Single clone 1-year-old branches of P. pygmaeus were selected as plant materials, which have been growing since 1982 at Nanjing Forestry University, Bamboo Garden (Nanjing, Jiangsu, China). For shoot production and shoot expansion, long nodal (10 mm) explants were grown under tissue culture conditions conducted in medium of Murashige and Skoog (1962) containing 0.5 ml, 4 ml, 7–10 g L⁻¹, and 30 g L⁻¹ kinetin, 6-benzyl amino purine, agar, and sucrose. In this regard, roots proliferated from young shoots that were used in glass petri dishes with a constant diameter (60 mm) to keep the MS medium containing 4 μM nicotinic acid, 0.6 mM myoinositol, 1.2 μM thiamine-HCl, 30 g L⁻¹ sucrose, 3 μM pyridoxine, 7–10 g L⁻¹ agar, and 1 mg L⁻¹ Indole-3-acetic acid (IAA) as growth hormones.

In this study, five concentrations of ZnO-NPs (0, 50, 100, 150, and 200 μM) alone or in a combination with two types of toxic metals/metalloids (150 μM As and 150 μM Hg) were used in a completely randomized design (CRD) through four replications. The total duration of the experiment was 3 weeks. Thus, different treatments including ZnO-NPs and As/Hg in a form of powder were added to 1 L MS medium with 30 g of sucrose. The As was prepared from sodium arsenate heptahydrate powder (Na₂HAsO₄.7H₂O) and the Hg was from the salt of white Hg powder (HgCl₂). After the regulation of the MS medium in an appropriate pH (5.8 ± 0.1), 7–10 g L⁻¹ agar was added. Then, the MS medium was transferred to a microwave oven (China Energy Label) for 30 min at an optimum temperature of 120°C. Sterilization of the MS medium was conducted by an autoclave (HiClave HVE-50; ZEALWAY). For the bamboo plantation, an inoculation hood (Air Tech), fluorescent white lamps, and UV light (wavelengths of 10–400 nm) were used at temperatures of 15 and 30°C. Finally, the planted bamboos were preserved in a special chamber room under controlled conditions for 3 weeks.

ZnO-NPs were provided by the Nanjing Jiancheng Company, Nanjing, Jiangsu Province, China. This material has the characteristics of a powder with high nano Zn purity and a diameter of less than 50 nm. In this study, the levels of ZnO-NPs and metals were selected according to the different ranges of bamboo tolerance obtained by our previous studies (Emamverdian et al., 2020a, 2021a,b).

At the end of the experiment, all bamboo samples were separated from the cleaned MS medium. For this study, antioxidant enzyme activities, thiobarbituric acid reactive substances (TBARS), H₂O₂ content, soluble protein (SP), proline (Pro), and glycine betaine (GB) content, total phenolics, flavonols, tocopherols, tyrosine ammonia-lyase (TAL) activity and phenylalanine ammonia-lyase (PAL) activity, electrolyte leakage (EL), lipoxygenase activity (LOX), Chl, carotenoid contents, ZnO-NPs, and metal accumulation in plant shoots, stems, and roots were measured carefully. The translocation factor (TF), shoot tolerance index, and root tolerance index (TI) were calculated. Then, a scanning electron microscopy (SEM) was used to observe the stomata in fresh leaves under metal stress. Finally, plant biomass indices such as root and shoot dry weight (DW) were measured.

Bamboo leaves (0.5 g) were crushed in a mortar and pestle, ground into powder, mixed into liquid nitrogen, and kept at 2°C–8°C. The obtained powder was dissolved in 2 mg of phosphate buffer (pH 7.8) inside a test tube. The obtained homogenate was centrifuged at 2,000–3,000 g for 20 min at 4°C. After centrifugation, the obtained supernatant was used for the measurement of antioxidant activities.

The activity of superoxide dismutase (SOD; E.C. 1.15.1.1) was quantified based on the method of Beauchamp and Fridovich (1971), which was achieved by the photoreduction obtained by nitroblue tetrazolium (NBT). Then, the supernatant (100 μl) was mixed with 50 mM phosphate buffer (pH 7.8), which was added to a solution containing 13 mM methionine, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 75 μM NBT, and 2 μM riboflavin. In the next step, the obtained solution was exposed to fluorescent lamps for 10 min. To determine the SOD, the absorbance was measured at 560 nm by a spectrometer. The activity of catalase (CAT; E.C. 1.11.1.6) was recorded by Urbanek et al. (1991). In this process, 0.1 ml of the extracted sample was dissolved in 3 ml phosphate buffer (pH 6.8) by using extinction coefficient of H₂O₂ in 39.4 M^-1 cm^-1. CAT activity was recorded by measuring the decline in absorbance at 240 nm. The activity of ascorbate peroxidase (APX; E.C. 1.11.1.11) was determined according to the method of Nakano and Asada (1981). Therefore, in this measurement, the reaction buffer solution including 50 mM potassium phosphate (pH 7.0) was added to the extracted sample, and then 0.1 mM EDTA, 0.1 mM H₂O₂, and 0.5 mM ascorbate were added to the solution. Hence, APX antioxidant activity was obtained by recording the reduction in absorbance at 290 nm (the coefficient of absorbance at 2.8 mM⁻ⁱ cm⁻¹). The activity of glutathione reductase (GR; E.C. 1.6.4.2) was measured by the method of Foyer and Halliwell (1976) with some modifications. For this index, the mixture consisted of extract samples which were added to 100 mM phosphate buffer (pH 7.8) and then mixed by 3.0 mM oxidized glutathione, 0.05 mM nicotinamide adenine dinucleotide phosphate (NADPH), 0.1 μM EDTA, 50 μl of the enzyme extract, and 1.0 ml NADPH oxidation was recorded in the absorbance of the 340 nm twice. The first time included the addition of H₂O₂, and the second time occurred 1 min later. The difference between the two data points was determined by the extinction coefficient of the NADPH molar (6.22 mM cm⁻¹). The activity of GR was obtained as U mg⁻¹ protein.

The activity of TAL was determined by the method of Berner et al. (2006). Therefore, 20 μl of the extracted sample was added to the solution that contained tyrosine (30 mM) and boric acid buffer (pH 8.5; 500 μl). Then, in the final step, the TAL activity was recorded by the absorbance at 310 nm after 30 min. p-Coumaric acid was used for the standard curve. PAL activity was measured according to the method of Berner et al. (2006). Therefore, 500 μl boric acid buffer (pH 8) was mixed in the 20 μl extracted samples, and the absorbance was recorded at 290 nm after 30 min. In this test, various levels of Ecinnamic acid were used as the standard curve.

The H₂O₂ content was determined by the method of Velikova et al. (2000). For this purpose, 0.1% (w/v) trichloroacetic acid (TCA; 5 ml) was homogenized with leaf samples (0.5 g). Then, it was centrifuged at 12,000g for 15 min. In the next step, supernatant (0.5 ml) was added to 10 mM potassium phosphate buffer (pH 7.0; 0.5 ml) and 1 M potassium iodide (1 ml). In final step, the absorbance at 390 nm was recorded. For the calculation of H₂O₂, one standard calibration curve was used. The estimation of lipid peroxidation was made by the TBARS content, which is used as a cell peroxidation indicator. In addition, it was used by the method of Cakmak and Horst (1991). In this experiment, 0.5 g of leaf samples were crushed in containers of 0.1% (w/v) TCA (5 ml). As for the next step, the mixture was centrifuged at 12,000g for 7 min. The obtained supernatant was added to 4 ml of 0.5% (w/v) TBA and 4 ml of 20% (w/v) TCA and kept at 90°C for 30 min. In the following step, the mixture was centrifuged at 10,000g for 5 min. Then, the TBARS content was recorded by absorbance at 532 nm. The TBARS content was determined as μM g⁻¹ leaf FW. The lipoxygenase (LOX) activity was determined based on the methods of Grossman and Zakut (1979) and Sekha and Reddy (1982). Using these methods, 25 ml of the 0.1 M sodium tetraborate containing 0.1% Tween 20 was added to 10 μl linoleic acid. Then, 0.1 ml of the solution was added to an optimal amount (2.9 ml) of 0.1 M phosphate buffer of pH 4–5. EL was measured according to the method of Valentovic et al. (2006). Using this method, 0.3 g of leaf sample were added to 15 ml of deionized water. Then, the samples were preserved at 27°C for 2 h. Then, the EC₁ (electrical conductivity) of the solution was recorded. In the next step, the samples were kept in one autoclaved at 120°C for 17 min. Finally, (EC₂) electrical conductivity was recorded again. The EC was obtained as follows:

The content of GB was determined by the method of Grieve and Grattan (1983). The samples (leaves) were placed in an oven to dry at a controlled temperature (80°C). The dried samples were finely ground with deionized water at 110°C for 40 min. The GB concentration was recorded at 365 nm, which was obtained by the mixture of dry leaf powder after reaction with KI–I₂.

The proline content was measured based on the ninhydrin method (Bates et al., 1973). Therefore, 400 mg of the samples (leaf) were homogenized in sulfosalicylic acid. In the next step, 3 ml glacial acetic acid and 3 ml acid ninhydrin were added to the mixture. Then, the solution was heated at 110°C. The supernatant was extracted with toluene, and the free toluene was measured by absorption at 528 nm. In this experiment, the standard used L-proline.

Dry leaf samples (0.5 g) were mixed in 80% methanol (5 ml) and then centrifuged at 7,000g for 15 min. Methanolic extract was obtained for use in the experiment. The content of total phenolics was measured by the Akkol method (Akkol et al., 2008). For this purpose, 2.5 ml of 10% Folin–Ciocalteu reagent was added to 0.1 ml of methanolic extract. Then, the obtained mixture was neutralized by 7% sodium bicarbonate. In the final step, the content of total phenolics was recorded by absorbance at 765 nm. The results were expressed by using gallic acid calibration (mg GAE g⁻¹ plant material). The flavonol content was determined according to the methods of Akkol (Akkol et al., 2008). In this test, 0.5 ml methanolic extract was homogenized with 1.5 ml of 5% sodium acetate and 2% 0.4 ml aluminum chloride. The obtained supernatant was preserved for 2.5 h at normal room temperature, and then flavonoid content was recorded by absorbance at 445 nm. The calibration for rutin was used for the calculations and the results were expressed as (mg RE/g of plant material). The content of tocopherol was determined by the method of Kayden et al. (1973). In this study, 0.1 g of leaves and samples were added to 3 ml ethanol, and then the mixture was transferred to a centrifuge machine and centrifuged at 10,000g for 10 min. Next, the ethanol extract (0.1 ml) was added to 0.2 ml ferric chloride (0.001 M), 0.2 ml bathophe-nanthroline (0.2%), and 0.2 ml phosphoric acid (1 mM). The tocopherol content was obtained by recording the absorbance at 534 nm. TAC was determined by comparison with the tocopherol acetate standard calibration curve. The amount of TAC was expressed for extract samples in mM tocopherol acetate equivalent/g plant material.

Chlorophyll pigments including Chl a, Chl b, total Chl, and carotenoid contents were recorded according to the Lichtenthaler method (Lichtenthaler and Buschmann, 2001). For this study, 0.5 g of bamboo samples (leaf) were exposed to liquid nitrogen in a mortar and then crushed. Then, the obtained powder was mixed with 20 ml of 80% acetone at a temperature of 0°C–5°C. The mixture was extracted and centrifuged at 7,000 × g for 15 min. Finally, the obtained supernatant was placed in a UV/vis spectrometer. Therefore, Chl a, Chl b, and carotenoid contents were measured by recording the absorbance at 663, 645, and 470 nm. To obtain the final data, the following formulae were used, which are displayed in units of mg g⁻¹ fresh weight:

The quantities of ZnO-NPs and As and Hg in leaves, stems, and roots of bamboo species were measured in Nanjing Forestry University lab, and the sample preparations were according to the method of Karimi et al. (2013) with some modification. The different plant organs, including leaves, stems, and roots, were carefully washed and then dried at an oven temperature of 110°C for 5–8 h. Then, nitric acid (70%) was added to the samples at a temperature of 80°C for 15 min. Then, this process was continued by centrifugation of the samples at 10,000g for 7 min. For the determination of ZnO-NP contents and As and Hg accumulation in leaves, stems, and roots, the recordings were performed by atomic absorption spectrometry (AAS), which was equipped with a Zeeman-effect background correction system and a graphite furnace (Analyst 800, Perkin Elmer). These instruments were used to determine the metal accumulation through analysis. Determination of metal standards was conducted based on nitric acid (2.5%) using a spectral scan. The machine calibration of the standard (Perkin Elmer), a standard including all of the elements in an inorganic target analyst list (TAL), was run at optimum intervals in an unattended automatic analysis run mode.

In the last step of the experiment, bamboo samples were separated from MS medium and divided into two types of roots and shoot samples. For the analysis of physiobiochemical treatments in the future, a tiny fraction of the fresh shoots was stored under cold conditions at −80°C. The other parts of the plant root and plant shoot were placed in an oven to dry at the optimum temperature of 70°C for 48 h. Then, the plant biomass, including shoot dry weight (SHDW) and root dry weight (RDW), were obtained after weighing the samples.

To determine the performance of various concentrations of ZnO-NPs in coping with metal toxicity and to identify the involved mechanisms, the TF and TI were obtained. This was calculated based on the Souri method (Souri et al., 2020). The value was calculated by the following formulae:

The bamboo leaves were observed based on method of Li et al. (2016b) with some corrections and modifications. The central section of fresh leaves of the bamboo species was used for SEM. For this purpose, after the leaves were cut into 6.0 mm × 6.0 mm parts, the pieces were transferred to an oven and dried at 70°C for 20 min. A gold anion sputter apparatus (Model E-1010 Hitachi Ion Sputter JEOL, Japan) operated at 16 mA for 60 s was used to coat the samples. The middle section of leaves was scanned using a scanning electron microscope (SEM) system (JSM-6380, JEOL, Tokyo, Japan) operated at a voltage of 15–25 kV.

Data were analyzed by a two-way factorial design with four replicates. ANOVA was conducted using the R statistical software package. Tukey’s test was used for comparison of the mean differences between treatments at the p < 0.05 probability level.

The ANOVA showed that there were significant differences between the various levels of ZnO-NPs alone and in combination with As and Hg and antioxidant activities (SOD, CAT, APX, and GR; p < 0.001). Therefore, ZnO-NPs could increase antioxidant enzyme activity under As and Hg (Figure 1). The greatest increase in antioxidant activity was related to high levels of ZnO-NPs (200 and 150 μM), with 69%, and 59% increases in SOD activity, 95%, and 85% increases in CAT activity, 84%, and 65% increases in APX activity and 69%, and 57% increases in GR activity in comparison with their control treatments, respectively. Additionally, the results indicated that the treatments with 150 μM As and 150 μM Hg produced the lowest stimulation of the antioxidants, as shown by the SOD, CAT, APX, and GR activities, which were reduced by 59, 52, 63, and 40% by As and 75, 68, 89, and 46% by Hg, respectively, compared with their control.

Tyrosine ammonia-lyase and PAL are two important antioxidants that were measured in this study. Therefore, the results showed that the addition of ZnO-NPs alone and in combination with 150 μM As and 150 μM Hg significantly increased both TAL and PAL activities (p < 0.001; Figure 2). In this study, the greatest increase in TAL and PAL activities was related to 200 μM ZnO-NPs, with 35%, and 46% increases in TAL and PAL activities, respectively, compared with their control treatment. The lowest one was related to a concentration of 150 μM As and 150 μM Hg with 32%, and 48% reduction in TAL activities, and 50 and 60% reductions in PAL activities in comparison with control treatments. Therefore, we suggested that ZnO-NPs have the ability to increase TAL and PAL activities under metal/metalloid stress (Figure 2).

The investigation of the effect of HMs on plant membrane and cell peroxidation is important. For estimation, the impact of ZnO-NPs on ROS compounds and lipid peroxidation and the rate of plant membrane injury, H₂O₂ contents, TBARS, LOX, and EL in bamboo species were measured. The results showed that there was a significant difference between the various levels of ZnO-NPs alone and in the form of combinations with As and Hg metals (p < 0.001; Figure 3). Therefore, the results revealed that ZnO-NPs can reduce oxidative stress and membrane injury caused by As and Hg (Figure 3). In this study, the greatest reduction in H₂O₂ content, TBARS and LOX activity, as well as EL percentage, was related to 200 μM ZnO-NPs with 52, 56, 62, and 73% reductions compared with their control treatments. This showed the ability of ZnO-NPs to reduce membrane injury. Therefore, the combination of ZnO-NPs with metal/metalloid has a strong ability to reduce oxidative stress in plants, which can be related to the stimulation of antioxidant activities by separate concentrations of ZnO-NPs.

According to the results, the total phenolic, flavonol, and tocopherol contents reacted with an increasing trend upon the addition of ZnO-NPs. Therefore, there was a significant difference between the various levels of ZnO-NPs alone and in the form of combinations with As and Hg (p < 0.001; Table 1). In this study, the greatest enhancement of total phenol, flavonol, and tocopherol contents was related to 200 and 150 μM ZnO-NPs with 81, and 73% increases in total phenol and 37%, and 27% increases in flavonol and 44%, and 38% increases in tocopherol content in comparison with their control treatments. According to the results, the addition of ZnO-NPs could significantly increase nonenzyme antioxidants in bamboo plants, which can help to reduce ROS compounds and oxidative stress in plants. On the other hand, the total phenolic, flavonol, and tocopherol contents showed 29, 42, and 36% reductions by 150 μM As and 40, 59, and 54% reductions by 150 μM Hg relative to their control, respectively. This shows the toxic role of metals in reducing nonenzyme activities in the present study.

Previously, it had been reported that Pro and GB can be suitable indicators to measure metal toxicity and plant defense mechanisms’ abilities to respond to stress conditions. The results showed that ZnO-NPs significantly increased GB and Pro (p < 0.001), which indicates the role of ZnO-NPs in the reduction of As and Hg (Table 1). According to the obtained results, the greatest increase in Pro and GB was related to 200 μM ZnO-NPs, with 56%, and 55% increases relative to the control treatments, respectively. The lowest amount was related to 150 μM As and 150 μM Hg, with 46 and 59% reductions in Pro and 40 and 54% reductions in GB in comparison with their control treatment.

To evaluate the impact of ZnO-NPs on plant photosynthesis and plant metabolism after exposure to As and Hg toxicity, Chl indices, including Chl a, Chl b, and total Chl as well as carotenoid contents, were measured. In this study, the data recorded a positive impact of various levels of ZnO-NPs on the quantity of Chl indices and carotenoid contents (Figure 4), which demonstrated a significant difference between the various levels of ZnO-NPs alone or in the form of combinations of As and Hg (p < 0.001). Therefore, the greatest enhancement of Chl a, Chl b, total Chl, and carotenoids was related to 200 μM ZnO-NPs with 16, 35, 25, and 57% increases compared with their control treatments, respectively. As expected, 150 μM As and 150 μM Hg had a deleterious impact on Chl and carotenoid indices, which reduced Chl a, Chl b, total Chl, and carotenoid contents by 23, 42, 32, and 59% by As and 32, 60, 46, and 43% reduction by Hg in comparison with their controls, respectively.

In this study, plant biomass indices were demonstrated as indicators of plant growth and development, including the bamboo shoot and root dry weight. According to the obtained data analyses, ZnO-NPs significantly increased the dry weight of shoots and roots in bamboo species under two As and Hg. Therefore, the greatest increase in shoots and roots DW was related to the high concentration of ZnO-NPs (150 and 200 μM) with 0.12 and 0.15 g increases in shoots DW and 0.38 and 0.54 g increases in DW of roots in comparison with their control treatments. However, the levels of ZnO-NPs in combination with 100 μM As and 100 μM Hg showed the ability to increase shoots and roots DW in bamboo species (Figure 5; Table 2).

The data analysis obtained by this study demonstrated that there was a significant difference between the various levels of ZnO-NPs alone and in the form of combinations with As and Hg (p < 0.001). Therefore, the various levels of ZnO-NPs in combination with 150 μM As and 150 μM Hg could reduce the accumulation of metal/metalloid in plant leaves, stems, and roots. The greatest reduction was related to 200 μM ZnO-NPs in combination with 150 μM As and 150 μM Hg, with 66 and 59% reductions in the leaves, 66 and 61% reductions in the stem, and 23 and 47% reductions in the root in comparison with their control, respectively (Table 3).

In this study, to investigate the involved mechanisms in the reduction of metal/metalloid by ZnO-NPs, TF, and TI were calculated. According to the data from Table 4, the tested levels of ZnO-NPs did not significantly reduce the TF value for Hg (although a positive downward trend could be observed); in the case of As, there was a clear decrease in translocation in response to the use of ZnO-NPs, but no dose–response correlation was observed (no differences between the different levels of ZnO-NPs). On the other hand, the investigation of the tolerance index in shoots and roots demonstrated that ZnO-NPs remarkably increased plant tolerance in combination with As and Hg, and the results showed that ZnO-NPs in combination with As and Hg increased the tolerance index in bamboo plants by 7 and 4% enhancement in shoots and 37 and 18% enhancement in roots, respectively (Table 4).

As shown in Table 3, Zn in the form of ZnO-NPs reduced As and Hg accumulation, with alterations in metal/metalloid translocation from the roots to the shoots of bamboo. This phenomenon impacted the opening and closing of stomata in the leaves. Thus, ZnO-NPs could regulate the opening and closing of stomata in plants under As and Hg, which might be attributed to an enhancement in the antioxidant capacity in cells (Figure 6). We suggest that optimal levels of ZnO-NPs may regulate the function of stomatal pores via certain mechanisms, such as electrochemical and hydraulic adjustments in guard cells and osmotic pressure regulation. These processes may be related to the ability of ZnO-NPs to scavenge H₂O₂ and reduce the extent of lipid peroxidation by increasing the antioxidant capacity.