Introduction

Nanoparticles (NPs) have been applied worldwide, posing substantial impacts on the environment and its living organisms. Plants as sessile organisms are always exposed to considerable fluctuations of NP concentrations in air, water, and soil, so that studies have reported effects of nanoparticles on higher plants, but most are focused on microorganisms and animals/human cells. Recently, TiO2 NPs have been found to induce seed germination and plant growth [1, 2]. Furthermore, the presence of these NPs was observed to increase the dry weight, chlorophyll synthesis, and metabolisms in photosynthetic organisms [3]. Therefore, there is potential for expanding the range of TiO2 NP use for improvement of physiological and morphological characteristics of crops [4–7]. However, some studies have reported negative effects of TiO2 NPs on higher plants [8, 9] that varied between plant tissues, growth stages, plant species, applied concentrations, and specific properties of NPs. Thus, the exploration of their extensive application in agriculture and plant science is still in debate [10, 11]. Also in the case of TiO2 and other NPs in most previous research, interactions of NPs with plants usually were studied under optimum conditions, and no studies have previously been conducted under various extreme factors like environmental stresses. Thus, to support wide applications of TiO2 NPs, their possible role must be evaluated based on new research under cold stress (CS) conditions. This is the first report on the effects of TiO2 NPs in plants under CS.

CS, like other types of abiotic and biotic stresses, induces oxidative processes in plant cells. These processes are initiated by reactive oxygen species (ROS), which interact nonspecifically with many cellular components, triggering peroxidative reactions and causing significant damage to essential macromolecules, such as photosynthetic pigments, proteins, nucleic acids, and lipids, and especially damaging the membranes as the primary site of cold injury [12]. Plasma membrane is the primary site of cell that plays a crucial role in the perception, transduction, and transport of environmental signals. Environmental changes affect the functions of cell membranes, and, as a result, either the cell adapts to these changes or it would kill it. For adapting to these changes, organisms use factors by which the cell membrane modulates its physical state. These responses are due to reprogramming of gene expression which results in the adjusted metabolic alterations [13]. It can be interesting to plant physiologists to study the effects of NPs on cell membranes following CS and also can be important for understanding how NPs can change plant responses under CS. Some reports showed NPs’ ability to participate in cellular redox reactions. Along with standard tests for evaluation of NP effects in plant species, membrane damage tests may be appropriate, because some studies showed that seed germination and root elongation may not be sensitive enough or appropriate when evaluating NPs to plant species [14]. Previous studies showed that TiO2 NPs by induction of plant antioxidant systems alleviate accumulation of malondialdehyde (MDA) content [15]. MDA, one of the final products of lipid peroxidation of unsaturated fatty acids can also be considered as an evaluation factor of membrane damage. Increased rates of solute and electrolyte leakage index (ELI) under CS can be used as membrane damage index because of phase transition of membrane lipids. Thus, detecting ELI and MDA levels can be helpful in comparing the amount of damage created by CS and the amount of NP concentrations in the plant. Given the positive effects of TiO2 NPs on the biochemical and physiological properties of plants that probably enhance chickpea tolerance to cold-induced oxidative stress, this research has studied the effect TiO2 NP concentrations on indexes of injury (ELI and MDA) following cold-induced oxidative stress in chickpea genotypes.

Materials and Methods

Characterization of TiO2 NPs

TiO2 nanoparticles were purchased from Nanoyo Group Pte, Singapore, Malaysia. The size of the TiO2 NPs was examined using FESEM (Hitachi, S-460, Japan) in the University of Tehran, Iran. For SEM analysis, TiO2 NP samples were directly mounted to the sample holder with a piece of electrically conductive glue. The size of NPs was estimated to be 7–40 nm (Fig. 1). The crystal properties of TiO2 NPs were examined by X-ray diffraction (XRD) (XPert PRO MPD, PANalytical) in the 2θ range of 30°–120° operated at a voltage of 40 kV and a current of 40 mA. The XRD measurement showed that used TiO2 NPs were all present in the anatase form (Fig. 2). This form was considered to be more toxic than an equivalent sample of rutile TiO2 [16].

Fig. 1
figure 1

SEM image of TiO2 NPs. The aggregate particle size (7–40 nm) in diameter of TiO2 NPs (bar, 300 nm)

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Fig. 2
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XRD pattern of TiO2 nanoparticles. XRD measurement showed that used TiO2 NPs were all in the anatase phase

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TiO2 Bioaccumulation in Chickpea Leaves

Treated leaves were cut, and samples were washed in distilled water to remove electrolytes and soil from their surface, then oven-dried at constant 70 °C for 24 h, and digested on a hot block with concentrated HNO3 for 1 h at 60 °C. The samples were cooled to room temperature and diluted with distilled water before analysis. TiO2 bioaccumulation in the digested samples was performed by using inductively coupled plasma mass spectrometry (ICP-MS, HP-4500, USA) [17, 18].

Transmission Electron Microscopy of Chickpea Cells

Leaf samples from experimental treatments were fixed in 2 % (v/v) glutaraldehyde in phosphate buffer (pH 7.2) for 4 h at 4 °C. Samples were washed three times with buffer at 30-min intervals and fixed in 1 % (m/v) osmium tetroxide (OsO4) solution for 1 h at 4 °C. The samples were then washed three times with buffer and dehydrated through a grade series of ethanol/water (50, 70, 95, and three times in 100 % ethanol, v/v) 10 min in each solution. Ethanol was replaced by acetone three times, 10 min each time. Specimens were then embedded in EPON (Shell, London, UK) via a series of resin/acetone mixtures (1:1, m/v), followed by one change of resin/acetone mixture (1:2, m/v). Finally, the specimens were embedded in 100 % EPON for 1 h at room temperature. Resin was polymerized at 35, 45, and 60 °C (12 h each). Silver–gold sections (60–90) were cut on Reichert-Jung Supernova ultramicrotome by using a diamond knife. The sections were double stained with 4 % uranyl acetate in 50 % ethanol for 10 min and then with lead citrate for 5 min at room temperature. Transmission electron microscopy was performed on Zeiss EM 900 operating at 80 kV [19].

Plant Material and Growth Conditions

Seeds of two chickpea (Cicer arietinum L.) genotypes, cold sensitive (ILC 533), and cold tolerant (Sel 11439), were provided by Dryland Agriculture Research Institute of Iran in Maragheh City of Azerbaijan province. Nanoparticles of TiO2 solutions were prepared at concentrations of 0, 2, 5, and 10 ppm with filtered, double-distilled water. Working solutions were made by vigorous vortexing when required. Seeds sterilized with 10 % sodium hypochlorite for 5 min were washed five times, soaked in distilled water for 30 min, and then germinated in Petri dishes on filter paper at 0, 2, 5, 10 ppm TiO2 NP solutions (concentration treatments) and distilled water (control treatment) for 72 h at 25 °C in darkness. Ten milliliters of TiO2 different solutions was added to the Petri dish. Subsequently, the seedlings were planted in pots containing soil, sand, and farmyard manure at the rate of five seeds per pot [20]. Plants were grown in a growth chamber at 25 °C, with an irradiance of 200 μmol m−2 s−1 from white light luminescent lamps, a 16-h photoperiod, and 75 % relative humidity for 21 days. These seedlings were approximately 20 cm in height with at least five branches of 5–8 cm. Nanoparticle solutions were sprayed twice in the 12th and 16th days on plant leaves. Plants were transferred to another growth chamber (Chilling chamber, Arvin Tajhiz Espadana, Isfahan, Iran) with low temperature 4 °C for 2 days, and then samplings were conducted. All measurements were made on the middle leaves from the apex of all plants in each treatment. The seedlings were considered to be of similar physiological age [21]. Samples from unstressed plants were collected as control condition sample. Physiological experiments consisting electrolyte leakage index and lipid peroxidation assay were conducted using fresh leaves.

Root Growth Inhibition Test and Determination of EC50

The root growth inhibition test was performed according to Ghosh et al. [22]. The seeds of chickpea were germinated in the Petri dish according to the mentioned method. The root lengths (10 seeds per Petri dish, with three replications) were recorded. The germinated seeds were then exposed to different concentrations of Tio2 NPs (0, 2, 5, 10, 50, 100, 150, 200, 300, 400, and 500 ppm). The test solutions were replaced by fresh solutions every 24 h of exposure with final root measurement taken at the end of the 72 h of exposure. The EC50 (50 % inhibition of root length) value of TiO2 NPs was found to be 500 ppm.

Electrolyte Leakage Index

Cold tolerance was assessed by ELI in damaged tissues harvested in thermal treatments [23]. The leaf samples of 80-mg fresh mass (FM) were cut into two pieces, and then they were placed in glass tubes inclusive of 10 ml of distilled water. Samples were subjected to vacuum infiltration by the time all mesophil parts of the leaves were filled by water. Tubes were capped and placed on a shaker (150 rpm) for 30 min. The electrical conductivity (micro-Siemens per centimeter) of the extract containing released ions was measured by a digital conductivity meter (WTW TetraCon 325, InoLab Cond Level 1, Weilheim, Germany) at 25 °C. In the second stage, the tubes with their contents transferred to a boiling water bath for 10 min followed by shaking for 30 min, and their electrical conductivity was measured. The ELI (I%) was calculated according to the following formula: \( I=\left( {{{{\left( {{L_{\mathrm{t}}}-{L_0}} \right)}} \left/ {{\left( {{L_{\mathrm{b}}}-{L_0}} \right)}} \right.}} \right) \times 100 \), where L t is electrical conductivity of the sample after thermal treatments, L 0 is electrical conductivity of the sample under control conditions, and L b is an electrical conductivity of the same sample after boiling.

Analysis of Lipid Peroxidation in Chickpea Leaves

The measurement of lipid peroxidation in leaves, the thiobarbituric acid (TBA) test, which determines MDA as an end product of lipid peroxidation, was assessed [24]. Leaflets of 250 mg FM were hemogenated in 2-ml extraction buffer TCA 1 % (w/v), centrifuged at 13,000×g for 15 min. One milliliter of the supernatant was added to 2 ml of 5 % (w/v) TBA in 20 % (w/v) TCA. The mixture was incubated in boiling water for 30 min, and the reaction was stopped by placing the samples in an ice bath. Then the samples were centrifuged at 10,000×g for 10 min, and the absorbance of the supernatants was measured at 532 nm with a spectrophotometer (Shimadzu UV-160, Shimadzu Corporation, Kyoto, Japan). The amount of MDA was calculated using \( \mathrm{C}={{\mathrm{D}} \left/ {\mathrm{EL}} \right.} \), where C is the concentration of MDA, D is the optical density, E is the coefficient of molar extinction (1.56 × 105 cm−1 M−1), and L is the thickness of the layer of solution in the vessel (1 cm). Content of MDA was expressed in micromole per gram FM.

Statistical Analysis

Recorded data were processed by the analysis of variance in a 4 (concentration treatments) × 2 (genotypes) factorial experiment with three replications on the basis of completely randomized design. The data were analyzed using computer SPSS.19.0 software, and the means were compared by Duncan’s multiple range tests. Because nonsignificant differences in interaction between genotypes × concentration treatments was observed, we presented the results in the form of separate factors, and not combination.

Results and Discussion

The chickpea genotypes were first compared with respect to their responses to TiO2 NP concentrations under optimum temperature. The plants were also subjected to CS to establish their level of cold tolerance. In this work, we demonstrate that low concentrations of commercial TiO2 NPs change some physiological responses of chickpea seedlings for cold tolerance. Intracellular penetration of TiO2 NPs tracked by TEM analysis confirmed entry and localization of NPs in cells. It implies that NPs passed through the cell wall and plasma membrane. Plant cell wall acts as a barrier for entry of any external agent including NPs into plant cells. The sieving properties are determined by pore diameter of cell wall ranging from 5 to 20 nm [25]. Hence, only NPs or their aggregates with diameter less than the pore diameter of the cell wall could easily pass through and reach the plasma membrane [26, 27]. There is also a chance for enlargement of pores or induction of new cell wall pores upon interaction with NPs which in turn enhances their uptake. Thus, accumulation of NPs with a diameter larger than the cell wall pores is possible [28, 29]. Due to small size of TiO2, NPs would possibly pass through cell wall pores and distribute in subcellular compartments. Aggregation of NPs within the vacuole and chloroplast confirmed these observations that NPs absorbed in seedlings (Fig. 3). The uptake of TiO2 NPs by plants has also been shown earlier [1, 29, 30]; however, these reports did not explore the biological effects of such an uptake under abiotic stress like CS. The contents of TiO2 NPs in digested plant samples increased with increasing NP concentrations. ICP-MS data showed that, under thermal treatments, high content of TiO2 NPs was observed in ILC533 (sensitive) compared to 11439 (tolerant) genotype at different concentrations of NPs (Table 1). It is possible that the higher accumulation of TiO2 NPs in sensitive genotype under different concentrations of NPs may be associated with more membrane permeability. Other report also showed that sensitive genotype has thinner epiderm and larger stoma or their stoma is more open than that of the resistant genotype [31]. The higher accumulation of TiO2 NPs in sensitive genotype also was observed under CS treatment and different concentrations of NPs, so that TiO2 content was higher compared to optimum temperature. Previously, Maali Amiri et al. [20] and Heidarvand et al. [32] have shown that plants with higher tolerance levels showed less membrane injury. These results showed that higher accumulation of TiO2 NPs in sensitive genotype could be due to the direct and indirect effects of CS on plasma membranes [33, 34] and morphoanatomical characters [31] of chickpea leaves. On the basis of these data, it is expected that uptake of TiO2 NPs by chickpea seedlings possibly have the potential to induce physiological responses especially damage indexes during CS.

Fig. 3
figure 3

TEM observations of TiO2 NP distribution at 5 ppm concentration in chickpea leaves subjected to 4 °C for 2 days, showing the presence of nanoparticles in vacuole (V) and chloroplast (Chl). NPs are identified by arrows

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Table 1 Bioaccumulation of TiO2 nanoparticles (in microgram per gram DM) in the leaves of tolerant genotype Sel 11439 and sensitive genotype ILC 533 of chickpea at different concentration treatments (0, 2, 5, and 10 ppm) under optimum temperature and after 2 days of incubation in cold stress
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Many studies showed that NPs have negative effects at high concentrations, while different effects on cellular compartments were reported at low concentrations. In this study, the EC50 value of TiO2 NPs was found to be 500 ppm as compared to the control test. It was found that in Allium cepa, toxic effects of TiO2 NPs inhibit the root growth at concentration 319 ppm [22]. Therefore, we used low concentrations of TiO2, so that these concentrations did not have toxic effects on physiological and possibly molecular characters in chickpea. Mechanism of toxicity of NPs is unknown, but generation of ROS and oxidative stress are used to explain the toxicity [35–37]. Some reports showed that low concentrations of TiO2 NPs did not induce any global oxidative stress in plants and animals [3, 29, 38, 39]. In our study, it is possible that low concentrations of NPs under CS protect plants against ROS and alleviate damage caused by CS secondary oxidative stress. Membranes as the primary site of cellular injury can be, as a result of lipid peroxidation, caused by ROS. Our data confirmed that there was a close relationship between ROS formation and oxidative stress-induced damages in chickpea membranes [33, 40]. To ascertain the effect of TiO2 NPs on membrane integrity, MDA accumulation and ELI content (as damage indexes) were determined when decrease of temperature also occurred.

Figure 4 shows the results of the ELI analysis in experimental treatments. The leakage of intracellular electrolytes from ILC533 genotype was substantially higher than the leakage 11439 genotype under optimum temperature and different concentrations of TiO2 NPs (Fig. 4). This finding is in good relationship with previous data that have presented that tolerant genotypes showed lower leakage from sensitive ones not only under optimum but also CS conditions [20]. It can be seen that, under optimum temperature, the different concentrations of TiO2 NPs changed ELI content in two genotypes. Under optimum temperature, only in ILC533 genotype a nonsignificant increase was observed. The ELI content after a nonsignificant increase from 27.4 to 28.9 % (at 2 ppm) decreased significantly as a function of the following exposure concentrations (5 and 10 ppm) in 11439 genotype under these conditions. During optimum temperature, a decrease from 27.4 to 20.4 and 21.73 % in the ELI can indicate activation of tolerance mechanisms after TiO2 NP treatments (0 ppm compared to 5 and 10 ppm, respectively) in 11439 plants. Nonsignificant rising in ELI under optimum temperature at different concentrations of TiO2 NPs could point to sensing and transduction of the NP signal in cell membranes. Thus, it can be noted that TiO2 NP concentrations did not induce oxidative damage in the leaves of chickpea under optimum temperature. The release of 50 % of total electrolytes from the tissue is considered the index of cell death [41]. Treatment of 11439 plants with CS caused a significant increase of ELI at 0 and 10 ppm TiO2 concentrations (from 27 to 36.9 % and 21.73 to 28.7 %, respectively), whereas, except a nonsignificant increase from 28.9 to 30.5 % at 2 ppm, the content of ELI significantly did not change at 5 ppm TiO2 NPs. It was expected that ELI content, because of direct and indirect effects of CS, increased, but during CS, the content of ELI in the leaves of treated with TiO2 NPs was lower than that in plants treated with 0 ppm TiO2 NPs. Such results also were observed under optimum temperature that showed positive effects of TiO2 NP treatments on cellular responses. During CS, the ELI content reached the least at 5 ppm TiO2 NPs that was similar with the content of ELI under optimum temperature at 5 ppm TiO2. Positive effects of TiO2 NP concentrations also were observed in ELI decrease of ILC533 plants. Thus, our results indicate an improved tolerance of these plants to CS damage and oxidative stress induced by CS, which probably depends on the composition, properties of cell membranes, and the efficiency of ROS scavenging at different concentrations TiO2 NPs [13, 20]. The increase in the ELI in plants treated with 0 ppm shows tissue damage and, thus, their sensitivity to CS. To additionally show the damage extent of membranes, we measured lipid peroxidation level (MDA content) in genotypes under experimental treatments (Fig. 5). Comparing experimental data means of MDA has shown differences of genotypes at different concentrations TiO2 NPs under optimum temperature. Decrease in MDA content at 5 and 10 ppm TiO2 NPs brightly illustrates the induction of defense systems and ROS detoxification in tissues in the two genotypes under optimum temperature [6]. However, the MDA content from ILC533 genotype was substantially higher than the MDA in 11439 genotype under optimum temperature and different concentrations of TiO2 NPs that this finding confirmed ELI data in this study. During CS, MDA content in seedlings treated with 0 ppm TiO2 NPs compared to optimum temperature increased that indicated their sensitivity to CS. During CS in 11439 plants, we did not observe any increase in the MDA content at 2 and 10 ppm TiO2 NPs, whereas at 5 ppm TiO2 NPs, it even significantly decreased from 2.6 to 2.13. A decrease from 3.05 to 2.69 at 5 ppm TiO2 NP treatment of ILC 533 plants also was observed under CS compared to optimum temperature; however, at 2 and 10 ppm TiO2 NP treatments, a nonsignificant increase was seen between 3.58 to 4.1 and 3.22 to 3.53, respectively, in MDA content under CS in ILC533 genotype. It can be noted that at 5 ppm TiO2 NP treatment, the level of MDA reached lower than the seedling treated with 0 ppm TiO2 NPs under optimum temperature in the two genotypes. Previously, Maali Amiri et al. [42] have shown that the decreased MDA content under CS was related to improved tolerance to oxidative stress induced by CS as the enhanced unsaturated fatty acids in potato transgenic lines carrying desA transgene. In our study, the decline in MDA level could possibly be associated with induction of tolerance mechanisms by TiO2 NPs. The MDA content in plants treated with 0 ppm TiO2 NPs confirmed these results. We supposed that the higher cold tolerance of genotypes at TiO2 concentrations might be due to stabilization of the composition and physical properties of their membranes [43], which must be studied in details. However, because of the complexity of the stress response network and unknown effects of TiO2 NPs, other hypotheses could be considerable. Thus, the electrolyte leakage and lipid peroxidation assays can be used as marker for cold tolerance at different concentrations TiO2 NPs. However, it is valuable to note that biochemical and molecular studies of these plants are important in such programs and must be performed in detailed studies.

Fig. 4
figure 4

Effects of TiO2 NP concentrations on electrolyte leakage index in the leaves of tolerant genotype 11439 (a) and sensitive genotype ILC533 (b) of chickpea incubated under optimum temperature and 2 days after cold stress. Gray and white bars indicate thermal treatments: optimum (25 °C); cold stress (4 °C) for 2 days, respectively. The error bars represent the standard deviation (±SD) for replicates

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Fig. 5
figure 5

Effects of TiO2 NP concentrations on malondialdehyde in the leaves of tolerant genotype 11439 (a) and sensitive genotype ILC533 (b) of chickpea incubated under optimum temperature and 2 days after cold stress. Gray and white bars indicate thermal treatments: optimum (25 °C); cold stress (4 °C) for 2 days, respectively. The error bars represent the standard deviation (±SD) for replicates

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Conclusion

Our results showed that not only TiO2 NPs in low concentrations were not found to be detrimental to cell membrane, but also these findings have highlighted their positive effects on chickpea cells when they exposed to CS. This could be inferred from a lower damage on membrane (decreased levels of ELI and MDA) that probably can be along with decreasing of ROS in chickpea [33, 34, 40, 44]. It should be noted that TiO2 NP concentrations did not have any effect on the morphological characters, possibly because of short-term thermal treatments or low concentrations TiO2 NPs that should be studied in details. Thus, low concentrations of TiO2 NPs (especially 5 ppm) alleviated cold-induced damages in sensitive and resistant chickpea genotypes. Such results raise important questions about the possible mechanisms that manage these effects. We think that after absorption and perception of TiO2 NPs, some defense mechanisms in chickpea seedlings activated that could support plants to CS. It is interesting that there are such mechanisms in sensitive and tolerant genotypes that highlight the existence of a wide range of genetic capacity in chickpea genotypes to increase cold tolerance when environmental conditions change. The comparison of these responses could be useful in identifying differences associated with the relative capability of chickpea to cope with CS. Also, new findings possibly would be reveal the use of NPs generally or TiO2 NPs especially for increase of cold tolerance in crops.