**1. Introduction**

In recent decades, increasing anthropogenic activities have led to increases in greenhouse gases in the natural environment, and chemical fertilization has led to increases in heavy metal contamination in forestland and agricultural soils, deleteriously contributing to global climate change [1]. Many reports have shown that heavy metals pose a major threat to agricultural land, animals, and plants, which can influence the human food chain, leading to negative effects on human health [2,3]. Copper (Cu) and cadmium (Cd) have been mentioned as being the most abundant toxic metals in Chinese farmland soils [4].

**Citation:** Emamverdian, A.; Ding, Y.; Barker, J.; Liu, G.; Hasanuzzaman, M.; Li, Y.; Ramakrishnan, M.; Mokhberdoran, F. Co-Application of 24-Epibrassinolide and Titanium Oxide Nanoparticles Promotes *Pleioblastus pygmaeus* Plant Tolerance to Cu and Cd Toxicity by Increasing Antioxidant Activity and Photosynthetic Capacity and Reducing Heavy Metal Accumulation and Translocation. *Antioxidants* **2022**, *11*, 451. https:// doi.org/10.3390/antiox11030451

Academic Editor: Nafees A. Khan

Received: 25 January 2022 Accepted: 20 February 2022 Published: 24 February 2022

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While there is no evidence of the biological activity of Cd in the plant growth process, trace amounts of Cu could have a positive impact as a dietary nutrient on plant growth; however, extreme Cu levels induce plant toxicity [5]. Cu, as a trace element, can enhance photosynthetic efficiency, such as electron transport. Additionally, Cu regulates structural proteins involved in cell wall metabolism and can elevate mitochondrial respiration to produce energy [6]. Conversely, surplus concentrations of Cu in the form of Cu2+ are responsible for oxidative stress due to the generation of reactive oxygen species (ROS) compounds [7], which can result in a reduction in plant growth with an altered functionality of the cell membrane, limitation of enzyme activities, and depression of photosynthetic efficiency, ultimately leading to plant death [8]. Cd, a nonessential element with high toxicity, is known as the most dispersed element in soils and irrigation water, which has a destructive influence on plant and human life [9]. Excess Cd in soil and the absorption of cadmium by plants increase ROS production, such as free radicals, which are the main factors in the initiation of oxidative stress in plants [9]. Cadmium has a damaging impact on plant cell functions and the metabolic pathways involved in the production of lipids, proteins, and nucleic acids. Cd injures the cell membrane, which leads to lipoperoxidation and oxidative toxicity in plants. It has been demonstrated that cadmium reduces the plant defense system with a reduction in antioxidant activity capacity. This phenomenon finally reduces plant photosynthesis and inhibits plant growth and development [9].

Nanoparticles with unique structures and sizes (1 to 100 nm) [10] have been observed to increase plant nutrients and crop production [11]. The diverse surface-to-volume ratios of nanoparticles could differ from their bulk counterparts [12]. Recently, some studies have reported that titanium nanoparticles (TiO2) have the ability to increase plant growth under metal stress [13–17]. Therefore, we suggest that titanium could be a good material to reduce plant stress. Brassinosteroids (BRs) are a new phytohormone and belong to the polyhydroxy steroidal group. There are 70 types of BRs in plants. Among them, 24-epibrassinolide (EBL) is known as the top bioactive BR that can promote plant growth under stressful conditions [18]. In addition, 24-epibrassinolide EBL induces antioxidant activity, plant photosynthesis, seed yield, and oxidative production under stressful conditions [19–21]. It has been indicated that the interaction of EBL with other cellular molecules can enhance signaling efficiency within the plant defense grid under stress conditions [22]. This phenomenon can boost antioxidant capacity in the face of multiple stressful factors, such as HMs [23]. This research study represents the individual and co-application of TiO2-NPs and EBL, as well as the investigation of their role in the alleviation of Cu and Cd toxicity in bamboo plants with an emphasis on antioxidant, photosynthetic, and plant growth parameters.

Bamboo (*Bambusoideae*) species occupy the largest portion of Chinese farmland (6 million hectares) [24,25]. This fast-growing plant provides nutrient sources for local family livelihoods in southern and western China [26]. *Pleioblastus pygmaeus* is a suitable species for landscape purposes, with a characteristic height of 30–50 cm. *Pleioblastus pygmaeus* originated in Japan but was transferred to China in the early 20th century. A desirable condition of this plant for this experiment was its adaptation to basic (alkaline), acidic, and neutral soils [27]. Conversely, the excess of heavy metals (frequently Cu and Cd) caused by anthropogenic activities has become a major dilemma for agricultural and forestry soils in this area [4], which can influence bamboo plant growth and development. Hence, it is essential to find appropriate biologic materials to reduce soil toxicity and increase plant tolerance under heavy metal toxicity. Therefore, we selected two applications of TiO2 NPs and 24-epibrassinolide, individually and in combination, against heavy metal toxicity, which could aid in understanding the involved mechanisms in the combined application of nanoparticles and phytohormones against heavy metal toxicity. To our knowledge, this is the first comprehensive study to investigate the combination of TiO2 NPs and EBL in the amelioration of Cu and Cd toxicity in bamboo species. Therefore, in this paper, we aim to investigate the impact of TiO2 NPs and EBL on enhancing plant tolerance under heavy metal toxicity with an emphasis on antioxidant and nonantioxidant enzyme capacity, ROS production, photosynthesis, and growth indices under Cu and Cd.

#### **2. Materials and Methods**

#### *2.1. Plant Material and In Vitro Conditions*

This research study was performed under in vitro conditions in a plant tissue culture laboratory using MS medium (Murashige and Skoog, 1962) [28] consisting of 6 benzylaminopurine (6-BA) (4 mL), micronutrients (10 mL), macronutrients (100 mL), kinetin (KT) (0.5 mL), sucrose (30 g) and agar (8 g) at pH 5.8 ± 0.1. For this purpose, a completely randomized design (CRD) was employed that contained 100 μM TiO2 NPs and 10−<sup>8</sup> M 24 epibrassinolide individually and in combination with 100 μM Cu as well as 100 μM Cd in four replications (Table 1). We adjusted the pH value in MS to 5.8 for two reasons: firstly, to optimize nutrient absorption, the availability of the nutrients to the plants was optimum at pH 5.8; and secondly, the preparation of the gelling of the agar-solidified medium should be completed at ca. pH 5.8. To proliferate bamboo roots, young shoots (10 mm long nodal explants) were planted in MS medium supplemented with pyridoxine (3 μM), nicotinic acid (4 μM), thiamine–HCl (1.2 μM), myo-inositol (0.6 mM), 30 *g* L−<sup>1</sup> sucrose, and 0.1 mg L−<sup>1</sup> indole-3-acetic acid (IAA) as a regulator hormone involved in plant growth. The appropriate amount of each treatment (100 μM TiO2 NPs and 10−<sup>8</sup> M 24-epibrassinolide) was mixed in 1 L MS medium, adjusted to pH 5.8 ± 0.1, and then applied to 8–10 *g*/L agar. The solution was placed in 60 mm diameter glass petri dishes containing 100 mL of culture, and sterilization of the intended MS medium was conducted in an autoclave (HiClave HVE-50, ZEALWAY-USA, Delaware, DE, USA) at the optimum temperature of 110 ◦C for 40 min. The dishes were transferred to an Air Tech incubation hood with ultraviolet sterilization with white fluorescent lamps (wavelength between 10 and 420 nm) at a temperature of 25 ◦C for 4 h. In the final step, the plantlet treatments were preserved as research materials in a controlled tissue culture chamber with fluorescent lamps (white) at a wavelength between 10 and 420 nm. In terms of temperature, the growth was performed at 17/22 ◦C in the dark periods and 30/25 ◦C in the light periods for three weeks.


**Table 1.** The treatment combinations of the experiment.

Titanium nanoparticles were provided by Nanjing Jiancheng Company, Jiangsu Province, China, and consisted of a white powder with a purity of >99% nanotitanium and a diameter of 25 nm. The levels of Cu and Cd were chosen according to the previous studies, which displayed high and low levels of toxicity in bamboo plants [13,14]. Bamboo (*A. pygmaeus*) was selected from local species by the Bamboo Research Institute, which is located at Nanjing Forestry University.

EBL + Cu 10−<sup>8</sup> M EBL + 100 μM Cu EBL + Cd 10−<sup>8</sup> M EBL + 100 μM Cd TiO2 + EBL 100 μM TiO2 + 10−<sup>8</sup> M EBL TiO2 + EBL + Cu 100 μM TiO2 + 10−<sup>8</sup> M EBL + 100 μM Cu TiO2 + EBL + Cd 100 μM TiO2 + 10−<sup>8</sup> M EBL + 100 μM Cd

In this research study, biomass and growth indices, including root and shoot dry weight (DW) and shoot length, were quantified. To investigate photosynthesis pigments, total chlorophyll (Chl), Chl a and b, and carotenoid contents were measured. To determine the fluorescence parameters, 5 parameters were recorded, including: (i) actual photochemical efficiency of PSll (ϕPSll), (ii) maximum photochemical efficiency of PSll (Fv/Fm), (iii) photochemical quenching coefficient (qP), (iv) effective photochemical efficiency of

PSll (Fv /Fm ), and (v) nonphotochemical quenching (NPQ). Heavy metal accumulation and TiO2 NP contents were measured in leaves, stems, and roots. Plant defense enzymes and nonenzymatic antioxidants were measured. To assay cell membrane injury, ROS compounds, electron leakage, and malondialdehyde (MDA) content were estimated. Finally, the translocation factor (TF), bioaccumulation factor (BAF), and tolerance index (TI) of the shoots and roots were calculated.

#### *2.2. Preparation of Samples*

Leaf samples were collected from the different treatments, and then 0.5 g samples were placed in a container and crushed into a powder. An appropriate amount of liquid nitrogen was added to the samples, and the obtained powder was dissolved in PBS (pH 7.2–7.4) at 2–8 ◦C. The solution was centrifuged at 2500–3500× *g* for 17 min to extract the supernatant, which was kept for use in antioxidant enzyme activity tests.

#### *2.3. Protective Enzymes*

Superoxide dismutase (SOD, EC: 1.15.1.1) was measured based on the results of photoreduction obtained by nitro blue tetrazolium (NBT), which was conducted using the Zhang method [29]. Peroxidase (POX, EC: 1.11.1.7) was estimated by using the protocol of Upadhyaya [30]. Catalase (CAT, EC: 1.11.1.6) was estimated based on the results of reactions analyzing H2O2 at an absorbance of 240 nm, which was estimated by the Aebi protocol [31]. Glutathione reductase (GR, EC: 1.6.4.2) was estimated using the protocol reported by Foyer and Halliwell [32] with some modifications. Ascorbate peroxidase (APX, EC: 1.11.1.11) was measured using the Nakano and Asada method [33]. APX antioxidant activity was obtained by recording the reduction in absorbance at 290 nm (coefficient of absorbance at 2.8 mM−<sup>1</sup> cm−1). Phenylalanine ammonia-lyase (PAL, EC: 4.3.1.5) activity was assessed using the Berner [34] protocol.
