The Effects of Zinc Oxide Nanoparticles on Drought Stress in Moringa peregrina Populations

Introduction: Moringa peregrina (Forssk.) Fiori, is a nutritionally and medicinally important desert tree, which is constantly exposed to drought stress. This study was accomplished to alleviate the adverse effects of drought stress on M. peregrina populations through the foliar application of zinc oxide nanoparticles (ZnO-NPs) by monitoring some physiological and biochemical alterations. Methods: Moringa peregrina seeds were collected from the Southeast of Iran in 2014. Fourteen days after germination, the seedlings were subjected to drought stress by withholding watering until 50% field capacity (FC), followed by spraying 0.1% and 0.05% ZnO-NPs and no spraying

genotypes, severity, duration, and time course of drought stress. 4 Nowadays, the cultivation of medicinal plants is done in a drought stress condition to influence the secondary metabolites content such as phenols. 5 Phenolic compounds as the largest groups of phytochemicals are present in foods. Flavonoids and other phenolic compounds are potent antioxidants and anticarcinogenic agents. Polyphenols exist in many plants and are especially abundant in the Moringa tree, whose dried leaves are used as antioxidant. 6 Drought can also lead to pigment degradation, thus causing irreversible water-deficit damage to the photosynthetic apparatus. One of the indicators of the stress tolerance capacity of plants is chlorophyll stability index. 7 In addition, it has been demonstrated that antioxidants affect the chlorophyll content of plants as well. 8 The tolerance of plants to environmental stresses such as water deficit can be fortified by micronutrient fertilizers. Zinc (Zn) is an essential micronutrient required for metabolic activity, water balance, and stomatal regulation in plants. It adjusts various enzymatic functions and is required for biochemical reactions resulting in chlorophyll formation. 9 Currently, the use of nano-materials such as zinc oxide nanoparticles (ZnO-NPs) has been expanded. 10 It has been demonstrated that the use of micronutrient fertilizers in the form of NPs is an appropriate procedure to release required nutrients gradually and in a controlled way, which is essential to mitigate the problems of fertilizer pollutions. Due to high surface-to-volume ratio, highly active surface, unique size and shape, and physical, chemical, biological, and catalytic properties, NPs can modulate chemical and biological activities of cells. 11 It has been reported that ZnO-NPs can produce better results than the conventional ZnO. 10 Additionally, ZnO-NPs possess excellent electrical properties. 12 However, to the best of our knowledge, there is little or no reliable information about the physiological effects of foliar application of ZnO-NPs on mineral and biochemical content of medicinal plants under well-watered as well as drought stress conditions. Therefore, this study was carried out to evaluate the changes of mineral nutrients, chlorophylls as well as phenolic compounds, and antioxidant activity of different M. peregrina populations under well-watered and drought stress conditions in response to foliar application of ZnO-NPs.

Plant Materials and Growth Conditions
The experiment was conducted in a greenhouse at the Faculty of Agriculture, University of Sistan and Baluchestan, Zahedan, Iran (latitude of 29°27 ′ 34 ″ N, longitude of 60°51 ′ 10 ″ E, 1385 m altitude, mean annual temperature of 18.3°C and rainfall of 72 mm ), during the 2014 growing season. Ten populations of M. peregrine seeds were collected from different regions of Sistan and Baluchestan province, Southeast of Iran. The seeds were cleaned to remove damaged seeds, stones, leaves, wood, dust, and any other unknown materials. Cleaned seeds were stored in black plastic bags and labeled. The locations of all samples have been shown in Table 1. The regions where the populations were located are typically characterized by an arid and semi-arid climate. Seeds were surface-sterilized with 0.5% (v/v) sodium hypochlorite and treated with benomyl solution for 30 minutes at 24°C and then washed three times with sterilized deionized water. Twenty seeds were placed in each 9-cm Petri dish on two sheets of filter paper moistened. The seeds were kept in a germination chamber under a photoperiod of 12 hours at 25°C. After germination, the seedlings were kept in a growth chamber for 14 days at 25°C, 70% relative humidity with a 12-hour photoperiod and watered daily. Later, the young plants were sown in plastic pots (30 cm height and 20 cm diameter) filled with steam-sterilized soil containing washed sand (3:1:1, v/v/v), clay horizon (red earth), and organic horizon soil (black soil). They were fully randomized, kept in a greenhouse and watered daily. Young plants, 40 days after germination, were exposed to drought stress and ZnO-NPs treatment.

Treatments
The field capacity (FC) of the medium in the pot was measured prior to each treatment. Three pots were saturated with water, covered with plastic to avoid Sodium and Potassium Content Before the measurement of Na + and K + , leaf samples were exactly washed by tap water and then rinsed with deionized water to remove all surface remnants. Fruit flesh samples, after air-drying, were taken from the equatorial section of each fruit quarter, oven-dried at 70°C for 48 hours and milled to pass through a 40-mesh sieve. A portion of fine powder weighing 2 g was dry washed in a furnace at 550°C for 4 hours and then the ash was dissolved in 10 mL of 2 M hydrochloric acid (HCl). Through a Whatman No. 40 filter paper, the digested samples were filtered and used for the analysis of Na + and K + . Na + and K + content was determined by flame photometric (Biotech Engineering Management Co. Ltd., UK) method as already described. 14

Chlorophyll Assay
The content of chlorophyll a (Chl-a), chlorophyll b (Chl-b), and total chlorophyll (T-Chl) was determined by UV-visible spectrophotometry (PG Instrument Ltd., Leicester, UK) as described by Rajalakshmi and Banu. 15 Briefly, 10 mL of acetone 80% was added to 0.1 g of homogenized freeze-dried herbage samples. The samples were centrifuged at 6000 g for 10 minutes. The supernatant was filtered through a Whatman No.1 filter paper and the absorbances were read at 645 and 663 nm using UV-visible spectrophotometer. The amounts of Chl-a, Chl-b and T-Chl were calculated according to the following formulas: Where A 645 and A 663 are the absorbances at 645 and 663 nm, "V" is sample volume in absorbance, and "W" is the fresh weight (FW) of sample in grams.

Total Phenol Content
The total phenolic content (TPC) was determined using Folin-Ciocalteu method with minor modifications. 16 TPC extraction was carried out by 10 mL acidic methanol added to 1 g of the leaf powder with the mixture and then filtered through ordinary filter paper. Afterwards, 500 μL of this extract was diluted with 5 mL of Folin-Ciocalteu reagent (1:10 g mL -1 ), and then 4 mL of Na 2 CO 3 (1 M) was added to the mixture. This reaction solution was shaken in a shaker and kept in dark for 15 minutes. The absorbance of samples was taken at 765 nm using UV/ Visible spectrophotometer (model PG Instrument +80, Leicester, UK). Gallic acid was used as standard to obtain calibration curve. Data were expressed as milligram gallic acid equivalent (mg GAE) per 1 g of fruit FW.

Antioxidant Activity
The antioxidant activity was evaluated by 2, 2-diphenyl-1picrylhydrazyl (DPPH) free radical-scavenging method with some modifications. 16 The absorbance of the samples was measured at 515 nm and the antioxidant activity was expressed as the percentage of the decline of the absorbance, relative to the control, corresponding to the percentage of DPPH that was scavenged. The percentage of DPPH, which was scavenged (%DPPHsc), was calculated using: Where A cont is the absorbance of the control, and A samp is the absorbance of the sample.

Statistical Analysis
The experiments were carried out according to a 3-factor linear model based on a completely randomized design with three replications. Data were statistically analyzed using analysis of variance (ANOVA) by SAS software (version 9.1 2002-2003, SAS Institute, Cary, NC, USA). Before analysis of variance, data were tested for normality and homoscedasticity using the Shapiro-Wilk test. Least significant difference (LSD) test at P ≤ 0.01 probability level was considered as the statistical significance level.

Minerals Content
The results showed that drought stress reduced Na + and K + content in all M. peregrina populations ( Table  2). The highest Na and K contents were found at 50% FC in population number 7 (0.89 and 13.69 mg g -1 DW, respectively). Under well-watered conditions, untreated plants (controls) had higher Na content, whereas under drought conditions, ZnO-NPs treatment slightly enhanced Na content compared with control ( Figure 1). However, it was revealed that the interaction effects of drought stress and ZnO-NPs treatment on the K content of M. peregrina populations were not significant (P ≤ 0.01) according to the LSD test ( Figure 2). Our results showed that foliar spray of ZnO-NPs not only prevented chlorophylls degradation but also significantly enhanced Chl-a, Chl-b as well as T-Chl content in both drought-stressed and unstressed plants. The 0.10% ZnO-NPs concentration was most effective in enhancing chlorophylls content (Figure 3).

Discussion
The present study has revealed that ZnO-NPs spray can increase the tolerance of M. peregrina plant to drought stress by enhancing some secondary metabolites and antioxidant potential. Drought stress affects the minerals uptake by plant roots through influencing root growth and nutrient mobility in soil. Under drought stress, water availability is reduced, followed generally by reduction in total nutrient uptake and the concentrations of minerals in crops. 17 The most important effect of drought stress is observed on the transport of nutrients to the root which affects root growth and extension. The content of nutrient elements is balanced by nutrient uptake and unloading mechanisms as well as transpiration flow. 18 It has been revealed that drought stress significantly reduced Na and enhanced K uptake and ion uptake efficiency in different chickpea genotypes. 19 According to our results, drought stress reduced Na + and K + content in all M. peregrina populations. However, under drought conditions, ZnO-NPs treatment increased Na + content. Change in nutrients balance in response to Zn-NPs spray is in agreement with the findings of Soliman et al 20 who reported that foliar application of Zn-NPs significantly affects mineral nutrients content of M. peregrina. Zn seems to affect the capacity for water and nutrients uptake as well as transport in plants under different abiotic stresses. 21 Our results revealed that Zn-NPs spray enhanced Na content in drought stress conditions, which was in agreement with the findings of Martinez et al, 22 who reported that plant tolerances to water deficit is due to a common mechanism of Na uptake for osmotic adjustment.
Our results showed that foliar spray of ZnO-NPs not only prevented chlorophylls degradation but also significantly enhanced Chl-a, Chl-b as well as T-Chl content in both drought-stressed and unstressed plants. Photosynthetic pigments control the energy balance through chlorophylls and therefore, they involve in the adaptation and survival of plants in drought. 23 Inhibition of chlorophyll biosynthesis, activation of chlorophyllase and/or destruction of chloroplast structure lowered the pigment content under abiotic stress conditions. 20 In addition, Mejri et al 24 reported that drought stress prevented from chloroplast activity and led to the breakdown of chlorophyll as well as changes in chlorophyll a to b ratio. It has been reported that the decrease in chlorophyll under drought stress is generally the consequence of chloroplast damages resulting from reactive oxygen species (ROS). 25 Another reason for the decline in chlorophyll is the application of a glutamate precursor for the biosynthesis of proline. 26 Enhancing chlorophylls content by ZnO-NPs treatment are in agreement with the results obtained by Fathi et al 27 31 Based on the present study, TPC increased in response to drought stress. However, foliar application of ZnO-NPs increased TPC under both well-watered and drought conditions. The increase in TPC under drought stress condition is due to the soluble carbohydrates accumulation  in plant cells because transportation of soluble sugars is reduced under water deficit. 32 Phenolic compounds are biosynthesized through various mechanisms such as malonic acid and shikimic acid pathways. Simple carbohydrate precursors are converted into aromatic amino acids through shikimic acid pathway. 33 It has been reported that the increase of phenolic compounds is highly correlated with the balance between carbohydrate sources and sinks. 32,34 Moreover, the TPC enhancement under drought stress is greatly related to the production and distribution of various antioxidants in the plant and the intensity and duration of stress. 35 Similarly, we found that there was a significant positive correlation between TPC and antioxidant activity (r = 0.93). The increase of TPC by foliar application of ZnO-NPs is in agreement with findings of Oloumi et al 36 who mentioned that the phenolic compounds contents in seedlings of Glycyrrhiza glabra L. significantly increased in response to 10 μM ZnO-NPs. They suggested that it might be due to the ability of ZnO-NPs to affect metabolic activity.
In the present study, the antioxidant activity of M. peregrina populations remarkably increased under drought stress. Drought stress, at the cellular level, induces the accumulation of ROS such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals. The excess ROS are detrimental because they damage membranes, proteins, chlorophylls and nucleic acids. To reduce this oxidative damage, plants enhance their antioxidant defense systems including enzymatic and non-enzymatic scavenging mechanisms. 23 The antioxidant activity has a crucial role in maintaining the balance between the production and scavenging of free radicals, therefore, the number of antioxidants should be high to compensate and tolerate stress condition. 32 Our results indicated that ZnO-NPs treatment enhanced the antioxidant activity of M. peregrina populations as compared with untreated ones. It was revealed that the application of Zn under abiotic stress enhanced removing reactive oxygen species by increasing antioxidant enzymes activities. Zn ions bind to ligands containing sulfur, nitrogen, and to a lesser extent oxygen, and preferentially bind to the membrane proteins. 28 Our results are in agreement with the findings of Tavallali et al 37 who reported that Zn is able to facilitate the biosynthesis of antioxidant enzymes and enhance antioxidant activity in the leaves of pistachio under abiotic stress conditions. The balance between the generation and removal of free radicals determines the survival of the system. Therefore, Zn may have a role in modulating free radicals and their related damaging effects by enhancing the antioxidant activity of plants. 38 The antioxidant property of ZnO-NPs could be resulted from the transfer of electron density located at oxygen to the odd electron located at nitrogen atom in DPPH. 39 This characteristic is dependent on the structural configuration of oxygen atom, and it determines the thermal stability of nanoparticle. 40 In addition, the antioxidant efficacy of ZnO-NPs against DPPH is likely due to the electrostatic attraction between positively charged NPs (ZnO = Zn 2+ + O 2 ) of plant extracts and negatively charged bioactive compounds (COO -, O -). 41 Binding of ZnO-NPs to phytochemicals synergistically increase their bioactivity. 42

Conclusion
ZnO-NPs treatment effectively protected M. peregrina populations from drought stress by inhibiting the chlorophylls degradation and enhancing TCP and antioxidant activity. Hence, foliar application of ZnO-NPs is recommended for the growth of different M. peregrina populations under drought stress conditions.

Competing Interests
The authors declare that they have no competing financial, professional, or personal interests that might have influenced the performance or presentation of the study described in this manuscript.

Ethical Approval
Not applicable.