Enhanced Activity of Anti-oxidant Enzymes by Foliar Spray of Nanoscale Zinc Oxide under Drought Stress Conditions in Peanut (Arachis hypogaea L.)

Peanut (Arachis hypogaea L.) is an essential oil seed, confectionery, food and feed legume crop worldwide. Drought is one of the key environmental problems that hinder crop production and threatens global food security. Moisture stress causes many biochemical, molecular and physiological changes and reactions that affect various cellular and whole plant processes (Prasad et al., 2008). In the current context of global warming and climate change, improving peanut for drought is crucial to ensure high productivity. At the cellular level, one of the effects of salt stress is to hinder the cellular function due to excess production of reactive oxygen species (ROS) (Hasanuzzaman et al., 2012). To reduce ROS accumulation, plants produce antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX), keeping ROS lower than toxic limit (Gill et al., 2015).
       
Detoxification of excess ROS is achieved by antioxidant enzymes such as superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione reductase and other related enzymes. Super oxide dismutase acts as the first line of defense and detoxifies the super oxide radicals to hydrogen peroxide, while Catalase suppresses hydrogen peroxide. Under limited water availability, the photosynthetic process in the plant is greatly reduced and the continuous accumulation of photo-reducing energy causes more electrochemical forces in the tissues, forming superoxide radicals and hydrogen peroxide (Hernandez et al., 2001).
       
Zinc is one of the key nutrients in boosting the groundnut yields. Zn deficiency induce higher ROS levels in plants (Cakmak, 2000) and increases SOD, peroxidise (POD) and catalase (CAT) activities (Yu et al., 1998). Yield losses in groundnut due to Zn deficiency have been reported to be 13.3% to 20% (Singh et al., 2004).
       
Nanomaterials control the plant growth and development at multiple levels (Kwak et al., 2016). For example, the positive effect of nano-ZnO at low concentrations which is well documented in peanut (Prasad et al., 2012), mung bean (Mahajan et al., 2011) and tomato (Singh et al., 2016), suggest that nanoparticles generate tolerance to different abiotic stresses, viz., drought (Zaimenko et al., 2014), salinity (Almutairi, 2016) and low temperature stress (Hawrylak-Nowak et al., 2010). Mitigation of abiotic stress by nanoparticles was often associated with the increased activity of antioxidant enzymes (Sturikova et al., 2018).
               
Nanoparticles of less than 100 nm in size fall into the transition zone, producing both positive and negative biological effects on living cells (Nel et al., 2006). Studies related to the response of nanoscale zinc oxide particles on antioxidant enzyme activities in peanut due to drought stress are lacking. Therefore, the objective of the current study is to measure the levels of anti oxidant activity experienced by peanut crop under water deficit stress by the foliar application of nanoscale zinc oxide particles at different concentrations and to confirm the role of antioxidant enzymes in providing drought tolerance.

The present study was carried at Department of Crop Physiology, Institute of Frontier Technology (IFT), Regional Agricultural Research Station (RARS), Acharya N G Ranga Agricultural University (ANGRAU), Tirupati andhra Pradesh (A.P.) during 2015-16. ZnO nano particles of size 15-30 nm from the Department of Nanotechnology, IFT, RARS, ANGRAU, Tirupati, A.P. were used in the study. Nano crystalline zinc oxide has been prepared using an oxalate decomposition process. The samples are characterized by transmission electron microscopy (HRTEM, JEOL 3010), scanning electron microscopy (SEM, FEI quanta 200) and energy dispersive analysis X-rays (EDA X FEI quanta 200) (Prasad et al., 2012).
       
Peanut seeds of variety Narayani received from the Department of Genetics and Plant Breeding, RARS, ANGRAU, Tirupati, A.P. The pots (20 cm x 40 cm) were filled with equal quantity of soil and four seeds were sown per pot. Care was taken to use the same soil in all pots to reduce soil variability. The experimental design was a randomized block design with two water regimes as main treatments viz., well watered (WW) and water stress (WS) conditions, eight foliar sprays along with unsprayed treatment (control) as sub treatments and 3 replications. Water stress (WS) regime is imposed by withholding irrigation for 40 days i.e., from 40 to 80 days after sowing (DAS). The eight foliar spray treatments, includes nanoscale ZnO concentrations @ 10, 20, 40, 50, 100, 300, 1000 ppm, chelated bulk ZnSO₄ (EDTA based) @ 0.1% (recommended dose). The pots were maintained at field capacity (FC) until harvest under WW treatment, whereas under WS treatment, water stress was imposed @ 40% FC during flowering when 50% of the plants (at least two out of four plants) reached flowering. Foliar spray treatments were applied at 40 DAS and sampling was done after one week of foliar spray treatment.
       
SOD (EC 1.15.1.1) enzyme activity was estimated by its ability to inhibit photochemical reduction of Nitrobluete trazolium (NBT) method according to Madamanchi et al., 1994). In this assay, 1 unit of SOD is defined as the amount required to inhibit the photoreduction of NBT by 50% and SOD activity was measured  spectrophotometrically at 560 nm.
       
The activity of POD (EC 1.11.1.7) was measured by increased absorption of tetraguaiacol formation at 470 nm and the POD activity was measured as per extinction coefficient of its oxidation product, tetraguaiacol ε = 26.6 mM^-1 cm^-1 (Castillo et al., 1984). Enzyme activity was estimated by monitoring the increase in absorption at 470 nm due to guaiacol oxidation.
       
CAT activity (EC 1.11.1.6) was determined by measuring the H₂O₂ disappearance (Aebi, 1984) in the reaction compound. The decrease in absorption at 240 nm on a UV spectrophotometer was observed for 1 min. Enzyme activity was measured by calculating the amount of H₂O₂ decomposed.
               
At harvest, shoot dry weight (SDW) and pod yields were recorded after drying of individual plants and expressed in g plant^-1. The statistical analysis was assessed by Two Way Analysis of Variance (ANOVA) using GENSTAT software and the critical difference values were calculated at 5% significance level to compare the mean values.

Of the newest technological   innovations,   nanotechnology   offers   an important   opportunity and occupies a prominent position improving the existing crop management strategies and food production practices (Nair et al., 2010). The nano scale ZnO particles with mean diameter of 25 nm are crystalline as revealed by the high magnification image and the lattice of ZnO is clearly seen. Zhu et al., 2009 also reported lattice spacing of 0.26 nm to 0.28 nm of WURTZITE ZnO.
       
Application of nano-ZnO promoted melatonin synthesis and increased the antioxidant enzyme system, which reduces drought-induced damage to mitochondria and chloroplast in corn (Luying Sun et al., 2020). In the current study, water stress (WS) regime from 40 to 80 days after sowing (DAS) i.e., from pegging to pod formation stage, reduced soil moisture content by 42 % at 0-5 cm soil depth and 40 % at 15-30 cm soil depth compared to well watered (WW) plot where moisture level was maintained optimum throughout the crop growth period. Irrespective of the foliar treatments, the mean activity of SOD, POD and CAT increased significantly by 31.2%, 12.0 and 28.0% respectively under WS compared to WW (Table 1). Kusvuran and Yildiz Dasgan (2017) in bean and Koushik Chakraborty et al., (2015) in peanut reported higher activity of antioxidant enzymes under moistures stress compared to control conditions. Under WS conditions, the activity of SOD, POD and CAT increased by 11%, 12% and 6.0% respectively in chelated ZnSO₄ @ 1000 ppm treatment compared to unsprayed control. SOD value increased significantly even with ZnSO₄ spraying denotes the stimulating action of Zn as it is one of the cofactors of SOD (Cu/Zn SOD). Zinc may act as a scavenger of oxygen free radical production for mitigating the injury on biomembranes under salt stress and hence, treatment with zinc could reduce the effects of salinity stress in soybean plants and adequate zinc also prevents uptake and accumulation of Na in shoot, by increasing membrane integrity of root cells (Weria Weisany et al., 2012).
 

 
In the present study, among the different concentrations of nanoscale ZnO, significantly highest SOD, POD and CAT activity recorded in nanoscale ZnO @ 50 ppm treatment compared to unsprayed control and chelated bulk ZnSO4 under WW conditions. Similarly, foliar spray of nanoscale ZnO @ 50 ppm significantly recorded highest SOD, POD and CAT activity and increased by 25.1, 27.4 and 25.5 % respectively compared to unsprayed control whereas SOD, POD and CAT activity increased by 11.9, 13.7 and 22.6 % respectively compared to chelated bulk ZnSO₄ respectively under WS conditions (Table 1; Fig 1). Drought results in accumulation of ROS, leading to lipid peroxidation of the cell membrane system, which is mainly due to the production of malondialdehyde. Luying et al., (2020), documented that, nano-ZnO significantly increased the activities of SOD, CAT and APX, which reduced the accumulation of H₂O₂ under drought and in agreement with this, the relative transcript abundance of Fe/Mn SOD, Cu/Zn SOD, APX and CAT in nano-ZnO plants was significantly up-regulated higher than that of non ZnO treatment plants under drought.
 

       
Biomass accumulation improved in the ZnO nanoparticle treated Chickpea seedlings and this response was associated with lower activity of prominent antioxidant enzymes, compared to control (Burman et al., 2013). The study by García-López et al., (2018) on the effects of suspensions of zinc oxide nanoparticles on germination of Capsicum chinense seeds resulted in increased activities of peroxidase and catalase. Vigna mungo seeds treated with different concentrations of ZnO nano particles showed significant induction of the activities of Glutathione reductase, Guaiacol peroxidase and Catalase activity (Pavani et al., 2020).
       
Sharifi et al., (2012) reported that the tolerant wheat lines revealed high activity of POD and CAT enzyme under drought conditions with higher yields, thus showing positive correlation between enzyme activity and yield in drought conditions. Prasad et al., 2012 reported that, ZnO in the nanoscale form is absorbed by plants to a larger extent unlike bulk ZnSO₄ and these particles proved effective in enhancing plant growth, development and yield. 
       
The inherent small size and the associated large surface area of nanoscale ZnO fertilizer may increase the uptake of Zn. All these factors may be responsible to give higher yields for nanoscale ZnO compared to chelated ZnSO₄.
       
The present study reveals that, shoot biomass and pod yields recorded highest under WW compared to WS conditions. Shoot biomass and pod yields decreased by 37% under WS compared to WW conditions. Chelated ZnSO₄ foliar treatment recorded 5.0% increase in shoot biomass under both WW and WS conditions compared to unsprayed control whereas Chelated ZnSO₄ foliar treatment recorded 10.0% and 7.0% enhanced pod yields under WW and WS conditions compared to unsprayed control (Table 1). Under various stressed conditions, optimum Zn concentrations enhanced the plant growth. The lower dry weight in the control treatments may be due to water limited stress as water limited stress have been known to severely reduce the growth of plants (Adrees et al., 2020). In the present study, significantly highest shoot biomass and pod yields were recorded at foliar spray treatment of nanoscale ZnO @ 50 ppm compared to unsprayed control and chelated bulk ZnSO₄ under WW conditions. Foliar spray of nanoscale ZnO @ 50 ppm significantly recorded highest shoot biomass and pod yields and increased by 35.0 and 32.3 % respectively compared to unsprayed control increased by 17.6 and 23.8 % respectively compared to chelated bulk ZnSO₄ under WS conditions (Table 1). Adrees et al., (2021) also reported that, the shoot, root and grain dry weights enhanced more under water deficit stress compared to normal water conditions where 100 mg l^-1 ZnO NPs were applied, over their respective control treatments.
               
The study by Hesham Alharby et al., (2016) reveals that, lower concentration of nanoscale ZnO was beneficial compared to the higher concentration of nanoscale ZnO, at both salinity levels.  The current study also observed that the nanoparticles at higher concentrations exhibit inhibitory effects on plants in terms of enzymatic activity and promontory activity at lower concentrations. At 1000 ppm, the antioxidant enzyme activity, shoot weight and pod yield significantly decreased compared to unsprayed control under both WW and WS conditions respectively in terms of plant growth showing phytotoxicity (Table 1).

Foliar application of chelated bulk ZnSO₄ increased antioxidant enzyme activity, shoot biomass and pod yield compared to unsprayed control both under WW and WS conditions. Among different concentrations of nanoscale ZnO, foliar treatment of nanoscale ZnO @ 50 ppm increased the activity of antioxidant enzymes, shoot biomass and pod yields both under WW and WS conditions compared to unsprayed control and chelated bulk ZnSO₄. Nanoscale ZnO at higher concentrations showed inhibitory activity of antioxidants, shoot biomass and pod yield both under WW and WS conditions. The research is continuing for further exploitation of nano ZnO effects on other antioxidant enzymes in peanut.