystoid membrane structure, reduce chlorophyll content and inhibit plant growth [85].
Accumulating evidence from more detailed studies shows that the effects of MNPs on the photosynthetic system of Arabidopsis are complex. Sperdouli et al. exposed Arabidopsis leaves to CuZn NPs via foliar spraying and found that the photosystem II (PSII) function of young leaves was negatively affected, which could be attributed to the MNPs impeding the photosynthetic pathway by blocking the electron transport chain. In contrast, they observed a beneficial effect on PSII function in mature leaves and suggested that MNPs promoted the photosynthetic processes by improving light-harvesting complexes in plants [63]. Sergey Bombin et al. investigated the photosynthetic and related biochemical adaptations of IONPs in soil-grown Arabidopsis using a gas exchange system, carbon isotope ratio and chlorophyll content analysis. They observed that enhanced stomatal conductance of Arabidopsis promoted photosynthesis and increased biomass by 38% after treatment with a 500 mg/kg concentration. In addition, the uptake of iron by the roots and leaves was increased (Figure 9). Although this may be due to IONPs providing bioavailable iron as a nutrient or increasing phytohormone content and antioxidant enzyme activity, the underlying mechanism is not well-understood [73]. energy and severe lack of coordination, making it necessary to ensure the st NP dispersions in all relevant plant studies. Interestingly, it was found tha of tetracycline (TC) could significantly reduce the accumulation of Ti 4+ rel NPs in branches and roots [71]. Further, Arabidopsis could also mitigate chlo tive damage caused by low doses of TiO2 NPs through autophagy [70]. T provide essential information needed to understand the interaction betwee nanoparticles and contaminants, such as antibiotics, in the plant systems.
Additionally, MNPs have been found to affect the photosynthetic effi bidopsis in several other ways. For example, CuO NPs, CeO2 NPs and ZnO N the energy transfer or oxidation from the photosystem to the Calvin cycle a gas exchange dynamics [83]. Further, ZnO NPs can also alter the photosyn releasing Zn 2+ instead of Mg 2+ in the chlorophyll center, and exhibit tissue s concentration dependence. These observations corroborate experiments fr mone analysis [64,84]. Ag NPs, on the other hand, can accumulate in Ara through the particles themselves to further disrupt the cystoid membrane duce chlorophyll content and inhibit plant growth [85].
Accumulating evidence from more detailed studies shows that the ef on the photosynthetic system of Arabidopsis are complex. Sperdouli et al. bidopsis leaves to CuZn NPs via foliar spraying and found that the photosy function of young leaves was negatively affected, which could be attributed impeding the photosynthetic pathway by blocking the electron transport trast, they observed a beneficial effect on PSII function in mature leaves a that MNPs promoted the photosynthetic processes by improving light-ha plexes in plants [63]. Sergey Bombin et al. investigated the photosynthetic a ochemical adaptations of IONPs in soil-grown Arabidopsis using a gas exc carbon isotope ratio and chlorophyll content analysis. They observed that matal conductance of Arabidopsis promoted photosynthesis and increased bi after treatment with a 500 mg/kg concentration. In addition, the uptake of iro and leaves was increased (Figure 9). Although this may be due to IONPs p vailable iron as a nutrient or increasing phytohormone content and antiox activity, the underlying mechanism is not well-understood [73]. . Phenotype (a) and photo (b) of growth of control and nanoscale zerovale exposed Arabidopsis shoot at 21 days. Reprinted with permission from Ref. [86]. Thus, at the physiological level, the effects of MNPs on plants are mo on the particles themselves, for which the plants could have corresponding ures. In contrast, among other effects, ions released by MNPs regulate plan development by altering plant photosynthetic centers and reducing chloro but are mostly limited to the high ion concentration range [87]. . Phenotype (a) and photo (b) of growth of control and nanoscale zerovalent iron (nZVI)exposed Arabidopsis shoot at 21 days. Reprinted with permission from Ref. [86]. Thus, at the physiological level, the effects of MNPs on plants are more dependent on the particles themselves, for which the plants could have corresponding countermeasures. In contrast, among other effects, ions released by MNPs regulate plant growth and development by altering plant photosynthetic centers and reducing chlorophyll content, but are mostly limited to the high ion concentration range [87].

Toxic Effects at the Molecular Level
Au NPs with different surface charges can become adsorbed to the marginal cells of Arabidopsis roots, after which they can affect the growth and development of the plant. Correspondingly, downregulated expression of miR164, miR167, miR395, miR414, miR398 and miR408 in Arabidopsis further corroborates their involvement in the plant stress response and the complexity of their regulatory network via regulation of their target genes [62].
Sun et al. found that plants' response to Ag NPs was mainly associated with transcription, protein degradation, the cell wall, direct DNA/RNA/protein damage and cell division [60]. Ag NPs have not only been shown to activate genes associated with both metal and oxidative stress responses and induce the expression of genes related to the phytohormone abscisic acid (ABA) signaling pathway, but to also inhibit the upregulation of lateral root development growth hormone response genes, and downregulate genes associated with pathogen and hormone signaling responses [50,88]. When Arabidopsis roots were exposed to Ag NPs, expressions of homologous recombination (HR)-related genes and the alleviation of transcriptional gene silencing (TGS) in aerial leafy tissues were examined as genotoxic endpoints. It can be seen HR gene expression in aerial leaf tissue was upregulated, and TGS-silenced repetitive elements in aerial tissues could be observed. These observations suggest that the plant systemic response may involve distant induction of Ag NPs' genotoxicity ( Figure 10) [24].
Materials 2022, 15, x FOR PEER REVIEW 13 of stress response and the complexity of their regulatory network via regulation of their ta get genes [62]. Sun et al. found that plants' response to Ag NPs was mainly associated with tra scription, protein degradation, the cell wall, direct DNA/RNA/protein damage and ce division [60]. Ag NPs have not only been shown to activate genes associated with bo metal and oxidative stress responses and induce the expression of genes related to th phytohormone abscisic acid (ABA) signaling pathway, but to also inhibit the upregulatio of lateral root development growth hormone response genes, and downregulate gen associated with pathogen and hormone signaling responses [50,88]. When Arabidops roots were exposed to Ag NPs, expressions of homologous recombination (HR)-relate genes and the alleviation of transcriptional gene silencing (TGS) in aerial leafy tissu were examined as genotoxic endpoints. It can be seen HR gene expression in aerial le tissue was upregulated, and TGS-silenced repetitive elements in aerial tissues could b observed. These observations suggest that the plant systemic response may involve di tant induction of Ag NPs' genotoxicity ( Figure 10) [24]. The mRNA level of other HR-related genes in the aerial tissues of wil type plants, 7 days after root exposure to 1 μg/mL of Ag-NP. Results are the means ± SD (n ≥ 12 f GUS activity; n = 3 for RNA level, t-test ** p < 0.01). Reprinted with permission from Ref. [55].
Silver occurs naturally in several oxidation states, of which elemental silver (Ag and monovalent silver (Ag + ) are the two most common states. Previously, it was hypot esized that Ag + release was responsible for Ag0 toxicity [89,90]. However, in a study b Jane et al., who compared the toxic effects of Ag + and Ag NPs on Arabidopsis, the autho obtained inconclusive results. Studies on plant metabolism have confirmed that whi both Ag NPs and Ag + could induce glycolysis and affect the TCA cycle and aspartate fam ily pathways, there are some metabolic changes (shikimate-phenylpropanoid biosynth sis, tryptophan and galactose metabolism) that occur only upon Ag NPs treatment. B comparing the differences in Ag NPs and Ag + stresses, 111 genes responsible for the r sponse to fungal infection, anion transport and biological functions associated with th cell wall/plasma membrane were found to be unique to Ag NPs at the Arabidopsis g nome level (Figure 11) [24]. The mRNA level of other HR-related genes in the aerial tissues of wild-type plants, 7 days after root exposure to 1 µg/mL of Ag-NP. Results are the means ± SD (n ≥ 12 for GUS activity; n = 3 for RNA level, t-test ** p < 0.01). Reprinted with permission from Ref. [55].
Silver occurs naturally in several oxidation states, of which elemental silver (Ag0) and monovalent silver (Ag + ) are the two most common states. Previously, it was hypothesized that Ag + release was responsible for Ag0 toxicity [89,90]. However, in a study by Jane et al., who compared the toxic effects of Ag + and Ag NPs on Arabidopsis, the authors obtained inconclusive results. Studies on plant metabolism have confirmed that while both Ag NPs and Ag + could induce glycolysis and affect the TCA cycle and aspartate family pathways, there are some metabolic changes (shikimate-phenylpropanoid biosynthesis, tryptophan and galactose metabolism) that occur only upon Ag NPs treatment. By comparing the differences in Ag NPs and Ag + stresses, 111 genes responsible for the response to fungal infection, anion transport and biological functions associated with the cell wall/plasma membrane were found to be unique to Ag NPs at the Arabidopsis genome level (Figure 11) [24]. 022, 15, x FOR PEER REVIEW Figure 11. Venn diagrams of genes with more than two-fold expression chan the six stresses between Ag NPs and Ag + . Reprinted with permission from R Due to their different solubility, MNPs have been shown to affe development and gene expression by releasing metal ions or attachi sues. Jin et al. found that high concentrations of Al2O3 NPs stimulated Arabidopsis root-development-associated genes and nutrient-related root growth, but observed conflicting results when the same conce were used because Al ions were highly toxic to plant growth and caused severe oxidative stress [91].
ZnO NPs can downregulate the expression of microtubulin-re bidopsis under hydroponic conditions to promote the degradation of mers, which in turn affects the plant's cell division [92][93][94]. Wu et al low concentrations of ZnO NPs could increase Arabidopsis's genomic erating with other environmental stresses [95]. Under ZnO NPs, stres ulated the expression of cell-cycle-related genes and inhibited the gr Changes in sugar content, chlorophyll content, DAB and NBT staining defense system showed that ZnO NPs were toxic to all genotypes o Whether this generalized molecular level response originates from Zn of Zn 2+ is a hot topic of current research. Wan et al. compared the tran of Arabidopsis roots to ZnO NPs, bulk ZnO and ionic Zn 2+ . They obser ity of the transcriptional profiles and the increased number of transcr concentration of Zn 2+ in the culture medium suggested that the rele contributing to the toxic effects of ZnO NPs on the plant at the molecu [97]. Arabidopsis root-development-associated genes and nutrient-related genes to promote root growth, but observed conflicting results when the same concentrations of Al ions were used because Al ions were highly toxic to plant growth and photosynthesis, and caused severe oxidative stress [91].
ZnO NPs can downregulate the expression of microtubulin-related genes in Arabidopsis under hydroponic conditions to promote the degradation of microtubulin monomers, which in turn affects the plant's cell division [92][93][94]. Wu et al. demonstrated that low concentrations of ZnO NPs could increase Arabidopsis's genomic instability by cooperating with other environmental stresses [95]. Under ZnO NPs, stress ethylene downregulated the expression of cell-cycle-related genes and inhibited the growth of Arabidopsis. Changes in sugar content, chlorophyll content, DAB and NBT staining and the antioxidant defense system showed that ZnO NPs were toxic to all genotypes of Arabidopsis [65,96]. Whether this generalized molecular level response originates from ZnO NPs or the release of Zn 2+ is a hot topic of current research. Wan et al. compared the transcriptomic response of Arabidopsis roots to ZnO NPs, bulk ZnO and ionic Zn 2+ . They observed that the similarity of the transcriptional profiles and the increased number of transcripts with increasing concentration of Zn 2+ in the culture medium suggested that the release of Zn 2+ could be contributing to the toxic effects of ZnO NPs on the plant at the molecular level ( Figure 12) [97]. Figure 11. Venn diagrams of genes with more than two-fold expression the six stresses between Ag NPs and Ag + . Reprinted with permission fro Due to their different solubility, MNPs have been shown to development and gene expression by releasing metal ions or att sues. Jin et al. found that high concentrations of Al2O3 NPs stimu Arabidopsis root-development-associated genes and nutrient-re root growth, but observed conflicting results when the same c were used because Al ions were highly toxic to plant growth a caused severe oxidative stress [91].
ZnO NPs can downregulate the expression of microtubul bidopsis under hydroponic conditions to promote the degradatio mers, which in turn affects the plant's cell division [92][93][94]. Wu low concentrations of ZnO NPs could increase Arabidopsis's gen erating with other environmental stresses [95]. Under ZnO NPs, s ulated the expression of cell-cycle-related genes and inhibited th Changes in sugar content, chlorophyll content, DAB and NBT sta defense system showed that ZnO NPs were toxic to all genotyp Whether this generalized molecular level response originates from of Zn 2+ is a hot topic of current research. Wan et al. compared the of Arabidopsis roots to ZnO NPs, bulk ZnO and ionic Zn 2+ . They o ity of the transcriptional profiles and the increased number of tra concentration of Zn 2+ in the culture medium suggested that the contributing to the toxic effects of ZnO NPs on the plant at the m [97]. Consistent with the effects observed at the physiological l NPs by themselves had a much higher molecular level effect tha Consistent with the effects observed at the physiological level of Arabidopsis, CuO NPs by themselves had a much higher molecular level effect than Cu 2+ . After 2 h of exposure to CuO NPs treatment, global gene expression analysis showed much more robust upregulation of oxidative-stress-related genes than with corresponding Cu 2+ exposure [67].
Treatment of Arabidopsis mutants with TiO 2 NPs and CeO 2 NPs altered the regulation of 204 and 142 genes, respectively, and affected a range of metabolic processes, such as DNA metabolism, hormone metabolism, tetrapyrrole synthesis and photosynthesis, in the plant. Although the two nanoparticles differ significantly in the molecular mechanisms they use to promote sprouting growth, they altered oxidative stress reactions, as well as expression of genes that encode for responses to stimuli, localization and growth and development (Figure 13), suggesting the effect of each MNP on altered gene expression could be qualitatively and quantitatively different [98].
Materials 2022, 15, x FOR PEER REVIEW Treatment of Arabidopsis mutants with TiO2 NPs and CeO2 NPs altered the tion of 204 and 142 genes, respectively, and affected a range of metabolic process as DNA metabolism, hormone metabolism, tetrapyrrole synthesis and photosynth the plant. Although the two nanoparticles differ significantly in the molecular nisms they use to promote sprouting growth, they altered oxidative stress react well as expression of genes that encode for responses to stimuli, localization and and development (Figure 13), suggesting the effect of each MNP on altered gene sion could be qualitatively and quantitatively different [98]. Figure 13. Pie charts of top 5 categories of upregulated and downregulated genes in Ar thaliana germinants exposed to nano-titania and nano-ceria annotated to broad functions. R with permission from Ref. [82].
ZnSe QDs were also shown to cause oxidative stress in Arabidopsis leaves to cantly inhibit the growth of Agrobacterium rhizogenes in the vicinity of Arabidops Most genes were to be found repressed in roots treated with 100 μM of ZnSe QD was the first time that differential regulatory responses to ZnSe QDs exposure in A sis were observed by gene expression and metabolomic characterization [48]. Co to Cd 2+ ions, the regulation of genes related to ABTS and DPPH radicals, total p GSH redox status and lipid peroxidation in plants treated with CdS QDs was not cant, suggesting that this could be the reason why QDs only release Cd 2+ to a limite [99,100].
MNP exposure can affect plant growth via alterations in their gene expressi Figure 13. Pie charts of top 5 categories of upregulated and downregulated genes in Arabidopsis thaliana germinants exposed to nano-titania and nano-ceria annotated to broad functions. Reprinted with permission from Ref. [82].
ZnSe QDs were also shown to cause oxidative stress in Arabidopsis leaves to significantly inhibit the growth of Agrobacterium rhizogenes in the vicinity of Arabidopsis roots. Most genes were to be found repressed in roots treated with 100 µM of ZnSe QDs. This was the first time that differential regulatory responses to ZnSe QDs exposure in Arabidopsis were observed by gene expression and metabolomic characterization [48]. Compared to Cd 2+ ions, the regulation of genes related to ABTS and DPPH radicals, total phenols, GSH redox status and lipid peroxidation in plants treated with CdS QDs was not signif-icant, suggesting that this could be the reason why QDs only release Cd 2+ to a limited extent [99,100].
MNP exposure can affect plant growth via alterations in their gene expressions and metabolic genetics. Studies suggest that the effects of MNPs on Arabidopsis at the molecular level are largely consistent with those observed at the normal and physiological levels of uptake and transport in Arabidopsis. The effects discovered at the molecular level in Arabidopsis are related to the detoxification and transport of different metal elements in vivo, and the regulation of DNA replication, transcription and translation, manifested as the material specificity of MNPs. They can be summarized as the effects of the particle itself, which are considered to be the primary mechanism, and the effects of the release of ions in a way that causes differential kinetics and cytotoxicity, particularly through upregulation or downregulation of the expression levels of certain genes ( Table 2) [101]. Table 2. Gene expression of Arabidopsis thaliana exposed to different MNP treatments. Raise: respond to abiotic stress (oxidative stress, salt stress, osmotic stress and water stress) and biological stress (pathogen defense), and participate in Zn 2+ binding, transport and steady state [65] Lower: participate in cell tissue and biogenesis (tubulin, arabinogalactan glycoprotein), DNA or RNA metabolism (histone) ZnO 100 mg/L 7 d

Types of MMNPs
Raise: lateral roots develop in response to abiotic stress (oxidative stress, salt stress, osmotic stress and water stress) and biological stress (wound stimulation and pathogen defense) [36] Lower: participate in cell tissue and biogenesis (translation, nucleosome assembly, tubulin), electron transfer

Conclusions and Outlook
In summary, MNPs enter Arabidopsis via the plastid extracellular pathway but have a low in vivo internalization and transfer rate. Only a few MNPs can be readily transferred to the aboveground parts of Arabidopsis. In response to stresses from MNPs, changes in Arabidopsis can be observed at the following three levels: the morphological level, rep-resented by inhibition of root and leaf growth and decreased biomass; the physiological level, manifested by reduced chlorophyll content, affected photosynthetic efficiency and others; and the molecular level, comprising upregulation of antioxidant-related genes, upregulation of resistance signaling pathway genes, downregulation of mRNA expression and more. Further, the interactions between MNPs and Arabidopsis depend on the surface charge, exposure concentration, particle size and morphology of the MNPs. Most importantly, some MNPs can dissolve in biological fluids, release metal ions that interact with biomolecules and act in conjunction with the material itself to cause redox imbalance in Arabidopsis, the two main mechanisms by which MNPs can be toxic to Arabidopsis. To more comprehensively explore the effects on Arabidopsis and clarify the underlying mechanisms of action, the following should be systematically considered in future studies: (i) Currently available literature that investigated the effects of MNPs on Arabidopsis used experimental settings that differed considerably from actual environment settings. For instance, the medium used was water or sandy soil rather than actual soil. The treatment time was comparatively shorter than that observed in nature, and Arabidopsis was exposed in a relatively single manner to MNPs, i.e., the roots were mainly exposed to the soil in the presence of MNPs, and thus, the effects of using leaf sprays or hydroponics remain to be determined. (ii) The physicochemical properties, treatments and growth stages of the MNPs used in the experiments differed among studies. Therefore, the source of nanomaterials, preparation methods, testing equipment selection and design of exposure conditions should be standardized. (iii) The internalization of metal ions produces different levels of toxicity to plants than direct ingestion of metal ions. The toxic effect seems to be concentration-dependent. Further, as the toxic effects from MNPs could be from the released metal ions alone or the combined effect of nanoparticles themselves, the source of toxicity should be identified. (iv) As studies at different levels of the plant's response, i.e., subcellular, physiological and biochemical levels, are being performed, the interactions between MNPs and Arabidopsis should be designed in a way that combines the traditional toxicological research methods with histological techniques (transcriptomics, metabolomics and proteomics) to provide more accurate and in-depth elucidation of the mechanisms of MNP-based phytotoxicity.
Funding: This work was supported by the National Natural Science Foundation of China (31870486), the Natural Science Foundation of Jilin Province (YDZJ202101ZYTS092), the seventeenth batch of innovative entrepreneurial talent projects of Jilin Province (2021Y032) and the Natural Science Foundation of Changchun Normal University (KXK2020002).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data used in this research have been properly cited and reported in the main text.