Selenium and silica nanostructure-based recovery of strawberry plants subjected to drought stress

Drought is an important environmental stress that has negative effects on plant growth leading to a reduction in yield. In this study, the positive role of nanoparticles of SiO2, Se, and Se/SiO2 (SiO2-NPs, Se-NPs and Se/SiO2-NPs) has been investigated in modulating negative effects of drought on the growth and yield of strawberry plants. Spraying of solutions containing nanoparticles of SiO2, Se, and Se/SiO2 (50 and 100 mg L−1) improved the growth and yield parameters of strawberry plants grown under normal and drought stress conditions (30, 60, and 100%FC). Plants treated with Se/SiO2 (100 mg L−1) preserved more of their photosynthetic pigments compared with other treated plants and presented higher levels of key osmolytes such as carbohydrate and proline. This treatment also increased relative water content (RWC), membrane stability index (MSI) and water use efficiency (WUE). In addition, exogenous spraying of Se/SiO2 increased drought tolerance through increasing the activity of antioxidant enzymes including catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) as well as decreasing lipid peroxidation and hydrogen peroxide (H2O2) content. Increase in biochemical parameters of fruits such as anthocyanin, total phenolic compounds (TPC), vitamin C and antioxidant activity (DPPH) in strawberry plants treated with Se/SiO2 under drought stress revealed the positive effects of these nanoparticles in improving fruit quality and nutritional value. In general, our results supported the positive effect of the application of selenium and silicon nanoparticles, especially the absolute role of Se/SiO2 (100 mg L−1), on the management of harmful effects of soil drought stress not only in strawberry plants, but also in other agricultural crops.


Results
Fresh and dry weight of root and shoot. Fresh and dry weights of roots and shoots decreased with the increase of drought stress such that under severe drought stress, fresh and dry weights of roots as well of those of shoots decreased by 73/64/67/69%, respectively, compared with normal condition (Fig. 1A,B). At all drought stress levels, spraying with Se-, SiO 2 -and Se/SiO 2 -NPs increased the amounts of these parameters compared with water sprayed plants, with Se/SiO 2 -NPs having the stronger effect. Under non-stress conditions, spraying with Se/SiO 2 -NPs (100 mg L −1 ) increased fresh and dry weights of roots (42% and 113%, respectively) as well as those of shoots (29% and 43%, respectively) as compared to water sprayed plants. Under severe stress, spraying Se/SiO 2 -NPs (100 mg L −1 ) increased all four parameters by 144/167/87/112%, respectively.
Leaf photosynthetic pigments and chlorophyll fluorescence. Amounts of chl a, chl b, total chlorophyll and carotenoid decreased with increasing drought stress (Table 1). Under severe drought stress the amounts of chl a, chl b, total chl and carotenoid decreased significantly (61/70/63/67%, respectively) compared with normal conditions. At all stress levels, spraying a solution containing Se-, SiO 2 -and Se/SiO 2 -NPs on strawberry plants increased the concentrations of photosynthetic pigments compared to plants sprayed with only water (Table 1). At all three stress levels (control, moderate, severe) Se/SiO 2 -NPs (100 mg L −1 ) was most effective at increasing the contents of chl a, chl b, total chl and carotenoid as compared with plants sprayed with only water.
Regarding chlorophyll fluorescence parameters, with an increase in drought stress, F 0 increased but other photosynthetic parameters including F m , F v , and F v /F m decreased (Table 1). Drought stress increased F 0 (65%) and decreased F m , F v , F v /F m (68/93/78%, respectively) compared to normal condition. Under moderate and severe drought stress, foliar application of Se-, SiO 2 -and Se/SiO 2 -NPs increased chlorophyll fluorescence parameters, with Se/SiO 2 -NPs having better effects (Table 1). At all stress levels, spraying with Se/SiO 2 -NPs (100 mg L −1 ) increased the amounts of F 0 , F m , F v , and F m /F v by 0. 36 Leaf total soluble carbohydrates, proline, membrane stability index and relative water content. The amounts of leaf carbohydrate and proline increased with increasing drought stress ( Table 2). Under severe drought stress the amounts of carbohydrate and proline increased significantly (43 and 103%, respectively) compared with normal condition. At all drought stress levels, spraying of the plants with Se-, SiO 2 -and Se/SiO 2 -NPs increased the values of both parameters compared to spraying with water. Se/SiO 2 -NPs (100 mg L −1 ) had the most beneficial effects such that carbohydrate and proline increased in amounts by 91/84/79% and 20/62/49%, respectively, in sprayed plants.
The leaf MSI and RWC decreased with increasing drought stress ( Table 2). Severe drought stress significantly decreased MSI and RWC (65 and 66%, respectively) compared with control plants. At all drought stress levels, spraying of plants with Se-, SiO 2 -and Se/SiO 2 -NPs increased the values of these two parameters compared to spraying with water, with Se/SiO 2 -NPs (100 mg L −1 ) sprays having a better effect. At all stress levels, comparing  Table 1. Effect of foliar application of nanoparticles containing selenium and silica on contents of photosynthetic pigments and chlorophyll fluorescence in strawberry plants under drought stress conditions. Values represent the means ± standard errors of three independent replications (n = 3). Different letters within the same column indicate significant differences at P < 0.05 among the treatments, according to Duncan's multiple range test. Se/SiO 2 (1: 50 mg L −1 ); Se/SiO 2 (2: 100 mg L −1 ); Chlorophyll a, Chl a; Chlorophyll b, Chl b; Total chlorophyll, Total Chl; Carotenoids, CARs; Fresh weight, FW; Minimum fluorescence, F 0 ; Maximum fluorescence, F m ; Variable fluorescence, F v ; Maximum quantum efficiency of photosystem II, F v /F m .

Treatments
Chl a (mg g −1 FW) Chl b (mg g −1 FW) Total Chl (mg g −1 FW) CARs (mg g −1 FW) Growth, firmness and fruit yield parameters. The number of leaves and leaf area decreased with increasing drought stress and significant decreases in these parameters were noted under the most severe drought stress treatment (Table 3; Supplementary File S1). At all drought stress levels, spraying of plants with Se-, SiO 2 -and Se/SiO 2 -NPs increased the number and area of leaves compared with plants sprayed with only water. As with previously reported parameters, Se/SiO 2 -NPs (100 mg L −1 ) had the most beneficial effect. At all treatment levels, plants treated with Se/SiO 2 -NPs (100 mg L −1 ) had higher numbers leaves and more leaf area compared with those sprayed only with water by 15/51/96% and 17/41/110%, respectively. The numbers of inflorescences, flowers and fruit decreased with increases in drought stress (Table 3; Supplementary File S1). When compared with control plants, the amounts of decrease were 76/89/91%, respectively. At all drought stress levels, spraying of plants with Se-, SiO 2 -and Se/SiO 2 -NPs increased the amounts of all three parameters compared with plants sprayed with only water. Under the three conditions of normal and moderate and severe stress, comparing plants sprayed with Se/SiO 2 -NPs (100 mg L −1 ) and water revealed that the number of inflorescence, flower and fruit were increased by 160/169/233%, 54/80/272% and 104/89/500%, respectively.
Fruit size, fruit weight and yield also decreased with increasing levels of drought stress (Table 3). Under severe stress, the amount of decrease was 62/75/99% compared with normal condition. Under the three treatment conditions, spraying with Se/SiO 2 -NPs increased the amounts of all three parameters. Comparing control and Se/ SiO 2 -NPs (100 mg L −1 ) treated plants revealed spraying increased fruit size, fruit weight, and yield 186/115/168%, 23/48/190% and 155/190/426%, respectively.
Fruit firmness decreased with the increase in drought stress compared to fruit from control plants (Table 3). Severe stress decreased fruit firmness by 58% compared with control plants. At all drought stress levels, spraying with a Se/SiO 2 -NPs solution increased fruit firmness compared with water sprayed plants. Se/SiO 2 -NPs (100 mg L −1 ) spraying resulted in 48/32/50% increase at all three stress levels.
Fruit anthocyanin, total phenolic compounds, ascorbic acid content and antioxidant activity. The contents of anthocyanin and total phenolic compounds increased under severe stress compared to normal condition such that the values of these two parameters increased by 328 and 137%. Conversely, the contents of vitamin C and DPPH decreased under severe stress compared to normal condition such that the values of these parameters decreased by 62 and 48%, respectively (Table 4). Under control conditions, Se/ SiO 2 -NPs (100 mg L −1 ) spraying increased the contents of anthocyanin, total phenol, vitamin C, and DPPH by Table 3. Effect of foliar application of nanoparticles containing selenium and silica on the leaf and fruit yield parameters and firmness in strawberry plants under drought stress conditions. Values represent the means ± standard errors of three independent replications (n = 3). Different letters within the same column indicate significant differences at P < 0.05 among the treatments, according to Duncan's multiple range test. Se/ SiO 2 (1: 50 mg L −1 ); Se/SiO 2 (2: 100 mg L −1 ). Multivariate analysis of NPs-treated strawberry plants under normal and drought stress conditions. All 3C). Also, carbohydrate, proline and oxidative enzymes accompanied Se/SiO 2 -NPs sprayed plants exposed to severe drought stress.
Pearson correlation analysis showed a positive correlation between yield and yield parameters with photosynthetic pigments and morphological traits. Also, a positive correlation was observed between fruit biochemical and morphological traits as well as photosynthetic pigments. On the other hand, F 0 , H 2 O 2 , MDA and oxidative enzymes had a negative correction with morphological traits, photosynthetic pigments, yield parameters and yield. Water use efficiency was positively correlated with yield and yield parameters. It also had a direct and positive correlation with osmolytes, photosynthetic pigments and Fv (Fig. 4A).
Cluster analysis and dendrogram investigations showed two main classes for strawberry fruits sprayed with Se-, SiO 2 -and Se/SiO 2 -NPs under drought stress (Fig. 4B). Clade I contained strawberry plants grown under moderate and severe stress conditions and also plants grown under similar conditions sprayed with Se-and SiO 2 -NPs as well as Se/SiO 2 -NPs. Clade II contained control plant and those sprayed with Se-, SiO 2 -and Se/ SiO 2 -NPs under normal conditions. Table 4. Effect of foliar application of nanoparticles containing selenium and silica on the anthocyanin, total phenolic compounds, ascorbic acid (Vit C) content and antioxidant activity in strawberry plants under drought stress condition. Values represent the means ± standard errors of three independent replications (n = 3). Different letters within the same column indicate significant differences at P < 0.05 among the treatments, according to Duncan's multiple range test. Se/SiO 2 (1: 50 mg L −1 ); Se/SiO 2 (2: 100 mg L −1 ); Total phenolic compounds, TPC; Ascorbic acid, Vit C; antioxidant activity, DPPH.

Discussion
Drought is a harmful environmental 2 issue and is one of the main factors responsible for decreases in growth and yield of agricultural crops around the world 25 . Drought stress decreases the yield and quality of fruits through changing the amounts and activities of photosynthetic pigments as well as osmolyte content and enzyme activity 2 .
Recently, nanotechnology has found its way into agricultural systems. Application of minerals as nanoparticles in different shapes and sizes has high potential in increasing performance 4 and modulating environmental stress 5 .
Although the positive effects of SiO 2 -NPs and Se-NPs spraying on agricultural crops has been well established, the combinational effect of Se/SiO 2 -NPs has not yet been investigated.
Fresh and dry weight of root and shoot. Morphological parameters, including fresh and dry weight of root and shoot, decreased under drought stress. Spraying of Se-, SiO 2 -and Se/SiO 2 -NPs alleviated the negative effects of this stress. Significant decreases of growth parameters under drought stress have been reported for different species such as lettuce 26 and barley 27 . Decreases of growth parameters may be due to the decrease of RWC and consequent shrinkage of cells, decrease of meristematic cell division, leaf growth reduction, leaf production blockage, senescence acceleration and leaf drop 28 . Also, water stress can directly affect the biochemical processes involved in photosynthesis and indirectly decrease the entrance of carbon dioxide into stomata, which close when subjected to drought. Therefore, photosynthetic material transfer is affected by drought and photosynthesis is limited which decreases the vegetative growth of plants 29 . The positive effects of Si 30 and Se 31 have been reported in drought tolerance and fresh and dry weight of different species such as maize (Zea mays). Growth stimulation due to Si and Se under different environmental stress conditions has been assigned to increased photosynthesis and antioxidative enzyme activities 22 . Probably, in the presence of these elements, assimilate production is increased and the activity of antioxidant enzymes such as CAT and SOD are also increased resulting in the modulation of negative effects of stress and increase of vegetative growth 32 .
Leaf photosynthetic pigments and chlorophyll fluorescence. The amount of photosynthetic pigments under drought stress conditions was significantly decreased compared to control plants, but spraying with Se-, SiO 2 -and especially Se/SiO 2 -NPs decreased these factors to a lower extent under drought stress. One reason for chlorophyll reduction during drought stress is that drought stress induces the production of active oxygen species which in turn destroys and decreases pigments. On the other hand, chlorophyll molecules www.nature.com/scientificreports/ decompose within chloroplasts and the thylakoid structure disappears 33,34 . Previous studies showed that Si and Se increase chlorophyll pigment content in different plants under stress and normal conditions 33,35 . It seems that these elements protect chloroplast structure against severe oxidative damage such as destruction of both grana and stroma lamellae, and increase the biosynthesis of photosynthetic pigments by protecting chloroplastic enzymes 33 . Probably, these elements act as cofactors in many enzymatic reactions involved in the biosynthetic pathways of the chloroplast 36,37 . Fluorescence chlorophyll parameters, except F 0 , decreased significantly under drought stress; however, spraying with solutions containing Se-, SiO 2 -and especially Se/SiO 2 -NPs decreased them at a lower rate. To evaluate the effect of stress and estimate average quantum efficiency of photosystem II (QII), chlorophyll fluorescence parameters have been widely used. It seems that chlorophyll fluorescence indicates thylakoid membrane integrity and relative efficiency of electron transfer from photosystem II to photosystem I 38 . In fact, lower F 0 values mean better photosynthetic activity 39 45 and trifoliate orange rootstock 46 under drought stress have been previously reported. Under drought conditions and due to a decrease of available water content, photosynthesis decreased, consequently decreasing the production of dry matter and assimilates 47 . Also, proline acted as a nitrogen and carbon source in plants under drought stress and the tolerance of plants against stress was increased. Under drought conditions, proline plays essential roles in protecting osmotic potential, scavenging of free radical and ROS, protecting molecules against denaturation, and adjusting cell pH. These events depend on plant species, stress duration, growth stage and stress intensity 47 . The positive effect of Se 48 and Si 49 in adjusting the amounts of osmolytes for modulating harmful effects of stress have been reported. It seems that these inorganic elements increase the production of soluble sugars through enhancing photosynthesis, which is probably effective as osmolytes in preserving water equilibrium, in addition to general growth stimulation.
MSI and RWC decreased significantly through the increase in drought stress, but spraying solutions containing Se-, SiO 2 -and especially Se/SiO 2 -NPs, increased their contents. Membranes are the first place in the cell which is influenced under stress conditions and the ability of plants to protect the integrity of membranes under drought stress determines the tolerance of plant to drought stress. Under drought stress, the water potential of soil decreases and plants prevent transpiration phenomenon using different mechanisms such as closing stomata, increasing stomatal resistance, and decreasing stomatal conductivity 50 . It was observed that Se/SiO 2 -NPs spraying increased both RWC and MSI in strawberry plants. It seemed that the consumption of Se and Si increased the amount of antioxidants and decreased free radical activities in plants which in turn increased cell membrane stability and improved water potential under normal and stress conditions 51 . Research has shown that Si plays a key role in protecting RWC under stress conditions 52 . Mateos-Naranhi et al. showed that the improving effect of Si on hydration status of plants could help decrease transpiration or phytolith deposition under epidermis cells of leaves and stems, which decreased waste of water from cuticle layers 53 . It seems, a well-ticked layer of silicon dioxide should help postpone water loss; thereby, it can increase WUE 54 .
While severe drought induced oxidative stress and increased the levels of H 2 O 2 and MDA, spraying with Se-, SiO 2 -and especially Se/SiO 2 -NPs decreased the harmful effects of stress. Also, drought stress increased the activity of antioxidant enzymes including CAT, APX, GPX and SOD in the leaves of strawberry plants and spraying of the nanoparticles increased the activity of these enzymes. Research has shown that severe drought induces oxidative stress due to the accumulation of ROS, which in turn induces free radicals that could not be controlled in damaging cell components and, eventually leading to cell death 55 . Increase of MDA, H 2 O 2 and other antioxidant enzymes have been reported for different plant species under drought stress 55 . Some elements such as Si and Se can act as free radical scavengers, affecting ROS elimination and also act as an antioxidant, improving the activities of oxidative enzymes resulting in increased antioxidative capacity in plants 51 . Many studies have shown that the protective role of Se and Si against oxidative stress in plants corresponded with the increase of oxidant enzyme activity and decrease of lipid peroxidation 22 . Increase in the activity of antioxidant enzymes such as CAT and APX as well as scavenging of H 2 O 2 were also observed by this enzyme after Se and Si sprays 46 . Se increased plant growth, probably through increasing starch content in chloroplast, and due to its antioxidative properties, it protected cell membranes against lipid peroxidation 56 . Silicon also prevented access of proteases to internal membrane proteins and destruction and disturbance of cell membranes. Our findings further support those obtained by Jiang et al. and Tang et al. which reported that the application of exogenous Se and Si caused the expression of genes related to antioxidant defense and consequently increased the activities of SOD, CAT, and APX in maize and ramie plants ultimately increasing their drought tolerance 31,51 . These findings demonstrated that Se-and Si-mediated antioxidative defense enhancement was one of the important mechanisms protecting plants against oxidative stress due to drought. www.nature.com/scientificreports/ Growth, firmness and fruit yield parameters. Leaf number and leaf area in strawberry plants under drought stress were significantly decreased but spraying of nanoparticles reduced these negative effects. Decrease of leaf number and leaf area under drought stress has been reported in many species 57 . It is important to note these decreases result in reduced yield through a reduction in photosynthesis. Limitation of leaf area can be considered the first line of defense in dealing with drought; therefore, reduction of water potential during drought decreased water content of plant tissues which resulted in the decrease of leaf area 58 . The effect of water stress on cell development is more obvious because cell enlargement takes place after turgor pressure due to water absorption; therefore, any decrease in water content blocks growth 59 . Increase of cell number and area by Si 60 and Se 61 in some plant species has been reported. Hossain et al. 60 reported that the increase in leaf size was not due to cell number increase but due to increased cell dimensions which indicated the effect of Si on cell elongation. It was found that Si and Se caused water loss from epidermis through deposition in epidermis cells of leaf and therefore preserved and maintained water in cells and increased turgor pressure resulting in the increase of green area of plants 62 . The yield and yield parameters of strawberry plants decreased under drought stress although exogenous application of Se-, SiO 2 -and especially Se/SiO 2 -NPs modulated the negative effects of drought. Yield decreases in different species such as tomato 63 and olive 2 under drought stress has been reported. The decrease of yield under drought stress could be due to many reasons including decrease of photosynthesis efficiency, leaf area, assimilate production, and decrease of water and mineral absorption by the root which ultimately decrease developmental and vegetative growth 2 . Exogenous application of Si and Se modulated the unfavorable effects of environmental stresses on the yield of different species 4 . Research has shown that Se played a critical role in different plants, ultimately affecting plant yield, factors such as starch accumulation in chloroplast, resistance enhancement to oxidative stress, delaying of senescence, and water status adjustment under stress conditions and increase of antioxidative capacity 22 .
Drought stress decreased the firmness of strawberry fruits but spraying of nanoparticles increased this factor in plants under drought stress and normal conditions. This response could be due to the induction of ethylene production in fruits under drought stress 64 . Due to adjusting the expression of genes and enzymes involved in reactions related to cell walls, ethylene affects fruit firmness. Under the action of ethylene, the activity of polygalacturonase was increased and then decreasing fruit firmness. The positive effects of Si and Se on increasing fruit firmness has been reported for different species 65,66 . It seemed that the mechanical strength of plant tissues due to Si was generally because of amorphous solid Si deposition on cell wall layers 67 . On the other hand, the effect of Se on fruit firmness could be assigned to the prevention of different oxidative reactions in different fruits 68 . Fruit anthocyanin, total phenolic compounds, ascorbic acid content and antioxidant activity. The biochemical parameters of the fruit were severely affected by drought. Spraying Se-, SiO 2 -and especially Se/SiO 2 -NPs increased the contents of all three above mentioned biochemical parameters. Increase of anthocyanin and total phenolic contents under drought stress were observed in some plant species 69 . Anthocyanins are a group of phenolic compounds which comprise a large group of secondary metabolites and have antioxidant properties 70 . Polyphenol compounds are among antioxidant compounds which play their antioxidative roles through different mechanisms such as scavenging of free radicals, blocking oxidative reactions, hydrogen reduction, oxygen blocking reaction, metal ion chelating and acting as peroxidase enzyme substrate 71 . Increase of polyphenolic compounds under stress conditions is related to the genetic structure and growth environment of plants. Vitamin C is an organic acid; under drought stress, respiration is increased and therefore, these acids act as substrate in respiration phenomenon. This results in the decrease of acidity and consequently, decrease of vitamin C due to drought 72 . Increase in the amounts of biochemical parameters (anthocyanin, total phenolics, vitamin C and antioxidant activity) in fruits of different species by the addition of Si and Se has been reported 4 . Inorganic elements such as Si and Se play critical roles in determination of organoleptic properties and antioxidative capacity of fruits through adjusting the biosynthetic phenylpropanoid pathway which results in metabolite accumulation 73 . Antioxidants play protective roles against oxidative stress in plants, with free -OH groups attached to the aromatic ring reducing oxidative damage by scavenging ROS and chelating metals. Generally, increases of secondary metabolites contributed to the improvement of cell responses to oxidative stress in addition to antioxidant activity of fruits which improved their quality 74 .  www.nature.com/scientificreports/ In conclusion, spraying solutions containing Se-, SiO 2 -and especially Se/SiO 2 -NPs at 100 mg L −1 on strawberry plants is an efficient way to improve their drought tolerance and yield. Favorable effects of Se/SiO 2 -NPs on growth efficiency and yield parameters at different drought levels have been attributed to (1) protection of pigments to increase photosynthetic capacity, (2) accumulation of assimilates to protect osmosis, (3) activation of an antioxidative system to eliminate ROS, (4) enhancing water use efficiency level for improvement of root biomass and maintenance of proper osmotic status of the cells and (5) accumulation of fruit biochemical compounds (total phenolics, anthocyanin contents and antioxidant capacity) to increase fruit quality (Fig. 5). Since spraying of nanoparticles is easily performed, Se/SiO 2 -NPs sprays are advised for managing drought stress in strawberry and even other agricultural plants. However, many new studies are required for identification of the efficiency of Se/SiO 2 -NPs before practical programs can be realized on a large scale.

Materials and methods
Chemicals and reagents. The silicon dioxide (SiO 2 -NPs) and selenium (Se-NPs) nanoparticle used in this study were obtained from the NANOSANY Corporation (Mashhad, Iran) (Fig. 6A,B) Study site and treatments. The experiment was carried out from March 7, 2018 to June 21, 2018 in a greenhouse in Maragheh, Iran (37° 30′ N; 46° 12′ E, at an altitude of 1477.7 m; moderate to cold and relatively humid). The greenhouse photoperiod of 14/10 h (light/dark), temperature of 25/20 ± 5 °C (day/night) and air relative humidity of 80 ± 5% were maintained throughout the experiment. 'Gaviota' strawberry rooted plants (Fragaria × ananassa Duch.) were obtained from a strawberry nursery, Maragheh, Iran. These plants were planted in 7-L volume plastic pots, filled with about 525 g soil mixture (1:1:2 ratio of sand: animal manure: topsoil, respectively). Pots were irrigated by graduated cylinder daily to 100% field capacity (FC) for 15 days in the growing season (March 7th till March 22th). Then, three levels of watering were imposed that included normal irrigation (100% FC), moderate stress (60% FC), and severe stress (25% FC). The FC was determined by the gravimetric method following the methodology described by Souza et al. 75 . Preservation of the water treatments was made by daily weighing of the pots replacing the water lost by transpiration using a precision scale. These regimes were applied for 92 days in the growing season (March 22th till June 21th) until harvest. Plants (5 fully expanded leaves) were subjected to non-drought (control) and different levels of drought (moderate and severe). At day 35 after the start of the drought treatment plants did show stress symptoms. On that day, the upper leaf surfaces of control and drought-treated strawberry plants were sprayed until full wetting (ca. 25 mL plant −1 ) with solutions containing 0 (NPs-untreated control), Se-NPs (25 mg L −1 ), SiO 2 -NPs (125 mg L −1 ) and Se/SiO 2 -NPs (50 and 100 mg L −1 ) once a week until harvest (3 sprays in total). Foliar application was done before the flowering stage and at sunset. Before each exposure, solutions were prepared with NPs dispersed in ultrapure water and homogenized 20 min with an ultrasonic bath. Plants from each treatment were harvested for evaluating their morpho-physiological responses.

Leaf photosynthetic pigments and chlorophyll fluorescence. Chlorophyll a (Chl a), Chl b, total
Chl and carotenoids (CARs) were measured in leaf samples according to the method of Arnon (1949) using a spectrophotometer (Shimadzu, Model UV 1800, Kyoto, Japan) at 470, 663 and 645 nm, respectively 76 .
Chlorophyll fluorescence parameters were assessed using a portable photosynthesis meter (Walz GmbH Eichenring, 691090 Effeltrich, Germany) at the end of treatments. Minimal fluorescence, F 0 , was measured in 30 min dark-adapted leaves and maximal fluorescence, F m , in the same leaves in full light-adapted conditions. Maximal variable fluorescence (F v ) and the photochemical efficiency of PSII (F v /F m ) for dark adapted leaves were also calculated from the measured parameters 77 .
Leaf total soluble carbohydrates, relative water content, membrane stability index and proline. To measure soluble carbohydrates, 100 mg FW (fresh weight; FW) of leaf tissue was homogenized with 10 mL of 95% ethanol, and extracts were centrifuged at 6000×g for 15 min. Then, the upper phase of the centrifuged samples was supplemented with 3 mL of anthrone and maintained at 100 °C for 10 min in boiling water. Then, the absorbance at 630 nm was read using a spectrophotometer (Shimadzu, Model UV 1800, Kyoto,  www.nature.com/scientificreports/ Japan) 78 . Total soluble carbohydrates were determined using a glucose standard, and expressed as mg g −1 FW. Relative water content (RWC) was measured as described by Barrs and Weatherley 79 . The membrane stability index (MSI) was determined according to methods established by Sairam et al. 80 . Proline was measured spectrophotometrically by the method of Bates et al. 81 .   Leaf catalase, ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase. To determine catalase (CAT; EC 1.11.1.6) activity in leaf extracts, 30 µl of extract were added to 50 mM K phosphate buffer (pH 7.0) and 2% H 2 O 2 for a total volume of 3 mL. Enzyme activity was recorded at 240 nm for 2 min with a spectrophotometer (Shimadzu, Model UV 1800, Kyoto, Japan) 84 . Ascorbate peroxidase (APX; EC 1.11.1.11) activity was assayed according to the methods of Nakano and Asada 85  Growth, firmness and fruit yield parameters. The plant growth parameters were investigated at the end of experiment by recording the number of leaves and leaf area (using Image J Software). Also, the number of inflorescences, flowers and fruits as well as fruit size was recorded. Fruit yield was determined in grams per plant. Fruit weight was also measured by digital balance with accuracy of 0.1 g. Firmness was determined on the equatorial region of fruit, using texture analyzer (STEP Systems GmbH, Nuremberg, Germany; with an 8 mm probe).
Fruit anthocyanin, total phenolic compounds, ascorbic acid content and antioxidant activity (DPPH). Tissue samples were obtained from various fruit parts and frozen in liquid nitrogen. Then, 5 g tissue was homogenized in 10 mL 50 mmol L -1 phosphate buffer at pH 7.8. The homogenate was centrifuged at 15,000×g at 4 °C for 20 min and the supernatant (fruit extract) was collected to measure fruit quality parameters: Total anthocyanins were estimated by the pH differential method using two buffer systems: 25 mM KCl buffer (pH 1.0) and 0.4 M Na acetate buffer (pH 4.5). Samples were diluted with KCl buffer until A510 was within a linear range of the spectrophotometer. The same dilution factor was later used to dilute the sample with Na acetate buffer. Readings were performed after incubating for 15 min at 510 and 700 nm in the two buffers, five replications per sample, and total anthocyanin content was calculated as indicated by Giusti and Wrolstad 88 : where A = (A510 -A700); MW = molecular weight; DF = dilution factor; MA = molar absorptive coefficient of cyanidin-3-glucoside (C 3 G). Results were expressed as mg C 3 G 100 g -1 of juice.
For phenolic compound analysis, 100 μL of fruit extract was mixed with 400 μL phosphate buffer and 2.5 mL of Folin reagent (Sigma-Aldrich). After 1 min, 2 mL of Na 2 CO 3 (7.5%) was added to the mixture and the sample kept at 50 °C for 5 min, before measuring the absorbance at 760 nm with a spectrophotometer (Shimadzu, Model UV 1800, Kyoto, Japan). Gallic acid was used as a standard, and results were expressed as mg of gallic acid per 100 g FW 89 .
The ascorbic acid (Vit C) concentration in fruit extracts was determined by titration using a solution containing I and KI (16 g KI and 1.72 g I in 1 L water). The titration ended when the sample turned dark blue and color was stable. The volume of the I + KI solution was recorded and the concentration of ascorbic acid calculated according to the following equation as ([0.88 × V]/5 × 100), where V is the volume of the consumed I + KI solution 90 .