The combined effect of water deficit stress and TiO2 nanoparticles on cell membrane and antioxidant enzymes in Helianthus annuus L.

Nanotechnology has become one of the several approaches attempting to ameliorate the severe effect of drought on plant's production and to increase the plants tolerance against water deficit for the water economy. In this research, the effect of foliar application of TiO2, nanoparticles or ordinary TiO2, on Helianthus annuus subjected to different levels of water deficit was studied. Cell membrane injury increased by increasing the level of water deficit and TiO2 concentration, and both types of TiO2 affected the leaves in analogous manner. Ord-TiO2 increased H2O2 generation by 67–240% and lipid peroxidation by 4–67% in leaves. These increases were more than that induced by Nano-TiO2 and the effect was concentration dependent. Proline significantly increased in leaves by water deficit stress, reaching at 25% field capacity (FC) to more than fivefold compared to that in plants grown on full FC. Spraying plants with water significantly decreased the activities of enzymes in the water deficit stressed roots. The water deficit stress exerted the highest magnitude of effect on the changes of cell membrane injury, MDA, proline content, and activities of CAT and GPX. Nano-TiO2 was having the highest effect on contents of H2O2 and GPX activity. In roots, the level of water deficit causes highest effect on enzyme activities, but TiO2 influenced more on the changes of MDA and H2O2 contents. GPX activity increased by 283% in leaves of plants treated with 50 and 150 ppm Nano-TiO2, while increased by 170% in those treated with Ord-TiO2, but APX and CAT activities increased by 17–197%, in average, with Ord-TiO2. This study concluded that Nano-TiO2 didn’t ameliorate the effects of drought stress on H. annuus but additively increased the stress, so its use in nano-phytotechnology mustn’t be expanded without extensive studies. Supplementary Information The online version contains supplementary material available at 10.1007/s12298-022-01153-z.


Introduction
The response of plants to water deficit stress is complex and involves changes in their macro-and micro-morphology and all physiological processes. Water deficit stress, as most abiotic stresses, leads to accumulation of reactive oxygen species (ROS), generated mostly in chloroplast and mitochondria, causing oxidative stress. Amongst the defense responses against abiotic stresses, plants tend to activate their ROS scavenging mechanism (Vranová et al. 2002). Therefore, plants intrinsically develop different types of antioxidants reducing oxidative damage and conferring stress tolerance. From these types are the antioxidant enzymes which include superoxide dismutase, peroxidase, and catalase (Siddiqui and Khan 2018;Khan et al. 2020). Water deficit stress causes oxidative damage in plants which results in the disruption of photosynthetic apparatus, results in decreased chlorophyll content (Shivakrishna et al. 2018).
Titanium has significant biological effect on plants, being beneficial at low levels but toxic at higher concentrations. Variations in titanium's effect on plants will depend on the differences in environmental factors, diversity of plant species and the applied concentrations of titanium (Feizi et al. 2012;Tan et al. 2018;Wahid et al. 2020). Similar to other nanomaterials, the properties and concentration of Nano-TiO 2 have important roles in its application. On the contrary, several reports have presented the harmful effects of a high concentration of TiO 2 on plants that varied between plant tissues, growth stages and plant species (Nair et al. 2010;Feizi et al. 2012;Zulfiqar and Ashraf 2021). Therefore, properties, concentration, method of application, uptake by plants, reactivity and translocation of nanoparticles into different plant tissues could determine their interference with various physiological activities that lead to toxic impacts (Mattiello et al. 2015;Rastogi et al. 2017;Paramo et al. 2020). Furthermore, surface area of nanoparticles, their reactivity and tendency for aggregation are additional possible reasons for their harmful effects (Kobayashi et al. 2019). High concentrations of Nano-TiO 2 mainly elevate generation of ROS, followed by chlorophyll degradation and cellular toxicity (Rico et al. 2015). In addition, damage of the cell walls and plasma membranes by high nanoparticles concentration result in an interaction with various cellular process (Rastogi et al. 2017;Hou et al. 2019). Sompornpailin et al. (2020) showed that application of titanium nanoparticles increased the cell membrane stability, photosynthetic pigments, and antioxidant activities in Nicotiana tabacum. Khan et al. (2020) also showed that Nano-TiO 2 increased the production of enzyme and non-enzyme antioxidants in Vicia faba subjected to a shortage in water supply. Hong et al. (2005) and Liu et al. (2021) reported an increase in the activities of SOD, CAT, POD and a decrease in accumulation of ROS when plants were exposed to TiO 2 nanoparticles. Mohammadi et al. (2016) concluded that foliar treatment of dragonhead plant with high concentrations of Nano-TiO 2 increased injurious effects of water deficit stress on physiological processes due to increasing the levels of lipid peroxidation and H 2 O 2 generation, and decreasing the stability of photosynthetic pigments. Therefore, extensive use of Nano-TiO 2 in order to diminish adverse effects of water deficit stress even in the case of economic crops need further investigation. Besides, the influence of Nano-TiO 2 residues on the environment and human health should also be considered and elucidated. Servin et al. (2013) reported that CAT activity increased in cucumber plants grown in soil amended with 250-750 mg Nano-TiO 2 kg -1 but the activity of APX decreased at 500 mg kg -1 . Also, Laware and Raskar (2014) reported that CAT and GPX activities were enhanced in the presence of 10-30 lg ml -1 , but their activities decreased by higher concentrations of Nano-TiO 2 . Lei et al. (2008) showed that Nano-TiO 2 treatment activated SOD, CAT, APX and GPX of spinach chloroplasts. According to Harrison (1996), the exogenous application of Nano-TiO 2 increased CAT activity and plant resistance to environmental stresses. The APX is thought to play the most essential role in scavenging ROS and protecting cells in higher plants, algae, euglena and other organisms. APX is involved in scavenging H 2 O 2 in waterwater and ASH-GSH cycles and utilizes ASH as the electron donor Kohli et al. 2019).
Recently, nanotechnology has been a subject of great interest in various fields including botany. Titanium dioxide (Nano-TiO 2 ) is among the most highly manufactured and widely used nanoparticles in the world (Jomini et al. 2015). Due to the extensive use and applications, these nanomaterials have already entered the agroecosystems leading to exposure of plants to Nano-TiO 2 . As titanium enter the food chains, the human exposure to these nanomaterials will be increased. Therefore, evaluation of the toxic effects induced by Nano-TiO 2 and its effects on the human health was reviewed by some authors (Iavicoli et al. 2012;Shah et al. 2017). Though, this is not the objective of this study, it is worthwhile to mention that the International Agency for Research on Cancer (IARC) classified TiO 2 in 2006 and reassessed it in 2010 as ''possibly carcinogenic to humans''. In this study, we addressed four questions. First, does the foliar application of Nano-TiO 2 exert the same effect as Ord-TiO 2 on sunflower plants? Second, how increasing concentrations of TiO 2 affect the cell membranes and some enzyme activity? Third, can Nano-TiO 2 ameliorate the effect of drought to some extent? Fourth, does Nano-TiO 2 have a combined effect with drought as abiotic stressor on plants? As the TiO 2 is applied by foliar spraying, whether or not the root will be affected similarly as the shoot under different levels of water availability was another objective for this study.

Experimental design and treatments
This experiment was carried out to study the effect of different concentrations of nanoparticles or ordinary titanium dioxide (Nano-TiO 2 or Ord-TiO 2 ) on cell membrane integrity and some antioxidant enzymes of Helianthus annuus L. (sunflower) plants subjected to different levels of water availability. Seeds of sunflower (cultivar Giza 102) were obtained from the Agronomy Department, Faculty of Agriculture, Assiut University, Assiut, Egypt. The experiment started on 26-September 2019 at the Green house of the Botany Department, Assiut University under field conditions. Through September, October and November, the averages of maximum temperature was 32 ± 2, minimum temperature was 18 ± 1.7, and relative humidity was 31% ± 1. About 15 seeds were planted in pots containing 3 kg of soil (2:1 clay: sand by weight). The pots (120 pots) were irrigated by tap water to the field capacity which was determined to be about 24% of dry soil. After about one month from cultivation, the number of individuals was fixed to 7 homogeneous plants per pot. To avoid nutrient impoverishment, each pot received 50 ml one tenth strength of Hoagland's solution, 10 ml with irrigation water day after day.
A factorial experiment based on randomized complete block design was carried out with three replications. The 120 pots were divided into 10 groups, three pots as replicates of four water levels (100%, 75%, 50% and 25% of field capacity), the welting point of sunflower was 7%. The first group was the control, the second group was foliar sprayed with distilled water and the other 8 groups were sprayed with 50, 150, 300 and 600 ppm Nano-TiO 2 or Ord-TiO 2 . The foliar spraying was applied after the plants established under different water levels for 10 days, where the plants were with about three pairs of leaves. Each pot was sprayed twice at 5 days interval with a total of 15 ml spraying solution. After 25 days from the first spraying (the plants were with 6 pairs of leaves), the plants were harvested for different measurements.
The Nano-TiO 2 with particle size less than 25 nm (Fig. S1), purity 99.7% and surface area of 45-55 m 2 g -1 was purchased from Sigma-Aldrich Company. Nano-TiO 2 and Ord-TiO 2 particles, separately, were dissolved in distilled water just before using and scattered by ultrasonic vibration ''BANDELIN SONOPULS HD 2070'' homogenizer at 100 W and 40 kHz for 10 min.

Membrane stability index and electrolyte leakage:
Cell membrane stability was carried out as given by Premachandra et al. (1992). Fresh leaf discs (10 discs) were rinsed three times with double distilled water, then submerged in 25 ml double distilled water for 24 h at room temperature. The electric conductivity of the bathing solution was measured using conductometer (YSI Model 35 Yellow Springs, OH, USA). Then samples were autoclaved and the EC measured again after cooling to the room temperature. Cell membrane stability index, or membrane injury, was evaluated as percentage injury according to the following equation: In addition, the electrolyte leakage was calculated relative to that of control plants as following: where T 1 and T 2 are EC values of the treated plants, while C 1 and C 2 represent the EC values of control plants before and after autoclaving, respectively.

Determination of lipid peroxidation (malondialdehyde content)
The level of lipid peroxidation in sunflower leaves was determined as malondialdehyde (MDA) content according to the method of Hodges et al. (1999) with minor modifications. Content of MDA, which is an end product of lipid peroxidation, was determined using the thiobarbituric acid reaction. 0.2 g fresh leaves was homogenized with 4 ml ethanol (80%v/v) containing 2% dimethyl sulfoxide, and the homogenate was centrifuged at 10,000 rpm for 10 min. For every one ml aliquot, 3 ml of 20% TCA containing 0.65% thiobarbituric acid (TBA) was added. The mixture was heated at 95°C for 30 min, then cooled quickly on an ice-bath. After that, the mixture was centrifuged at 6000 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm using UV2000/2200 Spectrophotometer (Ray Wild Limited Compony, Germany). Blanks with the same weight from the same leaf or opposite one were proceeded as the samples but without TBA. After subtracting blank reading, the level of lipid peroxidation was expressed as nmol g -1 FW of MDA formed using an extinction coefficient of 155 mM -1 cm -1 .

Determination of H 2 O 2
The hydrogen peroxide content in the leaves or roots of sunflower was spectrophotometrically measured as described by Sellers (1980) with minor modifications. A defined weight (0.1 g) of the tissue was immediately extracted with 2 ml cold acetone followed by 3 ml double distilled water. After centrifugation at 6000 rpm for 10 min, 3 ml from the supernatant was mixed with 1 ml of 0.1 M potassium titanium (IV) oxalate dihydrate [K 2 TiO(C 2 O 4 ) 2 .Á2H 2 O] in 5 M sulphuric acid. The intensity of yellow-orange color was measured at 400 nm. A blank was prepared by boilling the same weight of leaves or roots for one minute and proceeded as the fresh sample. The molar absorptivity 935 L mol -1 cm -1 was used in calculation of H 2 O 2 as lmole g -1 FW.

Determination of proline
Proline concentration was determined using the acid-ninhydrin method according to Bates et al. (1973). Exactly, 0.2 g fresh leaves were homogenized in 3 ml of 3% sulfosalicylic acid, then the homogenate was centrifuged for 10 min. at 10,000 rpm. One ml of the mixture was reacted with 400 ll acid-ninhydrin and 400 ll of glacial acetic acid and 200 ll of 3% sulfosalicylic acid in a test tube for one hour at 96°C, and the reaction was terminated in an ice bath. The reaction mixture was extracted with 4 ml toluene and mixed vigorously. The chromophore containing toluene was aspirated from the aqueous phase, warmed to room temperature and the absorbance at 520 nm was measured using toluene as a blank. The proline concentration was determined from a standard curve and calculated as mg g -1 FW.

Preparation of enzyme extract
Root or leaf tissues (0.5 g) were ground to a fine powder in liquid N 2 , and then homogenized in 5 ml of 100 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM ethylenediamine tetraacetic acid-disodium salt (Na 2 -EDTA) and 0.1 g polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 18,000 rpm for 10 min at 4°C and the supernatants collected and used for the assay of catalase, guaiacol peroxidase and ascorbate peroxidase. All colorimetric measurements were made at 20°C using spectrophotometer. The specific activity was expressed as units mg -1 protein min -1 . Catalase (CAT; EC 1.11.1.6) activity was determined by measuring the rate of H 2 O 2 dissociation to O 2 and water for one minute according to modified method of Aebi (1984). The assay medium (3 ml) consisted 2.8 ml 50 mM potassium phosphate buffer (pH 7), 100 ll of enzyme extract and the reaction was initiated by addition of 100 ll 10 mM H 2 O 2 . The decrease in absorbance at 240 nm was recorded for one minute.
Ascorbate peroxidase (APX; EC 1.11.1.11) activity was measured according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm. The reaction mixture contained 1.6 ml of 50 mM potassium phosphate buffer (pH = 7), 0.1 mM Na 2 -EDTA, 5 mM H 2 O 2 , 0.5 mM ascorbic acid and 50 ll enzyme extract. The decrease in absorbance at 290 nm was monitored to calculate the activity of APX.

Statistical analysis
Data were subjected to statistical analysis using SPSS (version 21). One-way ANOVA was performed following when the effect was significant at P B 0.05, by the post hoc Duncan's multiple-range test (at P B 0.05) for comparison between means of parameters at each level of water availability or at each concentration of the spraying solution. Two-way ANOVA was carried to achieve the effect of water availability, Nano-or Ord-TiO 2 and their interaction on different parameters estimated in leaves or roots and eta square ''g 2 '' was calculated as: g 2 = SS Effect / SS Total to achieve the size effects of each factor or the interaction between factors. In addition, Pearson correlation analysis was performed to obtain the relation between some parameters.

Results
Effect of nano-and ord-TiO 2 on the cell membranes of sunflower Electrolyte leakage and cell membrane injury of sunflower leaves (Fig. 1a, b) were matching, following the same trend, and both were decreased by spraying plants with water, then increased gradually and mostly significantly by either Nano-or Ord-TiO 2 or by decreasing the levels of water availability. Electrolyte leakage increased significantly in plants sprayed with Nano-or Ord-TiO 2 , and the leakage was more pronounced with Nano-TiO 2 treatments (Fig. 1a). The damage by spraying plants with 600 ppm Nano-TiO 2 was magnified by the shortage in water supply reaching to 2-, 4-and sixfold at 75%, 50% and 25% FC, respectively, compared to that at full FC. The cell membranes were significantly injured by lowering water availability to 50% and 25% FC. In plants grown on 25% FC and sprayed with 150 ppm Nano-TiO 2 , the membrane injury was magnified to 11-fold of that at full FC. In comparison, the membrane damage was generally lower by Ord-TiO 2 treatments, especially at the lowest level of water availability (Fig. 1B). The highest recorded membrane damage or electrolyte leakage by spraying plants grown on 25% FC with 600 ppm Nano-TiO 2 did not exceed significantly compared to those of 150 and 300 ppm Nano-TiO 2 at the same water level. Based on the two-way ANOVA, all concentrations of TiO 2 and all levels of FC significantly affected the electrolyte leakage and membrane injury of sunflower leaves. Only, 50 and 150 ppm Ord-TiO 2 were having similar effects (Fig. 1). The membrane injury of sunflower leaves was significantly correlated with the contents of H 2 O 2 either by spraying with Nano-TiO 2 (rvalue = 0.891**) or with Ord-TiO 2 (r-value = 0.844**).

Effect on H 2 O 2 generation, lipid peroxidation and proline accumulation
Data illustrated in Fig. 2a showed that leaf H 2 O 2 increased continuously with decreasing water availability at all treatments of TiO 2 . In leaves of plants grown on full FC, increasing concentration of the spraying solution (Nano-or Ord-TiO 2 ) did not induce significant differences in H 2 O 2 generation, except at 600 ppm Nano-TiO 2 . At 25% FC, H 2 O 2 content increased significantly in leaves of plants treated with Nano-TiO 2 up to 300 ppm, then decreased by spraying plants with 600 ppm Nano-TiO 2 . The maximum H 2 O 2 content was observed in roots of most plants treated with 50 and 150 ppm Nano-TiO 2 . In contrast, H 2 O 2 in the leaves increased by increasing the concentration of Ord-TiO 2 . At full FC, H 2 O 2 content did not change significantly in plants treated with Ord-TiO 2 . Contents of H 2 O 2 in sunflower leaves sprayed with Ord-TiO 2 were generally more compared to those sprayed with Nano-TiO 2 . At the lower level of water availability (25% FC), H 2 O 2 in plants treated with 300 and 600 ppm Ord-TiO 2 increased to 2.5-3.5-fold compared to that in the control ( Fig. 2A).
Electrolyte leakage (% of control) Compared to leaves, H 2 O 2 in sunflower roots showed different trend toward decreasing by 300 and 600 ppm Nano-TiO 2 , and this was opposite for the same treatments of Ord-TiO 2 (Fig. 2b). Roots H 2 O 2 significantly increased up to 150 ppm Nano-TiO 2 then reduced gradually till 600 ppm. An opposite trend was achieved with Ord-TiO 2 , i.e., the lowest H 2 O 2 content was detected at 50 and 150 ppm of Ord-TiO 2 , while the highest content was observed at 600 ppm for all levels of water availability. Compared to control, H 2 O 2 in roots of plants sprayed with 600 ppm Nano-TiO 2 was reduced by 40-80%, but was increased to 130-150% by 600 ppm Ord-TiO 2 . The results of two-way ANOVA revealed to significant differences between the levels of each main factor. In roots, change in the content of H 2 O 2 by increasing the water deficit stress was depending on the concentration of TiO 2 . Generally, foliar spraying of sunflower plants with Ord-TiO 2 increased H 2 O 2 generation (Fig. 2a) and concomitantly the lipid peroxidation in its leaves (Fig. 3a) more than Nano-TiO 2 . Also, the level of H 2 O 2 or lipid peroxidation increased by the concentration of TiO 2 (Nano or Ord) treatment. The maximum levels of H 2 O 2 and lipid peroxidation were shown in plants treated with 600 ppm Ord-TiO 2 . The minimum content of MDA was detected in plants treated with 50 ppm Nano-TiO 2 , while the maximum content was observed in plants treated with 600 ppm Ord-TiO 2 . However, the MDA content in sunflower leaves were significantly correlated with the contents of H 2 O 2 either by spraying with Nano-TiO 2 (r-value = 0.726**) or with Ord-TiO 2 (r-value = 0.915**), but the correlation was negative in the roots with r-values = -0.578* and -0.408, respectively.
Lipid peroxidation of leaf cells was generally increased proportionally and significantly with decreasing soil-water potential (i.e. from Full FC to 25% FC), especially by treatment with Ord-TiO 2 (Fig. 3a). This trend was different with high concentrations of Nano-TiO 2 , i.e., lipid peroxidation was unchanged from 300 to 600 ppm Nano-TiO 2 in plants grown on Full and 75% FC, while it was reduced by 600 ppm Nano-TiO 2 in plants grown on 25% FC. At all levels of water availability, spraying plants with 50 and 150 ppm Nano-TiO 2 did not induce any significant difference in MDA. In contrast, 300 and 600 ppm Ord-TiO 2 increased lipid peroxidation process to 130-170% compared to control, especially at 50% FC.
As in leaves, lipid peroxidation of the root cells of sunflower was the worst at 25% and 50% FC, compared to plants grown on 75% and full FC (Fig. 3B). At this shortage of water supply, lipid peroxidation increased by 2-3 fold compared to control. The levels of water availability 50% and above showed the peak of lipid peroxidation by 600 ppm Nano-TiO 2 . On the other hand, 600 ppm Ord-TiO 2 , compared to the other applied concentrations, significantly decreased the content of MDA at all levels of water availability. Comparing between groups by two-way ANOVA showed that there are non-significant differences between the effects of Nano-TiO 2 concentrations above 50 ppm on the lipid peroxidation in leaves, while there are significant differences between all treatments of Ord-TiO 2 (Fig. 3a). In roots, the effect of low concentrations of either Nano-or Ord-TiO 2 on lipid peroxidation was significantly different from that of high concentrations (Fig. 3b).
Changes of proline contents in leaves of sunflower plants responded differentially to TiO 2 concentrations and water availability (Fig. 4). The proline content in leaves of plants grown under shortage of water supply, whether sprayed with TiO 2 or not, increased significantly in comparison with plants grown under full FC. Proline dramatically increased in plants grown on 25% available water reaching more than fivefold compared to that in plants grown on full FC. On the other hand, concentrations of Nano-TiO 2 up to 150 ppm exerted weak changes in proline content that increased significantly at 300 and 600 ppm. In addition, plants sprayed with Nano-TiO 2 , especially 600 ppm, accumulated more proline compared to those sprayed with Ord-TiO 2 . In plants grown on 75% and 50% FC, despite the proline content was increased significantly by 600 ppm Nano-TiO 2 , it was decreased by Ord-TiO 2 . However, the adverse effect of water deficit stress on sunflower, as indicated by proline accumulation, is exacerbated by the highest concentration of TiO 2 where the combined effect of both main factors is higher compared to either factor alone.

Antioxidant enzymes
To evaluate if Nano-or Ord-TiO 2 have a role in the activity of antioxidant enzymes, catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) activities were assayed in leaves and roots of sunflower plants.
Results of CAT activity are illustrated in Fig. 5. Catalase activity significantly differed by water availability and TiO 2 material. The activity in leaves (Fig. 5a) gradually and significantly increased from full field capacity (Full FC) toward dryness (25% FC) under both types of TiO 2 treatments. This increasing activity by drought, especially at 25% FC, was relatively suppressed by high TiO 2 concentrations (300 and 600 ppm). The activity of CAT increased significantly in plants sprayed with 50 and 150 ppm Ord-TiO 2 compared to control or those sprayed with Nano-TiO 2 .
Generally, in roots (Fig. 5b), spraying plants with water significantly decreased the CAT activity and the other TiO 2 levels showed, with minor exceptions, slight or no changes compared to that of control. Notably there was unusual similarity in CAT activity at 150 ppm Ord-TiO 2 , i.e., there was no difference between the CAT activity among water availability gradients. The results in Fig. 5 showed that CAT activity is affected by changing water availability more than by changing the concentrations of spraying TiO 2 solutions. Also, at all levels of water supply, by spraying plants with water the CAT activity increased significantly in leaves but decreased in roots. As indicated by two-way ANOVA, the CAT (Fig. 5a) and GPX (Fig. 6a) activity in leaves increased significantly by all levels of both main factors. In roots, the activity increased significantly by 50% and 25% levels of FC, and by only the highest concentration of Nano-TiO 2 , but all concentrations of Ord-TiO 2 significantly decreased the CAT activity. The activity of GPX increased in sunflower leaves by increasing the concentration of Nano-or Ord-TiO 2 . In contrast to catalase, the GPX activity increased in plants treated with Nano-TiO 2 more than in plants treated with Ord-TiO 2 . The activity of GPX in sunflower leaves increased continuously and most significantly from control to 600 ppm of both types of TiO 2 treatments at all levels of water availability. Therefore, the highest activity was observed in plants treated with 300 and 600 ppm Nano-TiO 2 (Fig. 6). This increase, in leaves of plants grown on full FC, reached by nanoparticles treatment to about sevenfold, while it reached to about fourfold in plants grown on 50% and 25% FC. The same trend was shown for Ord-TiO 2 , but with less magnitude at 50% and 25% FC. The results in Fig. 6a refer to non-significant differences between the effect of 50 and 150 ppm or between 300 and 600 ppm Nano-TiO 2 . Mostly, at all treatments of TiO 2 , whether in leaves or roots, as the available water decreases the GPX activity increases.
Activity of GPX in roots followed approximately different trend from that of leaves (Fig. 6b). Generally, there was non-significant change in GPX activity by increasing TiO 2 concentration especially of Ord-TiO 2 . Except in plants grown on 50% and 25% FC and sprayed with 300 and 600 ppm Nano-TiO 2 , the activity of GPX was inhibited in sunflower roots compared to control. This reduction reached to 53% and 63% of control in plants grown on 50% FC and sprayed with 50 ppm Nano-TiO 2 and 150 ppm Ord-TiO 2 , respectively. Otherwise, unusual increase was observed in GPX activity by 600 ppm Nano-TiO 2 treatment in roots of plants grown on 50% and 25% FC.
Data illustrated in Fig. 7 showed that APX activity increased in sunflower leaves treated with Ord-TiO 2 more than in those treated with Nano-TiO 2 . In plants grown on full and 75% FC, APX activity did not change by Nano-TiO 2 treatments, but increased with Ord-TiO 2 . At all treatments, the activity of APX in leaves (Fig. 7a) or roots (Fig. 7b) increased by decreasing the water availability. However, APX activity increased in plants subjected to low soil-water potentials (at 50% and 25% FC) compared to Roots APX release due to TiO 2 treatments occurred after a notable reduction by spraying plants with water (Fig. 7b). However, its activity increased dramatically by increasing the level of Nano-TiO 2 . The APX activity in the sunflower roots due to Ord-TiO 2 treatments was changing within a narrow range at all levels of water availability, so the activity did not differ statistically compared to control. Also, the effect of drought stress (25% FC) was clearer on the enzyme activity in plant roots under all TiO 2 treatments. It is worthy to mention that foliar spraying on plants with water decreased the APX activity in the roots compared to control, and the decreases were significant at 50% and 25% FC. Data of two-way ANOVA revealed to a significant increase in the APX activity in leaves and roots of sunflower by 50% and 25% FC. The activity increased significantly by all concentrations of Nano-TiO 2 in roots and by 150 and 300 ppm in leaves. When averaged across all levels of water availability, the data in Table 1 showed that membrane injury, proline, and also the activity of CAT and GPX in sunflower leaves sprayed with Nano-TiO 2 mostly did not differ significantly from those sprayed with Ord-TiO 2 . In addition, lipid peroxidation, H 2 O 2 and APX activity increased significantly by most concentrations of Ord-TiO 2 compared to Nano-TiO 2 . Also, most measured parameters (averaged across all levels of FC) in sunflower leaves increased significantly by increasing concentration of Nano-or Ord-TiO 2 . Interestingly, the contents of H 2 O 2 increased but MDA decreased in the plant roots by Nano-TiO 2 up to 300 ppm, the situation was different at 600 ppm. Except MDA in roots of plants sprayed with Nano-or Ord-TiO 2 , all measured parameters (averaged across all concentrations of TiO 2 ) increased significantly and gradually by shortage in water availability. The content of H 2 O 2 increased significantly by 50 and 150 ppm Nano-TiO 2 compared to control group or other treatments.
To measure the effect size of each factor, g 2 was calculated by dividing the sums of squares for the effecting factor (SS effect ) on the total sums of squares for all effects including errors and interactions (SS total ) in the ANOVA. Values of g 2 in Table 2 showed that the shortage in water availability exerted the highest magnitude of effect on the changes of studied parameters in sunflower leaves treated with Nano-or Ord-TiO 2 except the content of H 2 O 2 and GPX activities where Nano-TiO 2 was having the highest effect. Compared to NPs, Ord-TiO 2 exerted the highest effect on changing lipid peroxidation, H 2 O 2 , GPX and APX activities in the leaves, while the shortage in water availability exerted the highest magnitude of effect on the changes of other parameters. In roots of sunflower treated with Nano-or Ord-TiO 2 , the levels of water availability mostly wielded the highest effect on enzyme activities, but TiO 2 influenced more on the changes of MDA and H 2 O 2 contents. The values of g 2 revealed that the effect of interaction between different levels of water availability and Nano-or Ord-TiO 2 is weak or negligible on all parameters either in leaves or roots. Based on the two-way ANOVA, the two main factors (water deficit stress, Nanoor Ord-TiO 2 ) and their interaction significantly affected approximately all measured parameters in leaves and roots of sunflower.

Discussion and conclusion
Water deficiency is one of the most severe environmental stresses which affects almost all plant functions. One of the inevitable consequences of water deficit stress is the The data are averages across all concentrations of TiO 2 in each level of water deficit (n = 18), and across all levels of water deficit iin control, H 2 O-sprayed group and each concentration of TiO 2 (n = 12). Comparison between Nano-and Ord-TiO 2 at each level of FC or concentration of TiO 2 was achieved from one-way ANOVA, while the comparison between different levels of each factor (water availability or TiO 2 ) was achieved from two-way ANOVA and Duncan's test at P B 0.05 For comparison between different levels of water deficit or different concentrations of TiO 2 either in leaves or roots, averages with different letters are significantly different according to Duncan's test * and ** = significant at P B 0.05 and 0.01, respectively; ns = non-significant Table 2 The eta squared (g 2 ) for the magnitude of effect of Nano-(NPs) or Ord-TiO 2 (OPs), water deficit stress (WD) and the interaction between them on changes of each studied parameter in sunflower leaves and roots. The significant effect of each factor on the parameters is achieved from the two-way ANOVA Organ Water deficit stressed plants sprayed with enhancement of ROS generation in the chloroplasts, peroxisomes and mitochondria of the plant cell. To keep the intracellular ROS level and set the redox-status of the cell, the enhanced ROS generation is mainly kept under tight control by a cooperative antioxidant systems. The enhanced ROS itself function as an alarm signal that triggers defense responses by specific signal transduction pathways that involve H 2 O 2 as secondary messenger (Cruz de Carvalho 2008;Ahmad et al. 2019). In this study, the shortage of water availability significantly increased electrolyte leakage and cell membrane injury, H 2 O 2 content and lipid peroxidation (which is a key indicator of oxidative stress) in sunflower plants. As the content of ROS increases, the damage of cellular membranes increases. In sunflower subjected to water deficit stress, an increase in thylakoid membrane electron leakage to O 2 was observed by Sgherri et al. (1996). However, it is difficult to assess the fraction of ROS generated due to electron leakage from thylakoid membrane from that generated by photorespiration which affect strongly on the oxidative load under drought stress. Noctor et al. (2002) estimated quantitatively the photorespiration oxidative load in the leaves of C 3plants and observed that about 70% of total H 2 O 2 is generated by photorespiration under drought stress. Titanium dioxide (Nano-TiO 2 ) is among the most often used nanoparticles in various fields of nano-phytotechnology. Because it has been used extensively, the exposure of plants to Nano-TiO 2 will be inevitable and concomitantly its transport through the food chains. Larue et al. (2012) reported that foliar application of nanoparticles can enter the leaves through stomatal apertures and then are translocated to various tissues via the symplast and/or apoplast pathways. In leaves of sunflower, TiO 2 whether nanoparticles or ordinary showed additive effect with shortage of water availability by injuring the cell membranes and increasing electrolyte leakage, lipid peroxidation, H 2 O 2 generation and proline accumulation. However, the adverse effect of water deficit stress on sunflower plants that was significant (especially under severe shortage in water availability) was aggravated by Nano-TiO 2 , and there combination was having a greater detrimental effect compared to either water deficit or nanoparticles separately. The effect was concentration dependent. Wang et al. (2010) observed that the Nano-TiO 2 particles caused an increase in lipid peroxidation of treated plants. Larue et al. (2012) reported that the mode of action of plant cell damage in certain concentrations of TiO 2 nanoparticles is unknown.
Both Nano-and Ord-TiO 2 differentially affected the contents of H 2 O 2 and MDA in the leaves and roots of sunflower, where both were positively and negatively correlated in the leaves and roots, respectively. This was due to the decreasing levels of H 2 O 2 in roots that did not exceed 12 lmole g -1 FW, while in leaves it was ranging between 8 and 38 lmole g -1 FW. In addition, the activities of assayed enzymes CAT, GPX and APX increased in roots by water deficit stress, Nano-or Ord-TiO 2 . The two main factors, levels of FC and concentrations of TiO 2 , and their interaction affected significantly on all measured parameters in leaves and roots of sunflower. Hydrogen peroxide generated in the plant cell organelles such as chloroplast, mitochondria, but mainly in peroxisomes. Normally, this ROS is going to be balanced between productions and scavenging in all cell sections. Sometimes this balance is disturbed by a number of contrary environmental stressors, including drought and unwanted metals associated with oxidative stress (Gupta and Sandalio 2012). Lei et al. (2008) reported that the application of Nano-TiO 2 enhanced the activities of rubisco and antioxidant enzyme activities, photosynthetic rate and chlorophyll formation that subsequently increased crop yield. TiO 2 could be considered as a stimulant for plants through the activation of different defense mechanisms involved in plant tolerance against various abiotic stresses. Zheng et al. (2005) showed that the growth of spinach plants was greatly improved at 250-4000 ppm Nano-TiO 2 , but no improvement was observed at higher concentrations when they used higher concentrations up to 8000 ppm. Regarding the negative effect of Nano-TiO 2 , Foltete et al. (2011) observed that these nanoparticles can attach to the Vicia faba root surface in 48 h upon exposure and subsequently inhibited the plant growth. The data of this study indicate that TiO 2 is a stressor at all concentrations, but the effect was concentration dependent. In addition, no stimulation of the growth parameters was observed by spraying sunflower plants with TiO 2 (Fig. S2). Shoemaker (2020) studied the effect of up to 1000 mg Nano-TiO 2 kg -1 soil on Sorghum bicolor and found no significant effect on physiology or biomass accumulation. He stated that further research will be necessary to identify if Nano-TiO 2 particles penetrate the leaf cuticle and epidermis and translocated by the plant.
The results in this study showed that the magnitude of effect of water deficit stress in increasing the content of proline and enzyme activities is generally higher compared to that of TiO 2 . Ashraf and Foolad (2007) reported that proline has a strong ability to hydrate, so it can play a protective role in cell structure. When the plant is challenged by adverse abiotic factors, proline accumulate and interacts with proteins to form a hydrophobic skeleton to stabilize and protect biological macromolecules and cell membrane structures. Proline is also a variety of free radical scavengers, and can reduce the ROS damage through chelating singlet oxygen and hydroxyl radical. Another way of proline to remove ROS is to stimulate the activity of peroxidase, catalase and other enzymes in plants (Yang et al. 2021). In this study, TiO 2 , either ordinary or nanoparticles, seemed to be not belonging to events that enhance proline accumulation in plants especially by treatments with low concentrations. The higher concentrations of TiO 2 when combined with the high level of water deficit affected significantly on proline accumulation. Our results contradict to those of Shah et al. (2021), who observed an enhanced accumulation of proline in salinized maize seedlings by priming its caryopses with TiO 2 , despite proline content reduced by salinity stress.
In contrast to proline, the magnitude of effect of Nanoor Ord-TiO 2 on increasing the generation of H 2 O 2 was higher compared to that of drought. There are numerous studies reporting the induction of oxidative stress by water deficit (Morán et al. 1994;Dat et al. 2000;Wang et al. 2016). The activities of antioxidative enzymes, like superoxide dismutase, peroxidase and catalase, were found to be correlated with tolerance to drought stress (Dat et al. 2000; Bartels and Sunkar 2005;Wang et al. 2016). Also, Mittler and Zilinskas (1994) observed that the activities of chloroplastic and cytosolic Cu, Zn-SODs and cytosolic APX increased during drought of pea plants. However, Dȏgarȏglu (2017) documented no effect of Nano-TiO 2 on the activities of antioxidant enzymes in wheat seedlings.
In this study, although the shortage in water availability and foliar application of TiO 2 exerted combined diverse effect on sunflower, both seem to be capable of enhancing the antioxidative enzyme defense reactions in the plants. Generally, the related antioxidative defense reactions including catalase capable of scavenging hydrogen peroxide are also activated. In leaves and roots of sunflower, despite the activity of CAT, GPX and APX increased by decreasing water availability, the activity varied according to the concentration of TiO 2 treatment solution. In addition the trend of activity was different from one enzyme to another. This may be due to the difference in the existing compartments of each enzyme in the cells. These enzymes are located throughout different compartments of the plant cell, with the exception of CAT that is exclusively located in peroxisomes. Its main function is to remove H 2 O 2 produced by photorespiration or fatty acid b-oxidation reaction (Willekens et al. 1997). However, as H 2 O 2 can easily diffuse across the cell membranes, H 2 O 2 generated by other parts of the cell can also be diffused into peroxisomes and decomposed by CAT. So, CAT is responsible for scavenging H 2 O 2 produced in or diffused into peroxisomes. The increase in activity of catalase, which was in the peroxisomes of plants, may be a manifestation of the adaptive response of plants to abiotic stress (Leung 2018). APX which needs a reductant (ascorbate) to scavenge H 2 O 2 has a high affinity for H 2 O 2 than CAT. APX is located in every cellular ROS producing compartment (Mittler 2002). This may explain why Nano-or Ord-TiO 2 affected APX activity in leaves and roots of sunflower in a different manner from that on CAT or GPX. The increase in APX activity in sunflower roots by high concentrations of Nano-TiO 2 was accompanied by a decrease in H 2 O 2 generation. This is documenting to the hypothesis that, APX may function as a fine regulator of intracellular ROS steady-state levels, possibly at signaling levels, whereas CAT may function mainly as a bulk scavenger for excess ROS generation under stress conditions (Willekens et al. 1997;Noctor and Foyer 1998;Mittler 2002). In leaves of sunflower, combination of high concentration of Nano-TiO 2 with high levels of water deficit inhibited the activity of APX. The combination of high levels of two or more stressors may damage some of the antioxidative systems in plants.
Unfortunately, there are many conflicting opinions and results about the effect of Nano-TiO 2 on the seed germination, growth parameters or physiological and enzyme activities of plants. Therefore, it is difficult to find an answer in the literature to the question whether or not application of Nano-TiO 2 is improving to the plants. According to the concentration applied, the particles size used, the target plant species and the environmental conditions, the effect of nano-TiO 2 varied from biostimulation to death of the cells and plants. Although membrane injury, lipid peroxidation and H 2 O 2 generation increased in sunflower leaves and roots by water deficit stress, they increased more by Nano-or Ord-TiO 2 , and concomitantly the activity of antioxidant enzymes enhanced. In conclusion, the foliar spraying of sunflower plants with Nano-TiO 2 did not ameliorate the stress caused by decreasing water availability, on the other hand it increased the stress and its use in nano-phytotechnology mustn't be expanded without extensive studies.
Author contributions TR Wrote the manuscript, interpreted the results and analyzed the data statistically; SAS designed the experiment and revised the manuscript; AMAA performed the experiments and participated in drafting the article; AKAAE carried out the practical experiments and drafted the article. All authors read and approved the final manuscript. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.