**4. Discussion**

Titanium, as a form of TiO2, has the ability to alter the bioavailability and behavior of metals in the environment [45]. The impact of TiO2 NPs on increasing antioxidant activity and plant growth has been reported in several studies [13,14,46]. This increase can be attributed to the inductive role of TiO2 NPs in enhancing signaling associated with the activation of antioxidant enzyme activity [13]. This finding is consistent with the reported results in the present study. Therefore, our results demonstrated that the individual levels of TiO2 NPs could increase antioxidant and nonantioxidant activity in bamboo plants under certain Cu and Cd levels. Conversely, the level of EBL regulates plant stress by stimulating antioxidant activity [47]. The increasing capacity of antioxidant activity based on the levels of EBL in plants under stress has been reported in many studies [48–50]. EBL seems to play a main role in the activation of genes responsible for antioxidants by stimulating the expression of genes responsible for SOD, CAT, and APX in plants exposed to heavy metal stress [51]. Hence, it is interesting to note that EBL has the ability to ameliorate oxidative stress caused by metals, which has previously been reported for many plant species, such as *Brassica juncea* [20], *Cicer arietinum* [52], and *Raphanus sativa* [53]. The main reason can be attributed to the role of BR signaling kinase (BSK 1) in the stimulation of salicylic acid levels against oxidative damage [54]. In our studies, the application of TiO2 and EBL individually and in combination enhanced antioxidant enzyme activities, including SOD, POD, CAT, GR, APX, and PAL. However, the combination of TiO2 NP and EBL was more effective in increasing antioxidant levels than TiO2 NPs and EBL alone. Phenolic compounds, as nonenzymatic antioxidant activities, alleviate the negative effect of reactive oxygen radicals and have a strong ability to chelate metals [55,56]. There seems to be a relationship between enhancing phenylalanine ammonia-lyase (PAL) and the total phenolic compound, and it has been reported that PAL is a key enzyme responsible for the activation of the synthesis of phenolic compounds under stress [57,58]. This phenomenon has been reported in some studies on the reduction of Cu and Cd toxicity [55,59]. Our results demonstrated that the application of TiO2 NPs and EBL individually and in combination increased nonantioxidant activity (total phenolics, flavonols, and tocopherols) under Cu and Cd toxicity. This phenomenon could be related to PAL gene transcript levels as well as increasing PAL activity in response to EBL levels under heavy metal stress, which ultimately reduces ROS compounds by synthesizing phenolic compounds.

When antioxidant activity increases, the plant experiences cellular injuries (H2O2, MDA, and EL) [60]. It has been reported that TiO2 NPs induce certain stress-combative mechanisms, such as an improvement of the defense mechanism against ROS accumulation in plant intercellular space, which has been shown to attenuate H2O2 induction [60]. An increase in MDA content has been described as the initial stage of plant injury, which shows the rate of membrane lipid peroxidation [61]. This reveals the extent to which plants face this serious problem. Based on the present findings, we suggest that TiO2 NPs protect the plant cell membrane from ROS, a phenomenon that has been related to the role of TiO2 NPs in boosting antioxidant activity. Conversely, EBL has the ability to positively alter the membrane structure and membrane stability in plants exposed to stresses, such as heavy metals, which leads to a reduction in membrane lipid peroxidation [62]. In one study, the level of EBL was observed to diminish the concentrations of H2O2 and MDA (20–60% reductions) in plants under Pb stress [63]. In another study, EBL diminished the oxidative toxicity in cowpea under Cd by reducing lipid peroxidation, MDA content, and electrolytes [64,65]. In the present study, the level of EBL improved the ROS content and reduced the plant cell membrane under heavy metal toxicity. It is interesting to note that in this study, the application of TiO2 NPs and EBL individually and in combination decreased ROS and lipid peroxidation, including H2O2, O2 •−, MDA, SP, and EL, in bamboo plants exposed to metal stress. One of the mechanisms underlying the amelioration of lipid peroxidation and ROS by EBL can be attributed to the increase in endogenous plant hormones that regulate plant growth, such as salicylic acid and ethylene, and the cross-talk between them. These mechanisms can improve plant tolerance under metal toxicity [66]. In this study, the co-application of TiO2 NPs and EBL was more efficient in reducing ROS compounds and ameliorating lipid peroxidation than TiO2 NPs and EBL alone.

Studies have indicated that TiO2 NPs enhance photosystem II in spinach by promoting oxygen evolution and energy transfer [67]. Additionally, TiO2 reduces Chl degradation and stimulates Chl biosynthesis, which can promote photosynthesis by stabilizing chlorophylls and carotenoids [60]. Chlorophylls are the most abundant component of the chloroplast and play an efficient role in the rate of photosynthesis [59]. The role of EBL in enhancing the cell number and photosynthetic pigment content (Chl a, Chl b, and carotenoids) has been demonstrated in some studies [68,69]. It seems that the increase in photosynthesis and Chl pigments by EBL is related to the stimulation activity of ribulose 1,5-bisphosphate carboxylase oxygenase as well as the increase in the Calvin cycle enzymes [20]. Therefore, the level of EBL with increasing carotenoids ameliorates photodamage during photosynthesis [70]. According to the above mechanisms, the co-application of EBL and Ti can improve photosynthesis and Chl pigment levels via Chl biosynthesis and reduce photodamage via the activation of the Calvin cycle enzymes in plants under heavy metal stress. Additionally, the results revealed a positive impact of the co-application of EBL and TiO2 NPs on fluorescence parameters, which showed an increase in the efficiency of 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). Therefore, we suggest that the co-application of EBL and TiO2 NPs can increase photosynthetic properties in plants exposed to heavy metals (Cu and Cd), and this phenomenon could be related to the increase in antioxidant activity and the reduction in heavy metal accumulation under metal toxicity stress.

Titanium is known to be the most abundant transition element after iron, with a level of 1–578 mg kg−<sup>1</sup> in different species of non-hyperaccumulator plants [71]. However, low mobility in the soil may impact its absorption by plants [72]. Our results showed that the root accumulation of TiO2 NPs was higher than that of stems and shoots, indicating that bamboo roots prefer to be storage organs of titanium, which has been reported in another study [73]. Therefore, the accumulation of titanium NPs led to the adsorption of heavy metals in roots, which, as a physical barrier, reduces metal translocation from roots to shoots. Conversely, EBL has the potential to reduce metal accumulation in plants by enhancing phytochelatin synthesis (PC) [48–50]. In a study on sugar beet, the levels of EBL reduced the absorption of heavy metals by plants by 50% [74–76]. It has also been reported [69] to reduce Cd accumulation in the roots, stems, and shoots of pea seedlings. Conversely, EBL levels reduce the accumulation of Cd by preserving ion homeostasis through the acceleration of calcium absorption [77,78]. Therefore, EBL leads to increased absorption of K+, mg+, and K+, which can be transported to the aerial parts of plants, such as leaves, and finally limit Cd and metal translocation from roots to shoots [79]. In the present study, the application of EBL and TiO2 could individually and in combination diminish the accumulation of heavy metals. These results are related to the role of EBL in preserving ion homeostasis, which can limit heavy metal uptake by coperception, which is associated with the role of TiO2 in the adsorption and absorption of heavy metals on the root surface. Similar to other transition metals, titanium, present in small fractions, absorbs to and accumulates in roots, as it is translocated through the xylem stream from roots to shoots [80]. The translocation of TiO2 NPs to aerial plant parts has been demonstrated [81,82]. As shown in Table 5, the co-application of TiO2 NPs and EBL significantly reduced metal translocation from roots to shoots, which is an important mechanism in increasing the tolerance of bamboo plants. However, the results showed that BAF in the roots was higher than in the stem and the leaves, which indicated that TiO2 NPs–EBL could effectively reduce BAF in the aerial parts of the bamboo plant. This could be explained by the mechanisms involved in the absorbance of Cu and Cd in the root surface by the co-application of TiO2 NPs and EBL. Therefore, we suggest that TiO2 NPs–EBL has an important role in the reduction of adsorption and uptake as well as the translocation of Cu and Cd to the aerial parts (leaves and stem). Thus, the application of TiO2 NPs–EBL can retain the heavy metals on the bamboo root surface. On the other hand, the results showed that the TF in the leaves was less than that in the stem, which was an indication that the heavy metals had been accumulated in the leaves less than in the stem (Figure 6).

**Figure 6.** The impact of the co-application of 24-epibrassinolide and titanium oxide nanoparticles on BAF and TF: (**a**) the comparison between bioaccumulation of heavy metals in the root, stem, and leaves; (**b**) the comparison between translocation factor in the stem and the leaves. Bioaccumulation factor (BAF) is obtained by the difference between the concentrations of heavy metals in the leaf, stem, or root and concentrations of heavy metals in the medium, while the translocation factor (TF) is obtained by the difference between the concentration of heavy metals (Cu, Cd) in the leaves or stem of plants and the concentration of the heavy metals (Cu, Cd) in the roots of plants. The capital letters (A–G) 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 between leaves, stem, and root at each treatment (the bars with various colors) based on Tukey s test (*p* < 0.05).

Titanium is a useful element that can aid plant growth by increasing plant photosynthesis and enzymatic activity as well as increasing plant uptake of other nutrients [72]. One study reported that TiO2 can enhance the absorption rate of micro-and macronutrients, which could be the main factor in plant growth and biomass [83]. In our study, the role of TiO2 in increasing the plant biomass was related to an increase in nutrient absorbance by the plants and reduced toxicity in response to an increase in antioxidant activity. Many studies have reported that EBL increases plant growth under heavy metal stress [21,84,85]. The reduction in plant growth by heavy metal toxicity is related to the number of intercellular metal ions bound to the surface of the cell [86,87]. Our results showed that EBL could increase the plant biomass and plant growth under heavy metal toxicity, which could be related to the role of EBL in the reduction in intercellular metal ions, a phenomenon that has been confirmed in a study on A. *obliquus* [63]. However, the role of EBL in plant growth regulation during the stimulation of plant defense mechanisms can also be considered. Therefore, we hypothesized that EBL increased the plant growth under heavy metal stress by promoting antioxidant capacity. The increase in Chl as well as carotenoid contents in response to EBL has been confirmed in many studies [88–91]. Conversely, EBL has the ability to control cell division and elongation by regulating xyloglucan endotransglucosylase [92,93], thus demonstrating the positive role of EBL on plant growth and development, especially under stressful conditions. In the present research, the role of the co-application of TiO2 NPs and EBL in improving plant biomass and plant growth seemed to be related to the ameliorative mechanisms activated by the levels of both TiO2 NPs and EBL. The application of small-sized nanoparticles in the range of 1–100 nm is a new strategy to maintain plant growth and development under heavy metals and other abiotic stresses. However, the build-up of TiO2 NPs within the plant organs can have dual effects of growth promotion and suppression. Titanium dioxide nanoparticles (TiO2 NPs) lead to several beneficial outcomes on the physiological, morphological, and biochemical traits of some plant species, which has been indicated in some studies [94,95] as well as our present study. Conversely, some researchers have reported the detrimental impacts of high levels of TiO2 NPs on plants [96,97]. These implications might arise due to various environmental conditions, different plant species, and the applied levels [97,98]. Therefore, the safety/danger of TiO2 NPs for plants depends on a myriad of factors including size, concentration, method of treatment application, plant type and growth pattern, uptake amount by plants, cellular chemical properties, translocation rate, and reactivity of TiO2 NPs in various tissues, which determine NP interaction with a wide array of metabolic activities of the plants that can thus lead to their advantageous or toxic effects [99,100]. Additionally, the TiO2 NP surface area, their predisposition for accumulation in tissues, and their inherent reactivity are the possible reasons for their toxicological repercussions [101]. Therefore, there is a great need to take the aforementioned variables into careful consideration while applying TiO2 NPs in the agriculture and food industry, which can minimize health risk for humans. This finding revealed the effective role of the co-application of TiO2 NPs and EBL in comparison with TiO2 NPs and EBL individually.

#### **5. Conclusions**

Heavy metals are deemed a considerable environmental safety hazard with inhibitory impacts on plant growth due to the induction of excessive levels of ROS compounds, which causes oxidative stress in cells and tissues. The use of nanoparticles and phytohormones as two possible agents that mitigate the deleterious effects of heavy metals has been on the rise in the recent years. Thus, conducting extensive research using various plant species with distinct growth and morphological characteristics is needed. Based on our experimental results, the individual application of EBL as a phytohormone and TiO2 NPs contributed to the amelioration of toxicity in bamboo plants under excess Cu and Cd. However, the co-application of EBL and TiO2 demonstrated a greater effective influence on increasing plant tolerance under metal toxicity. Therefore, our results indicated that while Cu and Cd stress led to increased ROS production, causing injury to the plant membrane, by boosting oxidative activity, the co-application of EBL and TiO2 significantly reduced the ROS content and oxidative stress in the plants, resulting in an increase in the photosynthetic properties and an enhancement in the plant growth and development. Conversely, the co-application of EBL and TiO2 increased the plant tolerance under metal toxicity by reducing the heavy metal accumulation within the plant and restricting the metal translocation from the roots to the shoots. Overall, our study revealed the cellular-and tissue-level mechanisms involved in increasing bamboo plant tolerance to Cu and Cd toxicity through the integrated use of EBL and TiO2. This result requires further investigation with different plant species.

**Author Contributions:** Conceptualization, A.E., Y.D. and J.B.; statistical analysis, A.E. and Y.L.; Investigation, A.E. and G.L.; Supervision, A.E., Y.D., and G.L.; Project Administration, A.E.; Funding Acquisition, A.E. and G.L.; writing—original draft and revised preparation, A.E., Y.D., J.B., F.M., M.R., and G.L.; writing—review and editing, J.B., F.M., and M.H.; visualization, M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received financial support from Nanjing Forestry University (Start-Up Research Fund) and the Bamboo Research Institute. Special Funding for this work was provided by Jiangsu Agricultural Science and Technology Innovation Fund, No. CX (18) 2031.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Nanjing Forestry University.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in article.

**Acknowledgments:** We would like to extend our sincere gratitude and appreciation to Peijian Shi, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu, China, for helping in the statistical analysis of the manuscript.

**Conflicts of Interest:** The authors declare that there is no conflict of interests regarding the publication of this paper.
