Effect of Metal Oxide Nanoparticles on Plant Enzymatic Activities Under Drought

The aim of this work was to study the effect of metal oxide nanoparticles (MO NPs) on enzymatic activities (EAs) of two main plant life forms under drought stress (DS). Foliar spray of silver oxide (AgO), iron oxide (FeO), zinc oxide (ZnO) and cadmium oxide (CdO) at levels of 30, 60, 90 and 120 mg.L -1 were used on aerial parts of forb Sanguisorba ocinalis L. and grass Agropyron cristatum (L.) Gaertn. under DS levels of 25-100% eld capacity (FC). Glutathione reductase (GR), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were measured after two weeks of the experiment. The results indicate that the EAs changes varied depending on the plant life form, levels of DS, type and levels of NPs. Overall, application of 30- 60 mg.L -1 MO NPs under 25-50%FC signicantly reduced EAs, especially in forb (P<0.001). Higher concentration of MO NPs signicantly increase EAs. A decrease in CAT (20.90%, 18.80%), POD (21.30%, 17.67%) and SOD (23.14%, 16.88%) was observed under levels of 60 mg.l -1 of ZnO NPs under 25-50%FC in forb and grass, respectively. High concentration of CdO NPs (90-120 mg.l -1 ) caused by increase (max= 24.0%) in EAs in both life forms under 25%FC.


Introduction
Today, metal oxide nanoparticles (MO NPs) have received much attention because of their effect on environmental biology (Baysal and Sayg\in 2018;Rajput et al. 2018). MO NPs directly/indirectly in uence the live organisms (Cheng 2003;Peng et al. 2016) and have great potential in agriculture as a means to deliver micronutrients to plants for increasing yield (Elmer et al. 2018). On one hand, to improve performance, it is imperative that we enhance plant drought tolerance (Ye et al. 2016). Among the availbe methods to improve the physiological condition of plants under stress, the use of MO NPs is a new method that has been used many times. In fact, MO NPs application is considered one of the effective methods for increasing plant resistance and biomass under drought stress (DS) by regulating their enzymatic activities (EAs). Also, proper use of MO NPs may lead to the maintenance/improvement of plants performance under DS.
Among the all enzymes, Glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) are the most important (Elmer et al. 2018) that play an important role against stress (Peyvandi et al. 2011;Parveen and Rao 2015;Ye et al. 2016;Trivedi et al. 2020). The plant's response to MO NPs varies according to the environmental factors. Since, different negative or positive results have been reported on the effect of MO NPs on EAs (Nair et al., 2011;Rajput et al., 2018;Akinyemi et al. 2017).
Among all the MO NPs, silver (Ag), iron (Fe), zinc (Zn), and cadmium (Cd) NPs have been widely used. Ag are prominent and are widely used in a growing number of applications (Ragam and Mathew 2020;Yu et al., 2013). Many studies have reported negative (Neal 2008;Bradford et al. 2009;Coleman et al. 2011) or positive (Yin et al. 2012;Parveen and Rao 2015) effects of AgNPs on EA. Fe also, is an essential nutrient for all organisms and plays a major role in plant EA and performance ( Miller et al., 1995;Mimmo et al., 2014;Sánchez-Alcalá et al., 2014). Fe components (i.e. FeO NPs) are auxiliary to over 140 enzymes that accelerate biochemical reactions. However, limited research has been done on the effects of iron oxide nanoparticles (FeO NPs) on plant EA (Peyvandi et al. 2011).
Moreover, Zn is a micronutrient that plays an important physiological role in plants (Dang et al. 2010;Ali et al. 2019;Shahzad et al. 2019). Zn NPs can easily penetrate into the plant cell and play an essential role in some cellular functions and metabolism in plant (Clarkson 1996;López-Millán et al. 2005;Dang et al. 2010;Jayarambabu et al. 2015). ZnO NPs are being widely used in numerous applications (Shahzad et al. 2019). Both negative and positive effects have been reported in EA by ZnO NPs application (Hernandez-Viezcas et al. 2011;Rao and Shekhawat 2014;Wang et al. 2016Wang et al. , 2019Abdel Latef et al. 2017;Khan et al. 2019). Furthermore, Cd is a metal that is released into the nature and affect live organisms (Bayçu et al. 2017;Khan et al. 2019). However, in some studies it is shown that CdO NPs signi cantly changes plant's EA (Guan et al. 2009;Manara 2012).
Although there are some reports about the MO effects on the physiological characteristics of plants, but, there is no detailed information on the effect of MO NPs on EAs of different plant life forms under DS. Hence, the purpose of this study was to investigate the effect of AgO, FeO, ZnO and CdO NPs on GR, CAT, POD and SOD in forb Sanguisorba o cinalis and grass Agropyron cristatum under DS. The results of this study can be used to determine the effective levels of MO NPs to improve production in grasses or forbes under DS.

Methods
The research was carried out using plots in a loamy soil (depth= 30 cm, pH=7.4±0.5; OM=1.2±0.35) on 18 May 2018 at Agricultural and Natural Resources Research and Education Center, Tehran, Iran (3776166.13 m N; 384363.39 m E).

Experiment
A eld experiment was conducted in a randomized complete block design under 4 levels of MO NPs and DS with 4 replications. Seeds (PLS=0.98) of Sanguisorba o cinalis L. and Agropyron cristatum (L.) Gaertn were obtained from the gene bank of the Institute of Forests and Rangelands Research and were analyzed for the germination test in the laboratory using standard test method. Numbers of 10 seeds of each species was cultivated in 1.5 m 2 plots at depth of 1-2 cm. Plots were irrigated to eld capacity (FC). After 40 days, plots were prepared for applying the treatments. Irrigation was considered as variable for plots at 25% of Field capacity (FC), 50% of FC, 75% of FC, and 100% FC (control). Then, after a week, samples were prepared for applying the MO NPs treatments.

MO NPs preparation
Silver oxide nanoparticles (AgO NPs; particle size= 50 nm), iron oxide nanoparticles (FeO NPs; particle size= <50 nm), zinc oxide nanoparticles (ZnO NPs; particle size= 20 nm), cadmium oxide nanoparticles (CdO NPs; particle size= <50 nm; purity of 99.8%) was purchased from Sigma Co. Previous characterization showed that CdO NPs have regular spherical shape and uniform size, with an average size of 50 nm (Aldwayyan et al. 2013); ZnO NPs were composed of homogeneous spherical particles that were single crystals with an average size of 20±5 nm (Fallah et al. 2017); AgO NPs have a spherical shape and an average size of 50 nm (Bamdad et al. 2018); FeO NPs look like a honeycomb and average size of 40-50 nm with smooth surface, but is nununiform (Srinivasarao et al. 2012). Therefore, con rming the seller's claim regarding MO NPs size, the research was conducted (Kim et al. 2011). MO NPs were used without any pre-treatment.
MO NPs concentrations were selected based on literatures (Mahil and Kumar 2019). MO NPs solutions were prepared using deionized water and then mechanical stirrer and ultra-sonicator (Wang et al. 2011). The required soluble concentrates were prepared adding 0.03, 0.06, 0.09 and 0.12 g of NPs powder per one liter of deionized water. Samples were placed in an ultrasonic homogenizer for 30 min to create a homogenous suspension. Before using the solution, an electromagnetic stirrer was used to prevent the accumulation of particles. ICP-AES spectrometer was used to verify the nominal values of MO NPs.
Analytical procedures were based on criteria of limits of detection and quanti cation (LOD/LOQ), linearity, sensitivity, recovery, and precision using the standard deviation of the blank signal multiplied by three and six, respectively. A 6-point calibration curve was prepared for each metal and comparing with the nominal concentrations, the measured metal concentrations in the prepared solutions showed good accuracy and statistical analysis con rmed no signi cant differences between the nominal and measured concentrations of metals. The ICP-AES spectrometer parameters and details of calibration curves described by Pokorska-Niewiada et al. (2018). Table 1 shows the detection and quanti cation limits and actual values of MO NPs in solution (30 mg.L -1 ).  (Hong et al. 2016) at levels of 120, 90, 60, 30 and 0 mg.L -1 . In order to prevent the sunburn of the leaves, MO NPs spray was applied to full foliage wetting at evening. Plants were harvested two weeks after the third stage of MO NPs spraying for EA measurement. Moreover, SPAD meter {SPAD-502, Konica-Minolta, Tokyo, Japan} was used for determination of the leaf chlorophyll concentration. Relative water content (RWC) was also measured for samples (Turner 1981).

Plant EAs measurent
Fresh leaves of the 4 plants were taken at random from each plot before owering at vegetative growth time. Samples were immediately frozen. Then, frozen leaves were ground to a ne powder with liquid nitrogen and extracted with ice-cold 50 nM phosphate buffer under neutral acidity condition (Meloni et al. 2003). CAT, SOD, GR and POD activity were measured using the methods described by Dazy et al. (2008), Giannopolitis and Ries (1977), Cribb et al. (1989), and Chance and Maehly (1955), respectively.

Data analysis
Two-way analysis of variance and compound analysis were performed to compare the measured factors. The data was then ranked and compared by Duncan's multiple range test (MRT) at the 5% level of error in SPSS v.17.01 software. Also, all graphs were drawn in Excel 2010 software.

Results
The following table shows the statistical analysis of the percentage of change in EAs and chlorophyll and relative water content in the two species by application of MO NPs under DS ( Table 2). The effect of factors individually and in combination has caused a signi cant change in the evaluated characteristics (P<0.001). Preliminary results of morphophysiological characteristics of plants under stress showed that the application of MO NPs signi cantly changed the photosynthetic and moisture content of the leaves ( Table 2) The CAT changes of A. cristatum, which was, of course, less pronounced than that of in S. o cinalis. Application 120 mg.l -1 CdO showed the most negative (increase) effect on EAs i.e. CAT under drought in both species (Fig. 2; A).
SOD activity also showed notable changes under MO NPs application and DS. The lowest SOD activity was observed under ZnO (L 2 ) in both S. o cinalis and A. cristatum. The most negative effect was observed under 120 mg.l -1 CdO and severe drought of 25%FC ( Fig. 2 and 3). Generally, using 30-60 mg.l -1 MO NPs particularly ZnO caused a signi cant reduction in SOD activity in grass A. cristatum and forb S. o cinalis.
Furthermore, POD showed signi cant changes under MO NPs and DS in which lower levels of MO NPs and DS had positive effects on increasing plant drought tolerance and led to decreased POD activity in S. o cinalis and A. cristatum (Fig. 2-3). Application of 60 mg.l -1 MO NPs had the most effect on POD activity under the severe DS (Fig. 3). Generally, lower concentration (30-60 mg.l -1 ; especially in ZnO) decrease and higher concentration (90-120 mg.l -1 ) of MO NPs increase POD activity in both species DS.
Generally, EAs changes varied depending on the plant species, type and concentration of MO NPs, and levels of DS. The most changes in EAs were related to forb S. o cinalis. Low concentration (<60 mg.l -1 ) of AgO, FeO, ZnO and CdO NPs had a positive effect on the reduction of EAs. And higher concentration (90-

Discussion
This study described the physiological properties of two plant forms including grass and broadleaf using different concentrations of MO Ns under drought conditions. The rst change involved a relative improvement in the moisture content of both plant forms. This improvement showed the greatest change in medium stresses and medium concentrations of nanoparticles. It seems that application of optimal amount of nanoparticles by controlling physiological activities and controlling moisture exchange in plant cell membranes helps maintain moisture in plant organs (Chakhchar et al. 2016).
RWC in plant organs is the result of the adaptation mechanism induction of investigated plants at drought conditions (Taran et al. 2017). However, the most change of RWC was observed under application of ZnO (60 mg.l -1 ) especially under severe drought in both studies grass and forb. It has been reported that foliar selenium application signi cantly lowered osmotic potential (13%) and increased RWC (Nawaz et al. 2015). In another study, also, RWC of Phaseolus vulgaris leaves was signi cantly affected under CeO2 sprayed and the most RWC (80%) was observed under 250 mg L−1 MO application and higher levels signi cantly decreased RWC of plant leaves (Salehi et al. 2018). Another study also indicated that Zn NPs decreased the negative effect of drought effects and increased RWC in wheat leaves (Taran et al. 2017). In fact, Zn improves the plant response under environmental stress . Therefore, MO NPs may have a positive effect on maintaining the water content of plant leaves.
Our results suggested that GR activity controlled and reduced under low concentration of MO NPs and levels of DS in both grass and forb. But higher levels of AgO, FeO, ZnO and particularly CdO NPs have negative effects and increase GR activity. Generally, GR activity will increase under stress. However, previous study indicated that ZnO NPs (100 and 200 mM) declined GR activity by 25 and 45% in wheat seedlings (Tripathi et al. 2017b). The GR plant chloroplasts controles physiological mechanisms, especially under DS (Yousuf et al. 2012) and therefore showed a decrease under low concentration of MO NPs.
Moreovere, the CAT changed under the in uence of type and concentration of MO NPs and as well as DS. Also, the rate of these changes was not the same between the two plant plant forms.The CAT plays an indispensable role in detoxi cation activity under stress (Yang et al. 2017) and could be changed by MO NPs (Du et al. 2017). A number of studies also showed that lower concentrations of Zn NPs changed the CAT activity and enhanced the plant defense system under stress (Raigond et al. 2017;Abdel-Aziz 2019;Du et al., 2017;Hernandez-Viezcas et al., 2011;Zhao et al., 2015). Another study also showed that lower concentrations of Zn NPs changed the CAT activity and indicated that the relatively low levels of NPs enhanced the plant defense system by increasing antioxidant EA in Lupine plant under stress (Abdel-Aziz 2019).
Similarly, Rao and Shekhawat (2014) disclosed that lower concentrations of ZnO hadn't signi cant effect on CAT but, higher concentration (>1000 mg/l) signi cantly increased EA in Brassica juncea (Rao and Shekhawat 2014). Such trend also was founded in the potato plant under higher levels of Zn NPs (Raigond et al. 2017). Simmilarly, using 25-200 mg L −l ZnO NPs has led to a decrease in the CAT activity in cotton (Venkatachalam et al. 2017). Therefore, CAT activity may affect by MO NPs. These changes will be different deponding on the plants species, the type and concentration of MO NPs and levels of DS.
Furthermore, the metalloenzyme SOD is the most effective antioxidant intracellular enzyme that various environmental stresses often lead to increased it (Chakhchar et al. 2016). Similar to our ndings, other researchers reported that high concentration of NPs signi cantly stimulated SOD activity in plants exposed to levels>100 mg/L ZnO NPs (Suman et al. 2015;Tripathi et al. 2017a). These results also have been con rmed in previous researches under the DS (Mustafa et al. 2015).
Simmilar to SOD, the POD activity showed signi cant changeds under MONs and DSs. Similarly, Siddiqi and Husen, (2017) reported that the POD activity was remarkably inhibited by the large quantity of ZnO NPs. However, the effects of different MO NPs depend upon the type and concentration of NP that change plant performance (Kanwar et al. 2019). Gupta et al., (2018) reported that AgNPs (10-40 ppm) application promoted the plant biomass and SOD, CAT and GR signi cantly decreased using 40 ppm.
Also, Çatav et al., (2020) showed that higher concentration of Cd severely increased GR, POD and SOD in Triticum aestivum. Similarly, Wang et al., (2019) disclosed that CAT, POD and SOD decreased by increase Zn concentration in wheat seedlings. EAs provide protection to plants by contributing to cellular osmotic adjustment, stabilization of protein structure, scavenging of hydroxyl radicals, and regulation of cytosolic pH under stress (Çatav et al. 2020). In fact, NPs application prompts activation of plant defense mechanism to combat damage under stress and very high concentrations of nanoparticles cause oxidative stress and impair the function of plant cells (Gupta et al. 2018).

Conclusion
This study demonstrates that the changes in the physiological characteristics (EAs of RG, SOD, CAT and POD) of each plant vary depending on the type and