Titanium dioxide nanoparticles (TiO2 NPs) promote growth and ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica

Considering titanium dioxide nanoparticles (TiO2 NPs) role in plant growth and especially in plant tolerance against abiotic stress, a greenhouse experiment was carried out to evaluate TiO2 NPs effects (0, 50, 100 and 200 mg L−1) on agronomic traits of Moldavian balm (Dracocephalum moldavica L.) plants grown under different salinity levels (0, 50 and 100 mM NaCl). Results demonstrated that all agronomic traits were negatively affected under all salinity levels but application of 100 mg L−1 TiO2 NPs mitigated these negative effects. TiO2 NPs application on Moldavian balm grown under salt stress conditions improved all agronomic traits and increased antioxidant enzyme activity compared with plants grown under salinity without TiO2 NP treatment. The application of TiO2 NPs significantly lowered H2O2 concentration. In addition, highest essential oil content (1.19%) was obtained in 100 mg L−1 TiO2 NP-treated plants under control conditions. Comprehensive GC/MS analysis of essential oils showed that geranial, z-citral, geranyl acetate and geraniol were the dominant essential oil components. The highest amounts for geranial, geraniol and z-citral were obtained in 100 mg L−1 TiO2 NP-treated plants under control conditions. In conclusion, application of 100 mg L−1 TiO2 NPs could significantly ameliorate the salinity effects in Moldavian balm.

(0, 50, 100 and 200 mg L −1 ). The seeds of Moldavian balm (Dracocephalum moldavica L.) were purchased from Pakanbazr Company, Isfahan, Iran. Regarding seed preparation, surface sterilization of the seeds was done with 1% (w/v) sodium hypochlorite (NaOCl) for 5 min, then washed three times with distilled water and finally soaked in distilled water for 10 min. The seeds were wetted with tap water and let to germinate for a week. Then, in each pot, eight plants were hydroponically grown in growth medium containing cocopite and perlite (2:1 ratio). Plants were irrigated daily with quarter-strength Hoagland solution with some modification 29 . After three weeks, salinity stress was imposed (eight-leaf stage), applied daily (in combination with quarter-strength Hoagland solution) and continued up to plant harvest (prolonged stress ≈ two months after applying salt stress) for the establishment of salinity effects on plant agronomic parameters. TiO 2 NPs were added three times (three continuous days) to quarter-strength Hoagland solution two weeks after salinity stress application. Control plants were irrigated daily with quarter-strength Hoagland solution until harvest and treated with 0 mM NaCl and 0 mg L −1 TiO 2 NPs. Agronomic parameters. Plant agronomic traits including plant height, shoot and leaf fresh and dry weights and leaf number were recorded at the harvest stage. For this purpose, five plants from each treatment were randomly sampled to measure the above traits. For fresh and dry weights, five samples were individually weighed for fresh weight and then kept in the oven (70 °C, 72 h) for dry weight measurements.
Chlorophyll a, b and carotenoid content. Chlorophyll (Chl) and carotenoids amounts were achieved by extracting 0.2 g of fresh leaves in 0.5 mL acetone (3% v/v). After centrifuging (10000 rpm, 10 min) and obtaining the supernatant, absorption was recorded at 645 nm (Chl b), 663 nm (Chl a) and 470 nm (carotenoids) by UV-Vis spectrophotometry (UV-1800 Shimadzu, Japan). The youngest and fully expended leaves (from growing point) were used for measurements. Photosynthetic pigment contents (Chl a, b and carotenoids) were calculated from the following equations as described by Sharma et al. 30 32 . Briefly, fresh leaves (0.2 g) were homogenized with 5 mL trichloroacetic acid (0.1% w/v) in an ice bath and then centrifuged (12000 rpm, 15 min). At that time, 0.5 mL of the supernatant was added to 0.5 mL potassium phosphate buffer (pH 6.8, 10 mM) and 1 mL potassium iodide (KI) (1 M Antioxidant enzyme activity assays. Young and fully expanded leaves were collected to assay antioxidant enzymes activities. For this purpose, samples were collected in an ice bucket and brought to the laboratory. All steps of enzyme extraction were carried out at 4 °C as follows: 0.5 g of the homogenized leaves were extracted with potassium phosphate buffer (pH 6.8, 10 mM) containing 1% polyvinylpyrrolidone (PVP) using magnetic stirrer for 10 min. The homogenate was centrifuged (6000 rpm, 20 min) and the supernatant was used for the assay of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and guaiacol peroxidase (GP) enzyme activities. In order to determine CAT activity, the mixture of 0.5 mL potassium phosphate buffer, 4.5 mL H 2 O 2 (3%) and 50 µL crude enzyme extract in a quartz cuvette was assayed using a UV-Vis spectrophotometer (UV-1800 Shimadzu, Japan) at 240 nm for 120 s 33 .
SOD activity was measured based on the method described by Sun et al. 34 with slight modifications. The reaction mixture consisted of 2.5 mL potassium phosphate buffer, 0.2 mL methionine (0.2 M), 0.1 mL EDTA (3 mM), nitro blue tetrazolium (NBT), 1 mL distilled water, 0.1 mL NaCa 3 (1.5 M), 0.1 mL riboflavin and 50 µL enzyme extract illuminating in glass tubes. The unilluminated mixtures were used as blanks. Test tubes were exposed to light by immersing in a beaker 2/3 filled with clean water, maintained at 27 °C. The increase in absorbance due to formazan formation was recorded at 560 nm. One unit of SOD was defined as the amount of enzyme that inhibited the rate of nitro blue tetrazolium reduction by 50%.
The assay mixture for the estimation of GP activity comprised of 1 mL potassium phosphate buffer, 250 µL EDTA, 1 mL guaiacol (5 mM), 1 mL H 2 O 2 (15 mM) and 50 µL enzyme extract. The rate of change in absorbance at 470 nm was determined according to Tang and Newton 36 . Statistical analysis. All obtained data analysis performed by SAS software and the means of each treatment were analyzed by Duncan's multiple range test at the 95% level of probability (SAS Institute Inc., ver. 9.1, Cary, NC, USA).

Results and Discussion
Characterization of TiO 2 NPs. FTIR spectrum was used for the chemical elucidation of the synthesized TiO 2 NPs. The existence of unresolved stretching vibrations of Ti-O-Ti could be assigned as broad band in the region of 400-900 cm −1 (Fig. 1). In addition, two bands at 1620 and 3427 cm −1 were related to bending and stretching vibrations of O-H groups 38 .
X-ray diffraction (XRD) pattern of TiO 2 NPs was investigated to study the structure and phase formation of the sample. According to Fig. 2, a well-crystallized anatase profile was observed for TiO 2 NPs, in good agreement with the JCPDS data (JCPDS data file No. 21-1272).
Surface, size and the particle morphology of TiO 2 NPs were imaged by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 3a,b). Based on the SEM image, spherical-like shapes with particle diameter of 70-90 nm could be seen for the synthesized TiO 2 NPs, while particle size was determined as 20-30 nm according to TEM. The difference in the size of nanoparticles obtained by SEM and TEM techniques may be related to the loss of stability of nanoparticles during the freezing-drying process as well as due to particle aggregation phenomena.
In the plants treated with high concentration of TiO 2 (200 mg L −1 ), the presence of NP aggregates was indicated by fluorescent light spots inside the root (Fig. 4B). No spots were observed in control (0 mg L −1 TiO 2 ) plants, as expected (Fig. 4A). Only a few studies exist on subcellular localization of TiO 2 in plants. Present results demonstrated that the high concentration of TiO 2 NPs increased their aggregations in the plant root. TiO 2 was actively taken up in Spirodela polyrrhiza roots and aggregated in the plant cells at toxic concentration 39 (See Supporting  www.nature.com/scientificreports www.nature.com/scientificreports/ Information Fig. S1). Both studies demonstrated the entry of TiO 2 NPs in the roots by markedly shiny spots at high inside the roots, representing aggregation. Similar to our observations, fluorescence microscopy imaging techniques were used to indicate the entrance of magnetic NPs into soybean plant tissues as shown in previous reports 26,39 . From an application point of view, various parameters such as size, concentration and aggregation of NPs are the most important issues in agriculture, playing important roles in determining reactivity, toxicity, fate, transport and risk in the environment 40 .
Assessments of agronomic parameters. Plant agronomic parameters was significantly influenced by application of TiO 2 NPs, salt stress and their interactions (Table 1).
Results demonstrated that the maximum plant height (≈62.33 cm) was observed in 100 mg L −1 TiO 2 -treated plants under control conditions. On the contrary, the lowest height was achieved in 100 mM NaCl without TiO 2 treatment. Regarding shoot fresh weight, the maximum and minimum values were recorded in 100 mg L −1 TiO 2 -treated plants under no salinity and 50 mg L −1 TiO 2 under 100 mM salinity conditions, respectively. In the case of shoot dry weight, 50 mg L −1 TiO 2 NPs under control conditions demonstrated the highest value, whereas 100 mM NaCl resulted in the lowest value. Application of 100 mg L −1 TiO 2 under no salinity conditions caused maximum leaf number (≈101.33), while 100 mM NaCl with no TiO 2 application had the lowest (≈37.33). Current results also showed that the highest and lowest amounts of leaf FW were achieved in plants treated with 100 mg L −1 TiO 2 without salinity and 50 mg L −1 TiO 2 under 100 mM salinity conditions, respectively. Furthermore, plants treated with 100 mg L −1 TiO 2 NPs without salinity stress had the highest leaf DW (≈3.15 g), as expected considering their FW. Lowest DW values were recorded in 100 mM NaCl-treated plants.
In total, plants treated with 100 mg L −1 TiO 2 displayed optimal performance for most agronomic traits, whereas worst-performing plants were the ones grown under severe salinity stress (100 mM NaCl). It is worth stating that TiO 2 application, especially in low and medium concentrations, also improved agronomic parameters under control conditions, thus rendering them as potential growth promoters. Contrarily, plants treated with 200 mg L −1 TiO 2 showed significant decrease in their agronomic attributes, indicative of toxicity effects. In  www.nature.com/scientificreports www.nature.com/scientificreports/ addition, application of TiO 2 NPs showed positive effects on the agronomic traits under salinity conditions and significantly ameliorated the stressor's negative effects. In detail, approximately all TiO 2 concentrations could reverse the negative effects of salinity stress by improving the agronomic parameters examined under different salinity levels; 100 mg L −1 TiO 2 under 50 mM NaCl and 200 mg L −1 TiO 2 under 100 mM NaCl achieved optimal performance in this regard.
Salt stress (NaCl) reduces plant growth due to its negative effect on photosynthesis rate, cell division and elongation, changes in enzymatic activity (subsequently affects protein synthesis), decrease in carbohydrates and growth hormone levels and disruption of biological and metabolic activities that finally could lead to growth inhibition 8 . Thus, plant height commonly decreases by increase in NaCl levels due to its destructive effects. Aziz et al. 41 previously reported a reduction in plant height by increasing salinity levels. Considering the result of TiO 2 application, all concentrations and 100 mg L −1 in particular, caused a significant increase in plant height, demonstrating that the application of TiO 2 NPs ameliorated the negative effects of salinity. The observed decrease in leaf number and FW in moderate and high salinity levels is attributed to a reduction in cell expansion due to low turgor controlled by cellular water uptake and cell-wall extension 42 . In addition, Kapoor and Pande 43 concluded that leaf numbers decreased under salinity conditions due to a reduction in branches per plant as a result of decrease in nutrient concentrations. Decline in fresh and dry weights, leaf numbers and abscissions under salinity stress were previously reported 44 . However, the positive effects of TiO 2 NPs were observed in leaf numbers as well as fresh and dry weights in the current study. In this regard, Rahneshan et al. 45 reported that TiO 2 application enhanced absorption rate of macro-and micro-nutrients, improved plant growth characteristics (e.g., plant height, leaf number) and reduced negative effects of salinity by affecting photosynthesis and absorption of essential elements.
Photosynthetic pigments. Application of TiO 2 NPs had significant effects on photosynthesis pigments (Table 2).  www.nature.com/scientificreports www.nature.com/scientificreports/ The highest contents of chl a, b and carotenoids were observed in 100 mg L −1 TiO 2 without salt stress. Furthermore, salinity stress decreased pigment content, but application of 100 mg L −1 TiO 2 increased chl a, b and carotenoid contents under both salinity levels. 200 mg L −1 TiO 2 led to significantly lower pigment contents compared with lower TiO 2 concentrations similar to salt-stressed samples, suggesting toxicity. The observed decrease in photosynthesis pigment content under salt stress conditions could be attributed to reduced biosynthesis or more likely increased breakdown due to ROS damage of the pigments in cells, functional disorders observed during stomatal movement and instability of the pigment protein complex under salinity stress. Salinity stress is known to result in pigment breakdown due to accumulation of toxic ions in chloroplasts and ROS-induced oxidative stress in plants 45 . In addition, Hernandez et al. 46 stated that pigment reduction in NaCl-sensitive plants happened due to changes in number and size of chloroplasts, starch content, disorganized chloroplast membranes, loss of envelope and disorganization of grana and thylakoids.  (Fig. 5A). Furthermore, all TiO 2 NP concentrations increased Fv / Fo with the highest value being recorded at 100 mg L −1 under control conditions, while NP pre-treatment ameliorated decreases recorded in this parameter under salt stress conditions (Fig. 5B). Similar findings were observed for Y (II) parameter, where NP application increased Y (II) under control conditions and reversed decreases observed in salt-stressed plants (Fig. 5C).
The significant decrease in these parameters was likely due to the dissipation of a major proportion of light energy as heat under salt stress 47 . Similar reduction in chlorophyll fluorescence parameters under salt stress was previously reported in maize 48 , as well as in sorghum 49 . Increase in the examined chlorophyll fluorescence parameters after TiO 2 NP application could be attributed to enhancement in light energy of PSI absorbed by chloroplast membrane to be transferred to PSII, promotion of light energy conversion to electron energy and electron transport and acceleration of water photolysis and oxygen evolution 50 . In addition, Rubisco enzyme activity increased after TiO 2 NP application due to increase in the expression of its mRNA 51 . Rubisco enzyme plays an important role in photosynthesis and optimal expression of this enzyme improves chlorophyll fluorescence parameters, while also increasing absorption of carbon dioxide in plants 52 . Overall, current findings suggest that TiO 2 NPs potentially ameliorated the negative effects of salinity stress through the improvement in chlorophyll fluorescence parameters and by maximizing PSII efficiency 46 (Fig. 6).
In addition, application of 100 mg L −1 TiO 2 in plants growing under moderate and high salinity stress as well as in 50 mg L −1 TiO 2 -treated plants under moderate NaCl stress decreased H 2 O 2 content in leaf tissues compared with plants subjected to similar stress conditions without any TiO 2 treatments. H 2 O 2 , produced in various vital processes of different organs cells, is highly toxic for cells and causes oxidative stress at high concentrations 53 , as well as damages to biological membranes via their peroxidation. Thus, the mentioned TiO 2 treatments could amplify plant performance under saline conditions likely by decreasing oxidative stress and lessening membrane damage. Superoxide dismutase (SOD), as the primary ROS scavenger localizing in chloroplasts, mitochondria, peroxisomes and cytosol, catalyzes the disproportion of two O 2 · − radicals to H 2 O 2 and O 2 54 . Moreover, H 2 O 2 is scavenged by ascorbate-peroxidase (APX) in ascorbate-glutathione cycle and through guaiacol peroxidase (GP) and catalase (CAT) in cytoplasm and divided into water and oxygen 55 . The increased activity of the mentioned enzymes by TiO 2 treatments in the present study might be another reason for the observed decrease in H 2 O 2 values under salinity stress compared with control conditions. Although all TiO 2 treatments increased H 2 O 2 values and the high concentration of TiO 2 might be considered as toxic, these increases were lower than those under salinity stress, demonstrating lower negative effects of NP treatments even at high concentration compared with salinity. Moreover, considering the positive impact of TiO 2 towards lowering H 2 O 2 content under salinity, NP treatments could be considered as beneficial for removing undesirable effects of salinity.
Evaluation of antioxidant enzymes. Application of TiO 2 NPs, salt stress and their interactions significantly affected superoxide dismutase (SOD) activity. The maximum and minimum activities were recorded in 100 mM NaCl-treated plants under no TiO 2 application and control samples, respectively. SOD activity of leaf tissues under moderate and high salinity stresses increased significantly compared with controls. Amongst treatments, the highest activity was observed in 200 mg L −1 TiO 2 under 50 mM NaCl, while the lowest was observed in 50 mg L −1 TiO 2 under no salinity and 200 mg L −1 TiO 2 under 100 mM NaCl (Fig. 7A).
In regard with catalase (CAT), enzymatic activity in leaf tissues under 50 and 100 mM NaCl increased significantly compared with control. Therefore, a positive regulation of CAT activity by salt concentration was observed; increasing NaCl levels resulted in increasing CAT activity, similar to SOD. The highest activity among treatments was achieved in 100 mg L −1 TiO 2 under 100 mM NaCl, while the lowest was in 50 mg L −1 TiO 2 -treated plants under no salt stress. Considering CAT activity, TiO 2 at 100 mg L −1 concentration generally increased enzymatic Scientific RepoRtS | (2020) 10:912 | https://doi.org/10.1038/s41598-020-57794-1 www.nature.com/scientificreports www.nature.com/scientificreports/ activity under both stress conditions compared with those plants at similar conditions without receiving any TiO 2 treatment (Fig. 7B).
The highest and lowest ascorbate peroxidase (APX) activities were observed in 100 and 200 mg L −1 TiO 2 -treated plants under 100 mM NaCl and the control and 50 mg L −1 TiO 2 under no salinity stress, respectively. Similar to SOD and CAT, increasing salinity levels lead to increasing APX activity, under no TiO 2 treatment. TiO 2 treatments increased APX activity under both non-stress and stress conditions, with these increases being higher than non-treated plants at the same conditions (Fig. 7C). www.nature.com/scientificreports www.nature.com/scientificreports/ Maximum guaiacol peroxidase (GP) activity was observed in 100 mg L −1 TiO 2 under 100 mM NaCl. In this regard, minimum activity was noticed in the control and 200 mg L −1 TiO 2 -treated samples under 100 mM NaCl. GP activity in leaf tissues under moderate and high salinity stress levels increased significantly compared with control samples. In fact, increase in salinity level increased GP activity. As well, TiO 2 treatments increased the activity in which TiO 2 -treated plants had higher activity that non-treated ones under both non-stress and stress conditions (Fig. 7D).
In total, the activity of GP, APX, CAT and SOD significantly increased under both moderate and high salinity levels. In addition, TiO 2 treatments at 100 and 200 mg L −1 concentrations increased antioxidant enzyme activities under control conditions. A similar increasing trend was observed in 50 and 100 mg L −1 TiO 2 -treated plants under both salinity levels for the above-mentioned enzymes. It is noteworthy that, although the applied salt stress increased enzymatic activities, highest levels were observed in 100 mg L −1 -treated plants for CAT, APX and GP. SOD enzyme was an interesting exception as its activity was enhanced by TiO 2 application under control conditions, whereas SOD activity decreased significantly in TiO 2-treated plants under moderate and severe salt stress compared with plants without any TiO 2 application. Additionally, the high concentration of TiO 2 (200 mg L −1 ) applied in plants, under both salinity levels, showed lowest antioxidant enzymatic activity levels overall in comparison with plants treated with 50 and 100 mg L −1 TiO 2 which could be correlated with toxicity phenomena.
Overall, it could be concluded that 100 mg L −1 TiO 2 application under moderate and high salt stress induces antioxidant enzyme activities, thus contributing in the effective protection of plants from salinity. This is likely through the detoxification of ROS, which is known to over accumulate in saline environments 56 . ROS compounds are generated by normal cellular activities (e.g., fatty acids β-oxidation), photorespiration and biotic or abiotic stress conditions. ROS elimination is mainly achieved by antioxidant mechanisms such as antioxidant enzymes (e.g., SOD, CAT, APX) 57 . SOD is the key enzyme for neutralizing ROS as the first line of defense mechanism against oxidative stress. Enhancement in SOD activity is tightlylinked with increased protection against negative effects of stress factors 58 . CAT, another important antioxidant enzyme, scavenges H 2 O 2 by converting it to water in peroxisomes and neutralizes its deleterious damages 59 . APX activity, yet another key antioxidant enzyme, eliminates H 2 O 2 activity and modulates its steady-state level in various subcellular compartments of plants 60 . Moreover, high levels of intercellular H 2 O 2 are known to induce cytosolic APX activity under salinity stress 61 . Thus, APX plays an important role in the collection and decomposition of H 2 O 2 during stress 62 . GP acts as an electron transmitter to H 2 O 2 , in an attempt to detoxify cells under stress conditions by converting H 2 O 2 into water 63 . Previous studies reported considerable induction of enzymatic antioxidants under salinity stress, thus preventing ROS-related damage (e.g. Filippou et al. 7 ). Our findings are in agreement with Weisany et al. 56 , who noted that CAT and APX enzymatic activities in soybean increased under salinity stress due to oxidative reactions caused by higher levels of H 2 O 2 . Regarding the enhancement in antioxidant enzyme activities of the plants treated with TiO 2 NPs under salt stress, positive interactions might take place which likely provide better signaling towards the activation of these defense enzymes. Moreover, the observed increases in SOD, CAT, APX and GP enzymatic activities under salinity in the present study might be related with the high intercellular H 2 O 2 levels in Moldavian balm leaf tissues. Likewise, enhancement in SOD, CAT, APX and GP activities was observed in plants treated with TiO 2 NPs. In addition, the lowest H 2 O 2 content was observed in 100 mg L −1 TiO 2 -treated plant. Therefore, it could be concluded that the lowest H 2 O 2 content recorded after 100 mg L −1 TiO 2 application was closely related to the significantly increased activities of CAT, APX and GP in the same samples. ROS detoxification after TiO 2 NP application might be due to stabilized composition of cells and improved physical properties of cell membranes. Lei et al. 11 reported that application of TiO 2 NPs under drought stress increased antioxidant enzyme activities in plants due to a reduction in lipid peroxidation and improvement in membrane integrity.  www.nature.com/scientificreports www.nature.com/scientificreports/ NaCl, as well as in control samples (Fig. 8). Salinity stress positively affected essential oil content. Generally, both salinity levels increased essential oil content, but maximal yield was achieved by 50 mM NaCl. Similarly, TiO 2 NP application had a positive impact in essential oil content, significantly increasing it under control conditions with optimal content recorded following 100 mg L −1 TiO 2 NP application. However, under salinity conditions, TiO 2 treatments had no considerable impact on this component compared with non-treated plants under stress.

Essential oil content and composition.
The essential oil composition of D. moldavica L. under different salt stresses and TiO 2 NPs applications is shown in Table 3. Base on the results, 29 constituents were identified by GC/MS analysis. Main components were geranial, z-citral, geranyl acetate and geraniol. 50 mM NaCl caused significant decrease in geranial and z-citral as well as minor decrease in geraniol, while it significantly increased geranyl acetate concentration. However, 100 mM NaCl had a different effect, as geranial concentration was not affected, geraniol and geranyl acetate content showed increase, while z-citral decreased compared with control samples.   www.nature.com/scientificreports www.nature.com/scientificreports/ condition. In addition, this treatment caused a decrease in z-citral and increase in geraniol content under both salinity levels. Geranial showed no significant difference under 50 mM salinity, while it lowered under 100 mM NaCl. Moreover, geranyl acetate content increased significantly following 200 mg L −1 TiO 2 treatment under both control and stress conditions with the highest increase being recorded at 50 mM NaCl application. Furthermore, in spite of considerable enhancement in myrtenol and germacrene D contents by 50 mM NaCl without TiO 2 application, their highest values were observed in 50 mg L −1 TiO 2 under 50 mM NaCl stress. Regarding nerol content, although salinity increased its content (increasing NaCl concentrations leading to increasing nerol content), the highest value was observed in 50 mg L −1 TiO 2 -treated plants under 100 mM NaCl stress. Nerol content was also increased following 100 mg L −1 TiO 2 under both control and stress conditions as well as following 200 mg L −1 TiO 2 under 100 mM NaCl.
Essential oils of Moldavian balm, as an important aromatic and medicinal plant, have various application in the pharmaceutical industry. Considering the importance of its essential oils, any treatment with positive effects on its essential oil content and dominant constituents could be of great value to growers. The positive effect of TiO 2 NPs was previously reported in Salvia officinalis essential oil content and constituents 55 . Taking into account these factors, the current study examined the effect on Moldavian balm under normal and salt stress conditions. Present results revealed that the essential oil content increased under both NaCl levels, in agreement with Khalid and Teixeira de Silva 64 and Neffati et al. 65 . However, a similar trend was not recorded for individual components of the essential oil profile, since the dominant constituents mostly decreased following salt stress particularly 50 mM NaCl. This decrease might be attributed to an impairment in photosynthesis, changes in metabolic systems and increase in osmotic pressure, which might then decrease nutrients and water uptake. Salinity stress has been previously shown to modify essential oil production and profile 41 . Current results demonstrated that salinity stress altered the content of specific essential oil components in Dracocephalum moldavica L. plants, in agreement with Khalid and Teixeira de Silva 64 and Neffati et al. 65 who attributed such changes to the regulation of the activity of essential oil biosynthetic enzymes following salt stress imposition.
TiO 2 application caused a remarkable increase in essential oil content under control conditions with maximum content being observed at 100 mg L −1 concentration. Results were in accordance to those reported by Ahmad et al. 66 , who demonstrated that TiO 2 NP application increased essential oil content in Mentha piperita   L. Furthermore, Lafmejani et al. 67 reported that Fe NPs foliar application increased essential oil content in M. piperita plants. Such an increase in essential oil content could be potentially explained by the observed increase in growth, photosynthesis, expression of secondary metabolite enzymes and size and distribution of oil glands as special sites for biosynthesizing essential oils following NP application 66 . In line with the increase in essential oil content, 100 mg L −1 TiO 2 NP application increased main components of essential oil profile. This could be the result of increased expression of specific biosynthetic enzymes involved in the production of components and availability of substrates, in line with previous findings by Ahmad et al. 66 and Lafmejani et al. 67 . The actual mechanism by which NP application modulates plant secondary metabolites is not yet fully elucidated. Recently, coordinated phytochemical and genomic studies confirmed that NPs might act as elicitors for secondary metabolite production in plants by inducing different cellular signal transduction pathways (e.g., mitogen-activated protein kinases, calcium flux and ROS metabolism). Accordingly, the observed changes in the above-mentioned pathways might lead to alterations in gene expression levels and metabolic enzyme activation that could alter secondary metabolite production 68 .
conclusion Nanotechnology is a highly promising novel approach that has great potential for application towards plant protection against different stress conditions. TiO 2 , recently developed nanoparticles with profound effects in plant morphological, physiological and biochemical properties, could improve overall plant performance. Its application in Moldavian balm plants demonstrated these positive effects under moderate and severe salinity stress as enhanced agronomic traits under both control and stress conditions. TiO 2 NP application additionally lowered H 2 O 2 content and increased antioxidant enzyme activities), thus ameliorating oxidative damage and demonstrating positive effects in plants under both conditions. Importantly, enhancement in essential oil content by TiO 2 treatments demonstrated another positive impact of TiO 2 NPs with implications in the potential for commercial application Interestingly, application of high concentration of TiO 2 (200 mg L −1 ) showed toxic symptoms in specific parameters, likely linked with NP aggregation in high concentrations which lead to increased ROS content. Consequently, TiO 2 might act as an inducer of secondary metabolite production (such as essential oils) and trigger for the activation of the enzymatic defense system, ultimately enhancing plant performance under control and stress conditions and thus acting as a promising stress protecting and growth promoting molecule.