**9. Effects of the copper toxicity and exogenous trehalose on antioxidant enzymes**

In agreement with Mostofa et al. [84], the regulating Cu homeostasis is crucial in maintaining theintracellularCuleveltoavoidtoxicity.Plantshavedevelopedvariousmechanisms torestrict Cu toxicity, such as inhibition of Cu uptake by binding with root exudates like organic acids, intracellular sequestration by strong ligands like cysteine-rich compounds and phytochela‐ tins, and exclusion of excessive Cu from the cells by sugar alcohols like trehalose (Tre) [26–28]. Tre, a non-reducing disaccharide of glucose, protects plant cells against long-term desiccation by stabilizing enzymes, proteins, and biological membranes under dehydration [29].

In relation to antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GST), the pretreatment with non-reducing disaccharide Tre induces increases in enzyme activities when compared with control or Cu stress treatments (Figure 4).

**Figure 3.** Electrolyte Leakage (A), Total Amino Acids (B), Proline (C) of young *Schizolobium parahyba* var. *amazonicum* plants subjected to boron toxicity. Different letters for boron levels indicate significant differences from the Skott-Knott test (P<0.05). Columns represent the mean values from 5 repetitions, and bars represent the standard deviations [75].

The proline concentration was maximized and this result is related to the amino acid acting in detoxification process of ROS and membrane protection against lipid peroxidation [78-79]; associated with ROS antagonist, it aims to attenuate the oxidative stress and to avoid the cell death. In other activities, the PRO protects the protein structure against denaturation and it will stabilize the cell membranes during interaction with phospholipids [80]. Contreras et al. [81] evaluating the B and NaCl effects in *Solanum lycopersicum* plants verified similar results

Proline is an amino acid synthetized into nitrogen metabolism with functions related to osmoprotection [82] and cellular homeostasis [83], which can contribute to improve the plant

tolerance under situations of abiotic stress, as B toxicity.

236 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

of this research.

**Figure 4.** Effect of exogenous trehalose on the activities of antioxidant enzymes in rice seedlings with or without Cu stress. (a) Superoxide Dismutase (SOD), (b) Catalase (CAT), (c) Glutathione Peroxidase (GPX), and (d) Glutathione Re‐ ductase (GST). Control, Tre, Cu, and Tre + Cu correspond to control, 10 mM trehalose, 100 μM CuSO4, and 10 mM trehalose + 100 μM CuSO4, respectively. Bars represent standard deviation (SD) of the mean (n = 3). Different letters indicate significant differences among treatments at p < 0.05, according to Duncan's multiple range test [84].

NBT staining indicated an increased amount of O2 – as scattered dark blue spots in the leaf plate of the Cu-stressed seedlings compared with the non-treated control (Figure 5a). Similarly, DAB staining confirmed a marked increase in brown polymerization products, which indicated the over-accumulation of H2O2 in the leaves of the Cu-stressed seedlings relative to control (Figure 5b) [84].

Results described by Mostofa et al. [84] indicate that prolonged exposure to excessive Cu resulted in serious toxic effects on the rice seedlings. In contrast, Tre pretreatment has been shown to be beneficial in alleviating Cu toxicity, which was mainly attributed to the ability of Tre (i) to restrict Cu uptake and accumulation to maintain Cu homeostasis, and (ii) to induce production of antioxidant and Gly enzymes to alleviate excessive Cu-triggered oxidative stress [84].

**Figure 5.** Effect of exogenous trehalose on ROS accumulation in leaves of rice seedlings with or without Cu stress. (a) Superoxide (O2 − ) and (b) Hydrogen Peroxide (H2O2) production in rice leaves were detected using nitro-blue tetrazoli‐ um (NBT) solution and 3,3'-diaminobenzidine (DAB), respectively, at day 7 of Cu stress. Control, Tre, Cu, and Tre + Cu correspond to control, 10 mM trehalose, 100 μM CuSO4, and 10 mM trehalose + 100 μM CuSO4, respectively [84].
