Introduction

Flaxseed or Linseed (Linum usitatissimum L., Linaceae), an herbaceous annual plant belonging to the family Lineaceae, is the sixth largest oilseed plant in the world, which has been used as a medicinal plant from a long time ago. Flaxseed oil contains a mixture of the fatty acids with a high linolenic acid component that improves their stability in food applications (Green and Marshall 1981). It has been reported that Flaxseed oil, because of its polymer-forming characteristics, can be used in the production of varnish and paints, oil cloth, and linoleum (Hatim and Abbasi 1994). This plant grows in a wide range of climates, but does best on moderate to cool regions, and tolerate many soils types and conditions (Hocking et al. 1987). Moreover, Flaxseed is a susceptible plant to drought stress conditions at the seedling stage, blooming, and early seed development time; however, it is not usually irrigated in Europe (Martin et al. 1976).

Drought stress is considered as one of the most significant factors limiting plant performance and yield (Dutta et al. 1995). Enhancement of seed yield following irrigation approves the positive effect of supplementary irrigation at sensitive growth stages to drought stress on plant productivity (Daun 1993). It is well known that water-deficit stress decreases the specific survival time and growth of plants, results in stomatal closure. This may cause oxidative stress, leading to reduces in photosynthetic electron chain and high production of reactive oxygen species (ROS) in the chloroplasts and mitochondria organelles (Fu and Huang 2001; Liu 2009; Ozkur et al. 2009; Ben-Ahmed et al. 2009). According to Fu and Huang (2001), high generation of ROS such as singlet oxygen (1O2), hydroxyl radical (HO−), superoxide (O2−), and hydrogen peroxide (H2O2) in plant tissues interrupt the regular cell metabolism through the oxidative damage to macromolecules, photosynthetic pigments, and enzymes.

Development of nanotechnology and nanoscience on agricultural section has spread out the domain area of nano-structured materials in various fields because of their unique physiochemical traits. Subsequently, different types of nanomaterials including metal- and carbon-based materials have been produced. Among the different nano materials created, nano-titanium dioxide (nano anataseTiO2) has great optical and biological performance, and therefore has newly caught the much attention of the modern plant physiologists.

The effects of nano-sized materials on plant performance are complex; even the same type of these materials may have various biological impacts (positive, negative, and or inconsequence) on various plant species, also, the same nanosacle materials that can be toxic in high concentrations may have positive role in low levels. Up to now, wide kinds of impacts of nano-TiO2 on seed germination and plants growth have been reported (Larue et al. 2011; Azimi et al. 2013). Nano-TiO2 particles are able to penetrate plant cell only smaller than a threshold of diameter, which specified as 36 nm (Laure et al. 2012). According to Foltete et al. (2011) TiO2 nanoparticles can attach to the Vicia faba root surface in 48 h upon exposure, which subsequently inhibits the plant growth. Also, it has been reported that the nano-TiO2 particles caused an increase in malondialdehyde (MDA) content of treated plants (Wang et al. 2010). Yang et al. (2006) reported that exposure to nano TiO2 significantly enhanced the activity of nitrate reductase, which in turn increased the conversion of inorganic nitrogen (nitrate and ammonium) to organic nitrogen (chlorophyll and protein).

Most previous researches were studied mainly in a laboratory conditions in vitro, and the mechanisms of the interactions between nanoparticles and whole plants are not well understood. Also, mechanism by which nanoparticulates affects plant growth and productivity requires further investigation and elucidation. Therefore, this study was performed to explore the possible inhibitory and/or stimulatory effects of different nanosized TiO2 concentrations on growth, photosynthetic pigments status, H2O2 and MDA levels, seed yield, seed oil, and protein contents in L. usitatissimum plant subjected to drought stress and water sufficient conditions.

Materials and methods

Characterization and scanning electron microscopy (SEM) image of nano TiO2

Nano TiO2 (namely nano-anatase) was provided from the Nanomaterials Pioneers Company, NANOSANY (Mashhad, IRAN). The provided pack was characterized by laboratory analytical methods. The specific surface area (SSA) of nano TiO2 was approximately 200–240 m2 g−1, with a pore size of 0.1 mL g and purity of >99 %. The sizes of TiO2 nanoparticles were specified by scanning electron microscope (SEM), and estimated to be 10–25 nm (Fig. 1). The crystal characteristics of nano TiO2 particles were determined by X-Ray Diffraction (XRD) method (XPert PRO MPD, PANalytical) in the 2θ range of 30°–120° operated at a voltage of 40 kV and a current of 40 mA with Cu Kα radiation. The XRD analytical procedure revealed that employed nanoTiO2 particles were all exhibit in the anatase form (Fig. 2).

Fig. 1
figure 1

SEM micrograph of nano TiO2, average size of the nanoparticle was 10–25 nm

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Fig. 2
figure 2

XRD pattern of nano TiO2 nanoparticles. XRD measurement showed that used TiO2 were all in the anatase phase

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Plant materials, treatments, and experimental setup

Linum usitatissimum cv. “Olajonzon” (Linaceae) seeds were prepared from Department of Medicinal Plants, Arak University, Iran. Seeds were surface sterilized with 1 % sodium hypochlorite (NaOCl) for 5 min and then were washed three times, soaked in distilled water for 10 min. Eight seeds were directly grown in pots, containing approximately 4 kg of soil comprising a mixture of clay, silt and sand in the ratios of 4.5, 71.5, and 24 %, respectively, with an electric conductivity (EC) of 1.52 dS.m−1 and pH 7.2. The concentrations of total N, P, and K were 0.07 %, 12.8, and 7.4 mg kg−1, respectively. At the 3–4 leaf stage, plants were thinned to five per pot. Supplementary light was provided in the greenhouse for 16 h per day with temperature of 28/18 °C (day/night).

A factorial experiment based on randomized complete block design was carried out with three replications (n = 3). Treatments were Nanoparticles of TiO2 solutions (4 levels), water stress (2 levels). Nanoparticles of TiO2 solutions were prepared at concentrations of 0, 10, 100, and 500 mg l−1 with filtered, double-distilled water. Working solutions were made by vigorous vortexing when required. Plants were exposed to water stress after initial seed-filling duration with daily weighting pots. Plants were irrigated every 2 days to achieve field capacity (FC) and 50 % of FC, for control and water-stressed plants, respectively. NanoTiO2 particles were applied at initial seed-filling duration. At this stage, nano TiO2 were sprayed at the rate of 50 ml per pot on the shoots daily for 3 days using hand atomizer. The plants sprayed with the same volume of distillated water were taken as a control. Leaves from each treatment were sampled at seed-filling duration after spraying. Samples were frozen in liquid nitrogen for 2 min then stored at −70 °C for all measurements such as plastid pigments, MDA and H2O2 contents. Plant morphological traits including plant height and branch number were recorded at full flowering stage and the number of capsules per plant, seed numbers per capsules, weight of 1000 seeds, seed yield, seed oil, and protein contents were determined after harvesting.

Plastid pigment measurements

Chlorophyll (Chl) and carotenoids were extracted from 0.5 g of fresh leaves by grounding in 0.5 mL of acetone (80 % V/V). The absorption was recorded at 645 nm (Chl α), 663 nm (Chl b), and 470 nm (carotenoids) in a spectrophotometer (PG Instrument LTD T80+UV/VIS. Measurements were carried on the youngest fully expended leaves from growing point. Photosynthetic pigment contents were calculated from the following equations as described by Lichtenthaler and Wellburn (1983).

$$ {\text{Chl}}\alpha\; \left( {{\text{mg}}\;{\text{g}}^{ - 1} {\text{fr}}.{\text{ Wt}}.} \right) = 1 1. 7 5 \times {\text{A}}_{ 6 6 3} - 2. 3 5 \times {\text{A}}_{ 6 4 5} $$
$$ {\text{Chl}}b\; \left( {{\text{mg}}\;{\text{g}}^{ - 1} {\text{fr}}.{\text{ Wt}}.} \right) = 1 8. 6 1 \times {\text{A}}_{ 6 4 5} - 3. 9 6 \times {\text{A}}_{ 6 6 3} $$
$$ {\text{Carotenoids }}\left( {{\text{mg}}\;{\text{g}}^{ - 1} {\text{fr}}.{\text{ Wt}}.} \right) = 4. 6 9 \times {\text{A}}_{ 4 70} - 0. 2 6 8 \times \left( { 20. 2 \times {\text{A}}_{ 6 4 5} + 8.0 2 \times {\text{A}}_{ 6 6 3} } \right) $$

Determination of H2O2 content

Hydrogen peroxide (H2O2) content in the leaves of Flaxseed plant was specified according to Velikova et al. (2000). Briefly, Flag leaf tissues (0.5 g) were homogenized in an ice bath with 5 ml of TCA (0.1 % w/v). The homogenate was centrifuged at 12,000×g for 15 min. Then 0.5 ml of the supernatant was supplemented to 0.5 ml of 10 mm potassium phosphate buffer (pH 7.0) and 1 ml of 1 m KI. Finally, the absorbance of the supernatant was recorded at 390 nm in a spectrophotometer (PG Instrument LTD T80+UV/VIS). The content of H2O2 was estimated by comparison with a standard calibration curve previously made by various H2O2 concentrations.

Determination of the MDA content

The level of MDA content (as an end product of lipid peroxidation) was assessed according to Heath and Packer (1968). Briefly, 0.5 g of fresh tissues of flag leaves were homogenized in 5 mL of 0.1 % (w/v) TCA solution and centrifuged at 12,000×g for 15 min at 25 °C. Then, 2 ml of supernatant was added to 2 mL of 0.6 % (w/v) TBA. The mixture was incubated at 95 °C for 30 min, then cooled on ice and then samples were centrifuged at 4000×g for 20 min. Thereafter, the absorbance of supernatant was recorded at 532 nm. The MDA content was calculated based on its extinction coefficient of 155 mM−1 cm−1.

Seed oil extraction and quantification

Oil was extracted from the crushed seed powder (20 g) of the plant by petroleum ether (300 ml) as solvent in 60 °C using soxhlet method according to AOCS (1983). The obtained extracts were filtered through the Whatman No. 1 filter paper under vacuum. Thereafter, solution was collected and concentrated with a rotary evaporator at 45 °C to reach the pure oil, and then weighted precisely and the percentage of seed oil determined accordingly.

Seed protein percentage

Nitrogen concentration of the seeds was determined according to the Kjeldahl method (Benton 1991). The amount of protein present is then estimated from nitrogen concentration of the seeds.

Statistical analysis

Data were analyzed using SAS statistical software. A factorial experiment based on randomized complete block design (RCBD) was carried out with three replicates (n = 3). Duncan’s Multiple Range Test (P < 0.01) was used to compare the means.

Results

Growth parameters and seed yield

Analysis of variance (ANOVA) showed that the plant height and the number of subsidiary branches per plant were not significantly (P > 0.05) changed under employed treatments (Table 1). However, the maximum plant height (49.8 cm) and the number of subsidiary branches per plant (8.1) were observed in plants exposed to 10 and 500 mg l−1 nano TiO2 particles, respectively (Table 2).

Table 1 Analysis of variance (ANOVA) for studied traits in Flaxseed plants under various irrigation and Nano-TiO2 treatments
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Table 2 Mean comparison of physio-morphological and biochemical traits in control and nano TiO2-treated Flaxseed plants under sufficient and scarce water conditions
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The number of capsules per plant significantly (P < 0.01) changed in Flaxseed plants under well watered and stress conditions (Table 1). At drought stress, the number of capsules per plant decreased up to 33.35 compared to well-watered situations (Table 2). Also, the number of capsules per plant significantly (P < 0.01) changed in plants exposed to different concentrations of nano TiO2 particles (Table 1). However, exposure to 10 and 500 mg l−1 nano TiO2 increased the number of capsules per plant by 22.5 and 9.98 % over untreated control plants under drought stress (Table 2).

Data analysis showed that the number of seeds per capsules significantly (P ≤ 0.05) affected by irrigation treatments (Table 1). Water stress caused a reduction (13.6 %) on seeds number compared to the normal watering.

For seeds number, there was a significant (P < 0.01) difference among nanoTiO2 particle concentrations under irrigation treatments. The highest (7.6) and the lowest (4.3) seeds number were observed in plants treated with 10 and 500 mgl−1 nano TiO2 particles under well-watered and drought stress conditions, respectively (Table 2).

1000-seed weight was significantly (P < 0.01) affected by reference treatments and their interactions (Table 1). The results showed that nano TiO2 particles generally enhanced the seed weight under both normal and stress conditions. The highest value of 1000-seed weight (5.66 g) was achieved in nano TiO2 application at 10 mg l−1 under well-watered treatment. Whereas, nano TiO2 concentrations up to 100 mg l−1 slightly increased the seed weight compared to untreated controls in drought-stressed plants (Table 2). However, drought stress caused a reduction (12.6 %) on seed weight in plants without nano TiO2 application.

Measurement of seed yield revealed that there were significant differences (P < 0.01) among the treatments (Table 1). Drought stress resulted in a 48.1 % reduction on seed yield (Table 2). The pattern of changes in seed yield was similar to that of the 1000-seed weight per plant. A significant positive correlation was found between seed weight (r 0.01 = 0.89), the number of seeds per capsules (r 0.01 = 0.90), and the number of capsules per plant (r 0.01 = 0.89) with seed yield.

Photosynthetic pigments

Data analysis showed that there were significant differences in the plastid pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) under different treatments applied except in chlorophyll b content under nano TiO2 application (Table 1). Whereas, the interactions of employed treatments were not significantly different at both probability levels for Photosynthetic pigments. Stressed plants decreased their chlorophyll a, chlorophyll b, and total chlorophyll contents by 34.8, 59.06, and 45.68 % over control plant (Table 2). Maximum content of chlorophyll a (2.76 mg g−1 fr.wt.) and chlorophyll b (1.18 mg g−1 fr.wt.) were obtained in well-watered plants treated with 100 and 10 mg l−1 nano TiO2 particles, respectively. In stress condition, plants exposed to 500 mg l−1 nano TiO2 triggered an increase in carotenoids content compared to normal condition and to other treatments. There was a significant correlation between chlorophyll a (r 0.05 = 0.51), chlorophyll b (r 0.05 = 0.50), total chlorophyll (r 0.01 = 0.56), and carotenoids (r 0.01 = 0.69) with seed yield in plants under experimental treatments.

Malondialdehyde (MDA) and hydrogen peroxide (H2O2)

MDA and H2O2 contents were significantly (P < 0.01) affected by reference treatments (Table 1). The minimum (1.36 nmol g−1 fr.wt.) and maximum (5.16 nmol g−1 fr.wt.) content of MDA were observed in plants treated with nano TiO2 particles at 10 mg l−1and non-treated ones in normal and stress conditions, respectively (Fig. 3). Similar trend as MDA changes was found for H2O2 accumulation under applied treatments. The level of H2O2 in nano TiO2-treated plants with 10 mg l−1 was lower than that of other plants, therefore, lipid peroxidation was less pronounced in such plants (Fig. 4). As can be seen, application of nano TiO2 particles at appropriate concentrations can ameliorate cell membrane damage from oxidative stress caused by drought conditions.

Fig. 3
figure 3

Effect of Nano-TiO2 concentrations (0, 10, 100, and 500 mg l−1) on malondialdehyde (MDA) content in Linum usitatissimum (Linaceae) plant. Bars with the same letters indicate non-significant differences (Duncan’s Multiple Range Test, n = 3)

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Fig. 4
figure 4

Effect of Nano-TiO2 concentrations (0, 10, 100, and 500 mg l−1) on hydrogen peroxide (H2O2) content in Linum usitatissimum (Linaceae) plant. Bars with the same letters indicate non-significant differences (Duncan’s Multiple Range Test, n = 3)

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Seed oil and protein

Seed oil and protein contents were significantly (P < 0.01) affected by the experimental treatments (Table 1). In both well-watered and drought stress conditions, the highest value of seed oil (37.9 and 35.1 %) (Fig. 5) and protein (19.8 and 22.28 %) (Table 2) contents were obtained in plants treated with nano TiO2 at 100 mg l−1, respectively. There was a significant positive correlation between seed oil and seed yield (r 0.01 = 0.81), seed weight (r 0.01 = 0.89), the number of seeds per capsules (r 0.01 = 0.75), and the number of capsules per plant (r 0.01 = 0.85) in plants under employed treatments. Also, a significant positive correlation (r 0.05 = 0.44) was found between seed oil and seed protein percentages in Flaxseed plants, which represent that increase in seed protein content cause an increase in seed oil, and subsequently enhance the seed yield.

Fig. 5
figure 5

Effect of Nano-TiO2 concentrations (0, 10, 100, and 500 mg l−1) on seed oil percentage in Linum usitatissimum (Linaceae) plant. Bars with the same letters indicate non-significant differences (Duncan’s Multiple Range Test, n = 3)

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Discussion

Nowadays, nanotechnology has expanded the application domain of particulate nanomaterials in numerous ways in field because of their unique physiochemical properties, which arise from their small size, shape, conductivity, surface area, and or surface chemistry compared to bulk materials. Subsequently, a number of nanoscience-based products including carbon-based materials (e.g., fullerene C70, single-and multi-walled carbon nanotubes) and metal-based materials (e.g., TiO2, CeO2, FeO, etc.) have been created (Rico et al. 2011). Nano-titanium dioxide (nano TiO2 or nanoanatase) is a very common metal-based nanoparticulate with strong oxidation ability, which can stimulate electrons on material surfaces and induce energy transition (Zhao et al. 2005). In this study, we found that application of nano TiO2 at specific concentrations can induce some morphological, physiological, and biochemical response of Flaxseed plants during normal and drought stress conditions. Similarly, it has been reported that drought stress decreased the quantitative and qualitative characteristics of the medicinal plant basil (Ocimum basilicum L.), while, the application of titanium nanoparticles enhanced the negative effects of drought stress to some extent (Kiapour et al. 2015).

Recently, it has been demonstrated that the same nanoparticulate that can be toxic for plants in high concentrations may have positive effects on physiological performance of plants in low concentrations (Khodakovskaya and Lahiani 2014).

Previous studies have shown that the overall growth and development of tobacco plants significantly decreased as nano-TiO2 concentrations increased, which mentioned could be due to expression of microRNAs (∼20 to 22 nt), responsible for plant development as well as plant tolerance to abiotic stresses such as drought and heavy metals (Frazier et al. 2014). According to Zheng et al. (2005) nano TiO2 may promote growth of spinach plants due to activation of photosynthesis and nitrogen metabolism. They also reported that TiO2 is a type of photocatalyst that can hydrolyze light into electrons, protons, and oxygen, in which the produced electron and proton go into an electron transfer chain of plants in the light reaction stage, therefore, enhancing the speed of photosynthesis. Mode of action of plant cell damage in certain concentrations of nano TiO2 particles is unknown (Laure et al. 2012). For this reason in our current study, lipid peroxidation and hydrogen peroxide content in leaves of Flaxseed plants were investigated under experimental treatments.

In our present study, plant exposed to lower concentrations of nano TiO2 resulted in an improvement of photosynthetic pigment contents when compared to the other concentrations applied. According to Priyadarshini et al. (2012) nanosilver particles at 100 mg l−1 increased the chlorophyll a and total chlorophyll contents in Brassica junceae seedlings up to 40 and 25 %, respectively. They reported that improved quantum efficiency in the leaves of treated seedlings significantly correlates with higher pigments values. Enhanced chlorophyll and carotenoids contents of the some plants through different nanomaterials application were previously reported in Pelargonium zonale cultivars with nanosilver (Hatami and Ghorbanpour 2014), in Zea maize plant with magnetic nanoparticles (Racuciu and Creanga 2006), and in Pelargonium graveolens with nano TiO2 particles (Ghorbanpour and Hatami 2015).

It is well known that MDA accumulation is an ultimate product and appropriate biomarker for lipid peroxidation, which increases in stress conditions (Debasis et al. 2007). In our current study, spray treatment of Flaxseed plants with nanTiO2 at specific concentrations showed that the treated plants inhibited H2O2 accumulation under both optimum and stress conditions. However, a significant increase in H2O2 level at some treated plants as well as control indicates the higher considerable development of oxidative stress. These findings are in agreement with the results of Lu et al. (2002), who reported that nanosilver treated plants enhance an efficient cellular electron exchange mechanism, which decreases the ROS generation and MDA accumulation as well. Recently, it has been reported that nano TiO2 not only induced oxidative injury but also decreased electrolyte leakage and subsequently ameliorated membrane damage index under cold stress conditions in Chickpea plants (Mohammadi et al. 2013). It has been suggested that increased level of MDA content at 4 mM dose of nano-TiO2 could be involved as one of the major mechanism leading to DNA damage in Allium cepa plant, which confirms the genotoxic potential of nano-TiO2 particles in plant systems (Ghosh et al. 2010).

Drought stress, similar to other environmental abiotic stresses cause oxidative injuries to plant cell via generation of ROS such as H2O2, OH−, and O2−. According to our data, MDA content in the leaves of Flaxseed plants significantly increased in drought stress conditions, although the lipid peroxidation level was very low in plants treated with nanoparticles and in well-watered conditions, which arised from the results of H2O2 content. It has been reported that application of engineered nanomaterials may decrease or eliminate the production of H2O2 through the enhanced efficiency of redox reactions and or activation of H2O2 metabolizing enzymes (Priyadarshini et al. 2012). Our results revealed that nano TiO2 particles at appropriate doses decreased H2O2 accumulation, which subsequently prevent chlorophyll degradation and or stimulate its biosynthesis. This phenomenon may protect photosynthetic processes of stressed plants. Significant negative correlations among photosynthetic pigment contents and oxidative damage features namely MDA and H2O2 verify such responses of Flaxseed plant.

No previous literature is available regarding the effects of nano-particulates on seed reserves and biochemical variations including protein and oil contents. Therefore, our findings suggest for the first time that nano TiO2 application enhanced seed oil and protein contents of Flaxseed plants under optimal and water-deficit stress conditions. Our previous study showed that nano-scale silver up to 60 mg l−1 significantly leaf protein content in Pelargonium zonale cultivars (Hatami and Ghorbanpour 2014). Also, we recently found that nano TiO2 may change the stage of Pelargonium graveolens growth and their secondary metabolites content including essential oils (Ghorbanpour and Hatami 2015). From the results obtained, it can be concluded that foliar treatment of Flaxseed plant with nano TiO2 at proper concentrations could act as modifier to alleviation of deleterious effects of drought stress on physiological processes through changing the levels of MDA and H2O2 and stability of plastid pigments. It also plays a key role to up-regulate the phytochemical production, which is critical for plant tolerance against stress.