Leaf Age-Dependent Effects of Foliar-Sprayed CuZn Nanoparticles on Photosynthetic Efficiency and ROS Generation in Arabidopsis thaliana

Young and mature leaves of Arabidopsis thaliana were exposed by foliar spray to 30 mg L−1 of CuZn nanoparticles (NPs). The NPs were synthesized by a microwave-assisted polyol process and characterized by dynamic light scattering (DLS), X-ray diffraction (XRD), and transmission electron microscopy (TEM). CuZn NPs effects in Arabidopsis leaves were evaluated by chlorophyll fluorescence imaging analysis that revealed spatiotemporal heterogeneity of the quantum efficiency of PSII photochemistry (ΦPSΙΙ) and the redox state of the plastoquinone (PQ) pool (qp), measured 30 min, 90 min, 180 min, and 240 min after spraying. Photosystem II (PSII) function in young leaves was observed to be negatively influenced, especially 30 min after spraying, at which point increased H2O2 generation was correlated to the lower oxidized state of the PQ pool. Recovery of young leaves photosynthetic efficiency appeared only after 240 min of NPs spray when also the level of ROS accumulation was similar to control leaves. On the contrary, a beneficial effect on PSII function in mature leaves after 30 min of the CuZn NPs spray was observed, with increased ΦPSΙΙ, an increased electron transport rate (ETR), decreased singlet oxygen (1O2) formation, and H2O2 production at the same level of control leaves.An explanation for this differential response is suggested.


Introduction
Both zinc (Zn) and copper (Cu) are essential elements for plant growth [1]. Zn deficiency results in a rapid inhibition of plant growth and development, while several physiological processes are impaired [1][2][3]. Zinc scarcity in arable soils [4] is a major problem worldwide [2], which is mainly due to the low Zn soil solubility resulting in Zn unavailability to plant roots [5]. Adequate Zn supply is suggested to improve productivity and nutrients in crops [6]. Low Zn concentrations in soils can be improved by adding Zn fertilizers, but this is a costly and ineffective policy [7]. However, to and α-brass (JCPDS no.  and no. , while no significant changes were observed from the previous reported by us CuZn NPs [38]. The composition analysis of the NPs by inductively coupled plasma (ICP) indicated a 52%/48% copper/zinc proportion, respectively, and thus an overall composition of α-Cu47Zn29/γ-Cu9Zn15 based on the X-ray diffractions. TEM images of CuZn NPs ( Figure 2) revealed small, spherical nanoparticles in the range of 20 nm to 30 nm in contrast to the formation of nanoclusters [38]. The different nanoarchitecture is attributed to the half amount of polyol (TrEG) that has been used in the present synthesis. CuZn NPs are hydrophilic, and thus readily disperse in water. The hydrodynamic diameter provided by DLS number measurements (Figure 1b) was 35 nm, matching well to the size provided by TEM and indicated monodispersity. Additionally, the amount of leached ions in a 30 mg L −1 aqueous suspension of CuZn NPs after 24 h of incubation was found to be 1.8 mg L −1 for Cu and 2.2 mg L −1 for Zn, respectively.

Changes in Lght Energy Partitioning at PSII in Young and Mature Leaves After Exposure to CuZn NPs
We estimated the light energy partitioning at PSII, that is, ΦPSII, ΦNPQ and ΦNO, which sum to one. The quantum yield of photochemical energy conversion (ΦPSΙΙ) at 30 min, 90 min, and 180 min after CuZn NPs are hydrophilic, and thus readily disperse in water. The hydrodynamic diameter provided by DLS number measurements (Figure 1b) was 35 nm, matching well to the size provided by TEM and indicated monodispersity. Additionally, the amount of leached ions in a 30 mg L −1 aqueous suspension of CuZn NPs after 24 h of incubation was found to be 1.8 mg L −1 for Cu and 2.2 mg L −1 for Zn, respectively. and α-brass (JCPDS no.  and no. , while no significant changes were observed from the previous reported by us CuZn NPs [38]. The composition analysis of the NPs by inductively coupled plasma (ICP) indicated a 52%/48% copper/zinc proportion, respectively, and thus an overall composition of α-Cu47Zn29/γ-Cu9Zn15 based on the X-ray diffractions. TEM images of CuZn NPs ( Figure 2) revealed small, spherical nanoparticles in the range of 20 nm to 30 nm in contrast to the formation of nanoclusters [38]. The different nanoarchitecture is attributed to the half amount of polyol (TrEG) that has been used in the present synthesis. CuZn NPs are hydrophilic, and thus readily disperse in water. The hydrodynamic diameter provided by DLS number measurements (Figure 1b) was 35 nm, matching well to the size provided by TEM and indicated monodispersity. Additionally, the amount of leached ions in a 30 mg L −1 aqueous suspension of CuZn NPs after 24 h of incubation was found to be 1.8 mg L −1 for Cu and 2.2 mg L −1 for Zn, respectively.

Changes in Lght Energy Partitioning at PSII in Young and Mature Leaves After Exposure to CuZn NPs
We estimated the light energy partitioning at PSII, that is, ΦPSII, ΦNPQ and ΦNO, which sum to one. The quantum yield of photochemical energy conversion (ΦPSΙΙ) at 30 min, 90 min, and 180 min after

Changes in Lght Energy Partitioning at PSII in Young and Mature Leaves After Exposure to CuZn NPs
We estimated the light energy partitioning at PSII, that is, Φ PSII , Φ NPQ and Φ NO , which sum to one. The quantum yield of photochemical energy conversion (Φ PSII ) at 30 min, 90 min, and 180 min after the foliar spray with 30 mg L −1 of CuZn NPs presented a significant decrease in young leaves, while in mature leaves, it increased significantly compared to controls (Figure 3a). Φ PSII recovered to control values in young leaves 240 min after the spray, while at the same time remaining significantly higher than controls in mature leaves (Figure 3a). Φ PSII in mature leaves at 30 min, 90 min, 180 min, and 240 min after spraying with CuZn NPs was significantly higher than young leaves (Figure 3a). values in young leaves 240 min after the spray, while at the same time remaining significantly higher than controls in mature leaves (Figure 3a). ΦPSΙΙ in mature leaves at 30 min, 90 min, 180 min, and 240 min after spraying with CuZn NPs was significantly higher than young leaves (Figure 3a). The quantum yield of regulated non-photochemical energy loss (ΦNPQ) at 30 min, 90 min, 180 min, and 240 min after the CuZn NPs spray increased significantly, compared to control, in young leaves (Figure 3b). ΦNPQ 30 min after the NPs spray decreased in mature leaves, while it increased significantly afterwards, compared to control (Figure 3b). ΦNPQ in young control leaves, and at 30 min and 90 min after the CuZn NPs spray was significantly higher than that in mature leaves (Figure 3b). In comparison, ΦNPQ was significantly higher in mature leaves 240 min after the spray with CuZn NPs (Figure 3b).  The quantum yield of non-regulated energy loss (ΦNO), which is a loss process due to PSII inactivity, increased significantly in young leaves 30 min after the CuZn NPs spray compared to control, while it remained unchanged in mature leaves, where it decreased significantly later on (90 min, 180 min, and 240 min after the foliar spray) ( Figure 4). In young leaves, ΦNO 90 min after the foliar spray with NPs decreased to control values, and increased later on (180 min), but retained control values 240 min after spraying with the NPs (Figure 4). ΦNO in mature control leaves was significantly higher than that in young leaves, but at 90 min, 180 min, and 240 min after the foliar spray with 30 mg L −1 of CuZn NPs, it decreased significantly compared to young leaves and control values ( Figure 4).  The quantum yield of regulated non-photochemical energy loss (Φ NPQ ) at 30 min, 90 min, 180 min, and 240 min after the CuZn NPs spray increased significantly, compared to control, in young leaves ( Figure 3b). Φ NPQ 30 min after the NPs spray decreased in mature leaves, while it increased significantly afterwards, compared to control (Figure 3b). Φ NPQ in young control leaves, and at 30 min and 90 min after the CuZn NPs spray was significantly higher than that in mature leaves ( Figure 3b). In comparison, Φ NPQ was significantly higher in mature leaves 240 min after the spray with CuZn NPs (Figure 3b).
The quantum yield of non-regulated energy loss (Φ NO ), which is a loss process due to PSII inactivity, increased significantly in young leaves 30 min after the CuZn NPs spray compared to control, while it remained unchanged in mature leaves, where it decreased significantly later on (90 min, 180 min, and 240 min after the foliar spray) (Figure 4). In young leaves, Φ NO 90 min after the foliar spray with NPs decreased to control values, and increased later on (180 min), but retained control values 240 min after spraying with the NPs (Figure 4). Φ NO in mature control leaves was significantly higher than that in young leaves, but at 90 min, 180 min, and 240 min after the foliar spray with 30 mg L −1 of CuZn NPs, it decreased significantly compared to young leaves and control values ( Figure 4). min, 180 min, and 240 min after the foliar spray) (Figure 4). In young leaves, ΦNO 90 min after the foliar spray with NPs decreased to control values, and increased later on (180 min), but retained control values 240 min after spraying with the NPs (Figure 4). ΦNO in mature control leaves was significantly higher than that in young leaves, but at 90 min, 180 min, and 240 min after the foliar spray with 30 mg L −1 of CuZn NPs, it decreased significantly compared to young leaves and control values ( Figure 4).  An asterisk (*) represents a significantly different mean of the same time treatment between young and mature leaves (p < 0.05).

Changes in the Photoprotective Energy Dissipation and the Electron Transport Rate in Young and Mature Leaves After Exposure to CuZn NPs
The non-photochemical quenching (NPQ) increased significantly at 30 min and 90 min after the CuZn NPs spray in young leaves, compared to the control, while it decreased 180 min after spraying, and increased again significantly 240 min after spraying (Figure 5a). NPQ in mature leaves decreased 30 min after spraying with NPs, but later on (90 min, 180 min, and 240 min after the foliar spray), it increased compared to control values ( Figure 5a). NPQ was significantly higher in young leaves compared to mature and control leaves and 30 min and 90 min after the CuZn NPs spray, but significantly lower than in mature leaves at 180 min and 240 min after the foliar spray ( Figure 5a). Columns with different letter (lower case for young leaves and capitals for mature) are statistically different (p < 0.05). An asterisk (*) represents a significantly different mean of the same time treatment between young and mature leaves (p < 0.05).

Changes in the Photoprotective Energy Dissipation and the Electron Transport Rate in Young and Mature Leaves After Exposure to CuZn NPs
The non-photochemical quenching (NPQ) increased significantly at 30 min and 90 min after the CuZn NPs spray in young leaves, compared to the control, while it decreased 180 min after spraying, and increased again significantly 240 min after spraying ( Figure 5a). NPQ in mature leaves decreased 30 min after spraying with NPs, but later on (90 min, 180 min, and 240 min after the foliar spray), it increased compared to control values ( Figure 5a). NPQ was significantly higher in young leaves compared to mature and control leaves and 30 min and 90 min after the CuZn NPs spray, but significantly lower than in mature leaves at 180 min and 240 min after the foliar spray ( Figure 5a). The relative electron transport rate at PSII (ETR) decreased significantly in young leaves 30 min, 90 min, and 180 min after the foliar spray with 30 mg L −1 of CuZn NPs, while at the same time it increased significantly in mature leaves compared to controls (Figure 5b). ETR recovered to control values in young leaves 240 min after the spray, while it remained significantly higher than controls in mature leaves ( Figure 5b). The ETR in mature leaves at 30 min, 90 min, 180 min, and 240 min after the spray with CuZn NPs was significantly higher than that in young leaves (Figure 5b).

Changes in the Redox State of Plastoquinone (PQ) Pool in Young and Mature Leaves After Exposure to CuZn NPs
The redox state of PQ pool (qP), which is a measure of the fraction of open PSII reaction centers, The relative electron transport rate at PSII (ETR) decreased significantly in young leaves 30 min, 90 min, and 180 min after the foliar spray with 30 mg L −1 of CuZn NPs, while at the same time it increased significantly in mature leaves compared to controls (Figure 5b). ETR recovered to control values in young leaves 240 min after the spray, while it remained significantly higher than controls in mature leaves (Figure 5b). The ETR in mature leaves at 30 min, 90 min, 180 min, and 240 min after the spray with CuZn NPs was significantly higher than that in young leaves (Figure 5b).

Changes in the Redox State of Plastoquinone (PQ) Pool in Young and Mature Leaves After Exposure to CuZn NPs
The redox state of PQ pool (q p ), which is a measure of the fraction of open PSII reaction centers, decreased significantly at 30 min and 180 min after the CuZn NPs spray in young leaves, compared to control; in contrast, it was at control values 90 min and 240 min after spraying ( Figure 6). At all the sampling periods (30 min, 90 min, 180 min, and 240 min after the NPs spray), the mature leaves were in a more oxidized state than control ( Figure 6).

Spatiotemporal Heterogeneity of the Quantum Efficiency of PSII Photochemistry and the Redox State of Plastoquinone (PQ) Pool in Young and Mature Leaves After Exposure to CuZn NPs
The quantum yield of photochemical energy conversion (ΦPSΙΙ) in control young leaves showed a spatial heterogeneity, with higher values in the midrib of the leaves than in the lamina (Figure 7a   At all the sampling periods (30 min, 90 min, 180 min, and 240 min after the NPs spray), the mature leaves were in a more oxidized state than control ( Figure 6). Mature control leaves (Figure 8a) presented less spatial heterogeneity in Φ PSII compared to control young leaves (Figure 7a), with higher values in the distal (tip) leaf area (Figure 8a). Higher values also occurred in the same area 30 min after the foliar spray with 30 mg L −1 CuZn NPs, which also caused the whole leaf Φ PSII to increase (Figure 8b). The spatiotemporal heterogeneity of Φ PSII in mature leaves was also evident 90 min after spraying with NPs (Figure 8c), but become less apparent 180 min after the foliar spray (Figure 8d). At 240 min after the foliar spray, Φ PSII decreased in mature leaves in the whole leaf area, but remained higher than in the control leaves (Figure 8e).  (Figure 7b). The spatiotemporal heterogeneity of ΦPSΙΙ in young leaves 90 min after the foliar spray was still evident due to an increase of whole leaf ΦPSΙΙ values (Figure 7c), and became amplified 180 min after spraying (Figure 7d). Then, 240 min after spraying with CuZn NPs, ΦPSΙΙ increased to the control whole leaf values, showing also a spatial heterogeneity, with higher ΦPSΙΙ values in the area where lower values were previously scored (distal leaf area) (Figure 7e).   (Figure 8a). Higher values also occurred in the same area 30 min after the foliar spray with 30 mg L −1 CuZn NPs, which also caused the whole leaf ΦPSΙΙ to increase (Figure 8b). The spatiotemporal heterogeneity of ΦPSΙΙ in mature leaves was also evident 90 min after spraying with NPs (Figure 8c), but become less apparent 180 min after the foliar spray (Figure 8d). At 240 min after the foliar spray, ΦPSΙΙ decreased in mature leaves in the whole leaf area, but remained higher than in the control leaves (Figure 8e).
Images of the redox state of the PQ pool (qP) of control young leaves showed a spatial heterogeneity, with higher values in the proximal (base) midrib of leaves (Figure 9a), as observed in the images of ΦPSΙΙ (Figure 7a). At 30 min after the foliar spray with 30 mg L −1 of CuZn NPs, a spatiotemporal heterogeneity of qP in young leaves was noticed, with lower values in the distal (tip) leaf area (Figure 9b) and significantly lower whole leaf qP values than those of the young control leaves (Figure 9a). At 90 min after the foliar spray with CuZn NPs, the qP images of young leaves (Figure 9c) were similar to the images of control young leaves (Figure 9a). At 180 min after the foliar spray with CuZn NPs, the whole leaf qP values in young leaves decreased (Figure 9d  Images of the redox state of the PQ pool (q P ) of control young leaves showed a spatial heterogeneity, with higher values in the proximal (base) midrib of leaves (Figure 9a), as observed in the images of Φ PSII (Figure 7a). At 30 min after the foliar spray with 30 mg L −1 of CuZn NPs, a spatiotemporal heterogeneity of q P in young leaves was noticed, with lower values in the distal (tip) leaf area (Figure 9b) and significantly lower whole leaf q P values than those of the young control leaves (Figure 9a). At 90 min after the foliar spray with CuZn NPs, the q P images of young leaves (Figure 9c) were similar to the images of control young leaves (Figure 9a). At 180 min after the foliar spray with CuZn NPs, the whole leaf q P values in young leaves decreased (Figure 9d   Images of the redox state of the PQ pool (q P ) of control mature leaves showed leaf homogeneity rather than leaf heterogeneity (Figure 10a). At 30 min after the foliar spray with 30 mg L −1 of CuZn NPs, a slight heterogeneity of q P was observed in mature leaves, with increased q P values in the whole leaf area (Figure 10b). Later on (90 min, 180 min, and 240 min after spraying with CuZn NPs), a further increase of q P values compared to the control mature leaves was observed in the whole leaf area (Figure 10c-e). Images of the redox state of the PQ pool (qP) of control mature leaves showed leaf homogeneity rather than leaf heterogeneity (Figure 10a). At 30 min after the foliar spray with 30 mg L −1 of CuZn NPs, a slight heterogeneity of qP was observed in mature leaves, with increased qP values in the whole leaf area (Figure 10b). Later on (90 min, 180 min, and 240 min after spraying with CuZn NPs), a further increase of qP values compared to the control mature leaves was observed in the whole leaf area (Figure 10c-e).

ROS Generation in Young and Mature Leaves After Exposure to CuZn NPs
ROS generation was quantified in young (Figure 11a-e) and mature (Figure 11f-j) A. thaliana leaves by the fluorescent probe DCF-DA. In both young (Figure 11a) and mature (Figure 11f) control leaves, no notable quantities of H2O2 could be observed. At 30 min after the foliar spray with CuZn NPs, the highest H2O2 generation was noticed in young leaves (Figure 11b), accompanying the lower measured qP values (Figure 9b). At the same time in mature leaves (Figure 11g), the level of ROS accumulation was similar to the control values (Figure 11f). At 90 min after the CuZn NPs spray, almost no H2O2 could be detected in young leaves (Figure 11c). In mature leaves, no H2O2 could be detected at 90 min, 180 min, and 240 min after the CuZn NPs spray, either (Figure 11h-j). At 180 min after spraying with CuZn NPs, a high H2O2 production (but substantially less than 30 min after spraying) was observed in young leaves (Figure 11d). At 240 min after spraying with CuZn NPs, the level of ROS accumulation in young leaves (Figure 11e) was similar to that of the control (Figure 11a).

Discussion
Inorganic NPs are emerging as novel agrochemicals due to their unique characteristics and high surface energy, which make them effective in lower doses compared to conventional inorganic ionic formulations. For instance, bulk brass has been utilized in the healthcare industry, while ionic forms of zinc and copper such as Bordeaux mixture, sulfate, and chloride salts are used in agrochemistry, but with adverse environmental effects and toxicity. However, due to the low water solubility, these ionic forms of agrochemicals are applied in relatively large amounts in order to effectively control   (Figure 11b), accompanying the lower measured q P values (Figure 9b). At the same time in mature leaves (Figure 11g), the level of ROS accumulation was similar to the control values (Figure 11f). At 90 min after the CuZn NPs spray, almost no H 2 O 2 could be detected in young leaves (Figure 11c). In mature leaves, no H 2 O 2 could be detected at 90 min, 180 min, and 240 min after the CuZn NPs spray, either (Figure 11h-j). At 180 min after spraying with CuZn NPs, a high H 2 O 2 production (but substantially less than 30 min after spraying) was observed in young leaves (Figure 11d). At 240 min after spraying with CuZn NPs, the level of ROS accumulation in young leaves (Figure 11e) was similar to that of the control (Figure 11a).  (Figures 7-10). Young leaves show a higher spatial heterogeneity (Figures 7 and 9) compared to mature leaves (Figures 8 and 10). Nevertheless, PSII function was not uniform for both leaf types, making conventional chlorophyll fluorescence

Discussion
Inorganic NPs are emerging as novel agrochemicals due to their unique characteristics and high surface energy, which make them effective in lower doses compared to conventional inorganic ionic formulations. For instance, bulk brass has been utilized in the healthcare industry, while ionic forms of zinc and copper such as Bordeaux mixture, sulfate, and chloride salts are used in agrochemistry, but with adverse environmental effects and toxicity. However, due to the low water solubility, these ionic forms of agrochemicals are applied in relatively large amounts in order to effectively control the phytopathogens when the spores vegetate, which is through causing the secretion of malic acid and amino acids and subsequently dissolving them [49]. As a consequence, the limit between plant protection and phytotoxicity is still a matter of discussion. A need exists for new products that are going to have high biological activity and less metal in the formulation. Under these perspectives, hydrophilic CuZn NPs retain the desired characteristics of bulk brass, while forming stable aqueous suspensions with minimal ionic dissolution that are effective in low doses.
Young leaves can utilize only a fraction of absorbed irradiance in photochemical reactions via CO 2 assimilation, since light capture ability develops earlier than CO 2 assimilation capacity [45,50,51]. When the absorbed light is not used in photochemistry, in order to avoid photodamage, the excess excitation energy has to be safely removed by a photoprotective mechanism called non-photochemical quenching (NPQ) [52,53]. Consequently, the ability to dissipate excess excitation energy by NPQ is higher in young leaves than in mature leaves [45,47], which means that under control growth conditions, NPQ is significantly higher in young leaves compared to mature leaves (Figure 5a).
Heterogeneity in PSII photochemistry has been frequently reported to depend on the leaf age [41][42][43]45,47,48]. We observed changes in light energy partitioning related to leaf age under control growth conditions mostly related to Φ NPQ and Φ NO . Control young leaves had higher Φ NPQ than mature leaves (Figure 3b), and without any significant difference in Φ PSII (Figure 3a), it resulted in significantly lower Φ NO (Figure 4). However, 90 min, 180 min, and 240 min after the NPs spray, Φ NO increased in young leaves compared to mature leaves (Figure 4) due to a decreased photochemical energy conversion (Φ PSII ) (Figure 3a) that could not be compensated by the increased Φ NPQ (Figure 3b). Φ NO consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of singlet oxygen ( 1 O 2 ) via the triplet state of chlorophyll ( 3 chl*) [41,[54][55][56], thus suggesting increased 1 O 2 formation in young leaves compared to mature leaves. NPQ is one of the most important photoprotective mechanisms in plants [25,41,57,58]. The enhancement of NPQ that reflects the dissipation of excess excitation energy in the form of harmless heat in young leaves (Figure 5a) at 30 min after spraying with CuZn NPs, could not protect young leaves from ROS generation at 30 min after the NPs spray ( Figure 11b). However, the increase of NPQ in young leaves 90 min after the CuZn NPs spray (Figure 5a) was effective at retaining the same redox state of the PQ pool with control leaves and reducing H 2 O 2 production at 90 min after the NPs spray to control levels ( Figure 11c). An effective photoprotection can be attained only if NPQ is adjusted in such a way that no changes occur in the redox state of the PQ pool [41,59]. Otherwise, an imbalance between energy supply and demand occurs, indicating excess excitation energy [57][58][59]. Under such circumstances, the generation of H 2 O 2 occurs (Figure 11b), which can be diffused through the leaf veins to act as a long-distance signaling molecule [38,41,[60][61][62]. The intracellular ROS signaling pathways are initiated by the redox state of the PQ pool that regulates photosynthetic gene expression, comprising also a mechanism of plant acclimation [38,63,64]. The redox state of the PQ pool is of unique significance for antioxidant defense and signaling [65]. It has been shown recently that ROS generation is influenced also by the circadian system [66,67]. We postulate that ROS generation at 30 min after the NPs spray (Figure 11b) possibly served as the signaling molecule to contribute to a more oxidized state of the PQ pool at 90 min after the NPs spray ( Figure 6, Figure 9c), resulting in a H 2 O 2 production similar to the control leaf level (Figure 11c).
The foliar spay of Arabidopsis thaliana young and mature leaves with 30 mg L −1 of CuZn NPs revealed a spatiotemporal heterogeneity of Φ PSII and q p measured (at 140 µmol photons m −2 s -1 ) 30 min, 90 min, 180 min, and 240 min after spraying (Figures 7-10). Young leaves show a higher spatial heterogeneity (Figures 7 and 9) compared to mature leaves (Figures 8 and 10). Nevertheless, PSII function was not uniform for both leaf types, making conventional chlorophyll fluorescence instruments not suitable for abiotic stress studies and pointing out the advantages of using chlorophyll fluorescence imaging analysis in the recognition of spatial heterogeneity at the leaf surface [29][30][31][32][33][34][35]. The response of cells to the same stress condition is not uniform, with some cells behaving more vulnerably than others [68].
In contrast to previous reports that young leaves acclimatize better to environmental changes and can maintain a better ROS homeostasis [43,45,69], mature leaves responded better. Thus, in disagreement with our hypothesis, the PSII photochemistry of young leaves seem to be negatively influenced when exposed to 30 mg L −1 of CuZn NPs. Young leaves could overcome the negative effects on the function of PSII only after 240 min of the NPs spray, at which point the level of ROS accumulation was also similar to that of control young leaves. On the contrary, a beneficial effect was observed in the PSII function of mature leaves 30 min after the CuZn NPs spray, which was through an increased quantum efficiency of PSII photochemistry (Φ PSII ), an increased electron transport rate (ETR), an increased fraction of open PSII reaction centers (q p ), decreased 1 O 2 formation, and no notable changes in H 2 O 2 generation.
Zinc and Cu are important micronutrients that are required for plant growth and development [1,38], but when they are in excess, they can cause toxicity effects on plant growth and development, affecting photosynthetic function [3,70]. In young leaves with sufficient Zn and Cu concentrations, the spray with 30 mg L −1 of CuZn NPs resulted in an excess supply of them, causing negative effects on PSII function and increased ROS production. Increased H 2 O 2 generation in young leaves after 30 min of spraying with 30 mg L −1 of CuZn NPs (Figure 11b) was correlated to a lower oxidized state of the PQ pool (Figure 9b). Zinc is involved in a wide variety of physiological processes, playing catalytic, regulatory, and structural roles with several crucial functions in the cell [1,[71][72][73], but excess Zn has to be detoxified in roots by sequestration to protect the sensitive photosynthetic leaf tissues [3,72]. Zn phytotoxicity varies extensively, depending on combinations with other heavy metals, the environmental conditions, the plant species, and the plant age [72], as well as by the leaf age, as shown here.
Leaf senescence turns leaves from units with a primary assimilation role into centers of nutrient mobilization [74,75]. During leaf senescence, new metabolic pathways are activated and others are de-activated, with nutrient and material remobilization, followed by a declining photosynthesis [74,75]. The A. thaliana rosette leaf 8 from six-week-old plants is a mature to senescing leaf; thus, nutrient remobilization occurs, resulting in nutrient deficiency. Spraying these leaves with 30 mg L −1 of CuZn NPs restores Zn and Cu deficiency and improves photosynthetic efficiency. Zinc is known to contribute to the repair processes of PSII by turning over the photodamaged D1 protein [72,76]. Copper is vital for photosynthesis, and more than half of Cu is found in chloroplasts participating in the light reactions [77]. The foliar spraying of Cu NPs induced stress tolerance by stimulating antioxidant mechanisms [78]. However, this nutrient remobilization explanation has yet to be established.
Although plants are producers and play a key role in the ecosystem, the impact of NPs upon them is not well studied [79]. In order to understand the uptake, transport, and also bioaccumulation of NPs in plants after foliar exposure, different qualitative and quantitative methods are still being developed with an unclear comparability of results among the different techniques [80,81]. Among the different techniques, inductively coupled plasma mass spectroscopy (ICP-MS) is one of the most reliable methods for the detection of NPs, offering a range of advantages in high detection limits and high sensitivity for many elements [79,[81][82][83].
Previously, both the positive and harmful impacts of NPs on terrestrial and aquatic plants have been established, which are mainly due to the concentration, size, and specific surface area of NPs, the exposure methodology, and the plant species that was examined [38,79,81,[84][85][86]. In the root uptake of NPs, translocation to the above-ground parts takes place in a unidirectional pathway through xylem vessels, while in the foliar uptake of NPs, translocation takes place in bidirectional pathways throughout the plant by the phloem [85]. The efficiency of uptake and translocation, and the effects of NPs on growth metabolism and photosynthesis vary from plant to plant [84]. Some studies report that the foliar application of NPs considerably increases the chlorophyll content in plants and results in a higher amount of light energy capture and photosynthesis enhancement [84], while another study reports that the root uptake of NPs decreases Φ PSII and q p , and increases Φ NO due to an ineffective photoprotective mechanism (NPQ) resulting from a significant decrease of the PsbS protein, which is the key regulator of the energy dissipation process [87]. Nevertheless, the investigation on NPs is still at an initial phase; more laborious work is required in order to understand their impact on the physiological, biochemical, and molecular mechanisms in plants [79,84].
The present study demonstrates that considering the leaf developmental stage is important for understanding the mechanisms underlying leaf growth responses to environmental stresses [41][42][43]88]; thus, it must be taken into account in environmental stress studies in order to compare leaves of the same developmental stage [41][42][43]48,89]. Leaves of distinct ages differentially control stress responses, and plant responses against biotic and abiotic stresses are balanced in a leaf age-dependent manner [90]. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.