*3.3. 24-Epibrasinolide and Titanium Oxide Nanoparticles Increase Nonenzymatic Antioxidant Activities (Flavonol, Tocopherol, and Total Phenolics) in Bamboo Species under Cu and Cd Toxicity*

The effects of TiO2 NPs and EBL concentrations on nonenzymatic activity (flavonol, tocopherol, and total phenolics) in the bamboo species revealed a significant difference between the co-application of 24-epibrassinolide and titanium oxide nanoparticles with Cu and Cd (*p* < 0.001). According to the results, the combination of TiO2-HMs and EBL-HMs significantly increased nonenzymatic antioxidant activities in our bamboo species. However, the greatest increase in nonenzymatic activity under heavy metal stress was related to the combination of TiO2 –EBL with Cu and TiO2–EBL with Cd, with a 1.55-fold and 1.51-fold enhancement in flavonols, 1.53-fold and 1.51-fold enhancement in tocopherols, and 1.68-fold and 1.58-fold increase in total phenolics, respectively, in comparison with the control treatment (Figure 3). Conversely, the concentrations of 100 μM Cu and 100 μM Cd clearly reduced nonantioxidant activity, as demonstrated by a 21% and 23% reduction in flavonols, 12% and 24% reduction in tocopherols, and 34% and 28% reduction in total phenolics, respectively, in comparison with the control treatment. We suggest that the combination of TiO2 and EBL has a positive impact on the reduction in heavy metal toxicity by stimulating nonenzymatic antioxidant activities (flavonols, tocopherols, and total phenolics).

**Figure 2.** The impact of the co-application of 24-epibrassinolide and titanium oxide nanoparticles individually and combined on malondialdehyde content (MDA) (**a**), hydrogen peroxide (H2O2) (**b**), superoxide radical (O2 •−) (**c**), soluble proteins (SP) (**d**), and electrolyte leakage (EL) (**e**) in bamboo species (*Pleioblastus pygmaeus*) with 100 μM Cu and 100 μM Cd. In this study, 1-year-old branches of

*P. pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide, individually and in combination with 100 μM Cu and 100 μM Cd using four replications. Planting of the treated bamboo was performed in an Air Tech inoculation hood with fluorescent white lamps and ultraviolet light (wavelengths of 10–400 nm) at 15 ◦C and 30 ◦C. The bamboo plants were constantly exposed to excess heavy metals for three weeks. Sampling for the measurement of MDA, H2O2, O2 •−, SP, and EL (**a**–**e**) was conducted after three weeks of plant exposure to the coapplication of 24-epibrassinolide and titanium oxide nanoparticles under 100 μM Cu and 100 μM Cd. The capital letters (A–C) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide involving individual or combined application of titanium oxide nanoparticles (EBL–TiO2 NPs) under 100 μM Cu and 100 μM Cd (the bars with similar colors), while the lowercase letters (a–c) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs, individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).

**Figure 3.** The impact of the co-application of 24-epibrassinolide and titanium oxide nanoparticles individually and combined on nonenzymatic antioxidant activities ((**a**) flavonols, (**b**) tocopherols, (**c**) total phenolics) in bamboo species (*Pleioblastus pygmaeus*) with 100 μM Cu and 100 μM Cd. In this study, 1-year-old branches of P. *pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide, individually and in combination with 100 μM Cu and 100 μM Cd,

using four replications. Planting of the treated bamboo was performed in an Air Tech inoculation hood with fluorescent white lamps and ultraviolet light (wavelengths of 10–400 nm) at 15 ◦C and 30 ◦C. The bamboo plants were constantly exposed to excess heavy metals for three weeks. Sampling for the measurement of flavonols, tocopherols, and total phenolics (**a**–**c**) was conducted after three weeks of plant exposure to the co-application of 24-epibrassinolide and titanium oxide nanoparticles under 100 μM Cu and 100 μM Cd. The capital letters (A–C) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide involving individual or combined application of titanium oxide nanoparticles (EBL–TiO2 NPs) under 100 μM Cu and 100 μM Cd (the bars with similar colors), while the lowercase letters (a,b) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs, individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).

#### *3.4. 24-Epibrassinolide and Titanium Oxide Nanoparticles Improve Photosynthetic Pigments and Fluorescence Parameters in Bamboo Species under Cu and Cd Toxicity*

Photosynthetic pigments and fluorescence parameters are important indices in the evaluation of photosynthetic efficiency in different species of plants under stress conditions. The indicators of plant photosynthesis performance, including photosynthetic pigments (Chl a, Chl b, and total Chl, as well carotenoid contents), and fluorescence indices, including the maximum photochemical efficiency of PSll (Fv/Fm), photochemical quenching coefficient (qP), effective photochemical efficiency of PSll (Fv /Fm ), actual photochemical efficiency of PSll (ϕPSll), and nonphotochemical quenching (NPQ), were measured. We found a significant difference between the co-application of 24-epibrassinolide and titanium oxide nanoparticles with Cu and Cd (*p* < 0.001). Based on the results, the levels of TiO2NPs and EBL alone and in combination with heavy metals (Cu and Cd) could increase photosynthetic pigments in bamboo under Cu and Cd. However, the greatest increase in photosynthetic pigments under Cu and Cd was attributed to the co-application of TiO2–EBL with Cu and the co-application of TiO2–EBL with Cd, which resulted in 21% and 17% increases in Chl a, 85% and 83% increases in Chl b, 42% and 38% increases in total Chl, and 46% and 39% increases in carotenoid, respectively, in comparison with their control treatments (Table 2). Conversely, the measurement of the fluorescence parameters demonstrated a significant difference between the combination of TiO2–EBL and Cu and Cd (*p* < 0.001). The data analysis revealed similar results, e.g., in the Chl and carotenoid contents and in the measurement of fluorescence parameters. Therefore, the greatest increase in fluorescence parameters was related to the combination of TiO2 and EBL, which resulted in a 50% increase in the maximum photochemical efficiency of PSll (Fv/Fm), 41% increase in the photochemical quenching coefficient (qP), 54% increase in the effective photochemical efficiency of PSll (Fv /Fm ), 56% increase in the actual photochemical efficiency of PSll (ϕPSll), and 58% increase in nonphotochemical quenching (NPQ) in comparison with their control treatments. We suggest that the combination of TiO2 NPs and EBL has a strong ability to increase photosynthesis parameters in plants exposed to heavy metal stress (Cu and Cd) (Figure 4).


**Table 2.** The effect of the co-application of 24-epibrassinolide and titanium oxide nanoparticles individually and combined on photosynthetic pigments (Chl a, Chl b, and total Chl, as well as carotenoid contents) in bamboo species (*Pleioblastus pygmaeus*) with 100 μM Cu and 100 μM Cd.

In this study, 1-year-old branches of *P. pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide individually and in combination with 100 μM Cu and 100 μM Cd using four replications. Planting of the treated bamboo was performed in an Air Tech inoculation hood with fluorescent white lamps and ultraviolet light (wavelengths of 10–400 nm) at 15 ◦C and 30 ◦C. The bamboo plants were constantly exposed to excess heavy metals for three weeks. Sampling for the measurement of photosynthesis pigments was conducted after three weeks of plant exposure to the co-application of 24-epibrassinolide and titanium oxide nanoparticles under 100 μM Cu and 100 μM Cd. The capital letters (A,B) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide with titanium oxide nanoparticles (EBL–TiO2 NPs) individually or in combination with 100 μM Cu as well as 100 μM Cd (the bars with similar colors), while the lowercase letters (a,b) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).

#### *3.5. 24-Epibrasinolide and Titanium Oxide Nanoparticles Reduce Heavy Metal Accumulation in Bamboo Leaves, Stems, and Roots*

The decrease in metal accumulation in different types of plants is one of the main mechanisms responsible for the reduction of metal toxicity and increase in plant resistance when exposed to oxidative stress. Our results showed that TiO2 and EBL had a positive impact on the reduction in heavy metal concentrations in bamboo species; therefore, TiO2 and EBL alone or in combination significantly reduced heavy metal accumulation in leaves, stems, and roots (Table 3). This phenomenon is related to the role of TiO2 as a physical barrier that leads to a reduction in metal translocation from roots to aerial parts. The roots in plants are typically the first contact points where exposure to heavy metals occurs. Therefore, root physical traits are extremely decisive in limiting metal entry into plants. The root-based cellular layers comprised of epiblema, endodermis, and exodermis form apoplastic barriers in the roots, which can restrict heavy metal uptake by plants. We hypothesized that TiO2 NPs, through strengthening the apoplastic barriers in the roots and enhancing their impermeability, can significantly diminish the uptake of heavy metals. On the other hand, TiO2 NPs with high adsorption capacity act as an efficient binder of metal ions. Hence, TiO2 NPs can restrain the movement of heavy metals within the extracellular or intercellular parts of roots, thereby restricting heavy metal translocation from the root to shoot. TiO2 NPs may also have the ability to influence the expansion of the epidermal layer in plants, preventing heavy metal accumulation in nonphotosynthetic tissues by providing additional physical resistance. Conversely, we indicated that EBL, a hormone that is involved in plant growth regulation alone and in combination with TiO2, plays a positive role in the stimulation of antioxidant activity, which can scavenge ROS components in plant organs and inhibit plant oxidative stress caused by heavy metal toxicity. As shown in Table 3, the combination of TiO2–EBL with heavy metals showed the greatest reduction in heavy metal accumulation in the leaves, stems, and roots of bamboo species (Table 3). We suggest that TiO2 and EBL can reduce heavy metal contents in plant leaves, stems, and

roots, thus demonstrating that the combination of TiO2 and EBL has the greatest impact on the decrease in metal toxicity.

**Figure 4.** The effect of the co-application of 24-epibrassinolide and titanium oxide nanoparticles individually and combined on fluorescence parameters, including the maximum photochemical efficiency of PSll (Fv/Fm) (**a**), effective photochemical efficiency of PSll (Fv /Fm ) (**b**), photochemical quenching coefficient (qP) (**c**), actual photochemical efficiency of PSll (ϕPSll) (**d**), and nonphotochemical quenching

(NPQ) (**e**) in bamboo species (*Pleioblastus pygmaeus*) with 100 μM Cu and 100 μM Cd. In this study, 1-year-old branches of *P. pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide individually and in combination with 100 μM Cu and 100 μM Cd using four replications. Planting of the treated bamboo was performed in an Air Tech inoculation hood with fluorescent white lamps and ultraviolet light (wavelengths of 10–400 nm) at 15 ◦C and 30 ◦C. The bamboo plants were constantly exposed to excess heavy metals for three weeks. Sampling for the measurement of fluorescence parameters (**a**–**e**) was conducted after three weeks of plant exposure to the co-application of 24-epibrassinolide and titanium oxide nanoparticles under 100 μM Cu and 100 μM Cd. The capital letters (A–C) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide involving individual or combined application of titanium oxide nanoparticles (EBL–TiO2 NPs) under 100 μM Cu and 100 μM Cd (the bars with similar colors), while the lowercase letters (a–c) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).

**Table 3.** The accumulation concentrations of titanium oxide nanoparticles and corresponding heavy metals (Cu and Cd) in bamboo shoots, stems, and roots.


In this study, 1-year-old branches of *P. pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide individually and combined with 100 μM Cu and 100 μM Cd using four replications. The capital letters (A–D) indicate significant differences between treatments of control (C), titanium (Ti), 24 epibrassinolide (EBL), and 24-epibrassinolide with titanium oxide nanoparticles (EBL–TiO2 NPs) individually or in combination with 100 μM Cu as well as 100 μM Cd (the bars with similar colors), while the lowercase letters ( a–c) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey's test (*p* < 0.05). 2–6 24-Epibrasinolide and titanium oxide nanoparticles increase plant biomass indices (root and shoot dry weight) and plant growth (length of shoot) in bamboo species under Cu and Cd toxicity.

To evaluate the plant growth rate under Cu and Cd toxicity, the plant biomass, including root and shoot dry weight, as well as the length of shoots, were measured. A significant difference was found for the co-application of TiO2–EBL and heavy metals (*p* < 0.001) (Figure 5). Therefore, TiO2 and EBL individually and in combination significantly increased plant growth and biomass under stressful conditions. Based on this result, the greatest increase in plant biomass and growth under heavy metal exposure was related to the combination of TiO2 and EBL, which resulted in a 21% increase in the dry weight of shoots, a 23% increase in the dry weight of roots, and a 19% increase in the length of shoots in comparison with their control treatments (Table 4). Conversely, the measurements showed that the lowest plant growth was recorded under 100 μM Cu and 100 μM Cd, which resulted in 0.5 g and 0.46 g dry weights of shoots, 0.59 g and 0.53 g dry weights of roots, and 10.04 cm and 9.21 cm shoot lengths, respectively (Table 4).

**Figure 5.** The impact of the co-application of 24-epibrassinolide and titanium oxide nanoparticles individually and combined on the dry weight of shoots (**a**), dry weight of roots (**b**), and shoot length (**c**) of bamboo species (*Pleioblastus pygmaeus*) with 100 μM Cu and 100 μM Cd. In this study, 1-year-old branches of *P. pygmaeus* were used as plant treatments together with 100 μM TiO2 NPs and 10−<sup>8</sup> M 24 epibrassinolide individually and combined with 100 μM Cu and 100 μM Cd through four replications. Planting of the treated bamboo was performed in an Air Tech inoculation hood with fluorescent white lamps and ultraviolet light (wavelengths of 10–400 nm) at 15 ◦C and 30 ◦C. The bamboo plants were constantly exposed to excess heavy metals for three weeks. Sampling for the measurement of biomass indexes and plant growth (**a**–**c**) was conducted after three weeks of plant exposure to the co-application of 24-epibrassinolide and titanium oxide nanoparticles under 100 μM Cu and 100 μM Cd. The capital letters (A–C) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide involving individual or combined application of titanium oxide nanoparticles (EBL–TiO2 NPs) under 100 μM Cu and 100 μM Cd (the bars with similar colors), while the lowercase letters (a–c) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).


**Table 4.** The changes in bamboo biomass in root and shoot dry weight as well as shoot length with 24-epibrassinolide and titanium oxide nanoparticles individually or combined with 100 μM Cu and 100 μM Cd in comparison with the control treatment.

3–7 24-Epibrasinolide and titanium oxide nanoparticles reduce the translocation factor (TF) and bioaccumulation factor (BAF) and improve the tolerance index (TI) in roots and shoots of bamboo species. ↑ indicates increases and ↓ indicates decreases.

The translocation factor (TF) is one of the main mechanisms used to evaluate the remediation efficiency of heavy metals in plant organs and the reduction of toxicity in plants under heavy metal stress. Therefore, it is calculated according to differences in the accumulation of Cu and Cd in shoots and roots, and it serves as an important factor in increasing plant tolerance to toxicity. In the present study, the addition of 24-epibrassinolide and titanium oxide nanoparticles significantly reduced Cu and Cd translocation from roots to shoots, which led to a reduction in toxicity by limiting metal accumulation in the plant aerial organs. Therefore, according to the results, the co-application of 24-epibrassinolide and titanium oxide nanoparticles in combination with heavy metals (Cu and Cd) resulted in a low level of translocation factor, which could reduce metal toxicity in the aerial parts of bamboo plants (Table 5). Additionally, the result showed that the co-application of EBL and TiO2 NPs significantly reduced Cu and Cd concentration in the leaves (*p* < 0.001), implicating the positive role of EBL–TiO2NPs in the reduction of heavy metal toxicity in the aerial parts of the bamboo plant (Table 5). Conversely, the calculation of the tolerance indices in shoots and roots revealed a significant difference between the co-application of TiO2 NPs and EBL alone under Cu and Cd (*p* < 0.001). Therefore, the levels of TiO2 NPs and EBL indicated an increase in shoot and root tolerance under heavy metal stress, which was obtained by the amelioration mechanism of the co-application of TiO2 and EBL against heavy metal toxicity, such as the stimulation of antioxidant activity and the increase in plant biomass. We suggest that TiO2 NP and EBL concentrations alone increase plant tolerance under metal stress; however, the most positive effect was more pronounced with the co-application of TiO2 NPs and EBL under Cu and Cd toxicity (Table 5).

**Table 5.** Changes in the translocation factor and tolerance index of shoots and roots in response to 24-epibrassinolide and titanium oxide nanoparticles individually or in combination with 100 μM Cu and 100 μM Cd compared with the control treatment. Each data point is the mean ± SE of four replicates. The capital letters (A–C) indicate significant differences between treatments of control (C), titanium (Ti), 24-epibrassinolide (EBL), and 24-epibrassinolide involving individual or combined application of titanium oxide nanoparticles (EBL–TiO2 NPs) under 100 μM Cu and 100 μM Cd (the bars with similar colors), while the lowercase letters (a–c) denote statistically significant differences at each concentration of the co-application of EBL and TiO2 NPs individually or in combination with 100 μM Cu and 100 μM Cd (the bars with various colors) based on Tukey s test (*p* < 0.05).

