Soil Treatment with Nitric Oxide-Releasing Chitosan Nanoparticles Protects the Root System and Promotes the Growth of Soybean Plants under Copper Stress

Abstract

The nanoencapsulation of nitric oxide (NO) donors is an attractive technique to protect these molecules from rapid degradation, expanding, and enabling their use in agriculture. Here, we evaluated the effect of the soil application of chitosan nanoparticles containing S-nitroso-MSA (a S-nitrosothiol) on the protection of soybeans (Glycine max cv. BRS 257) against copper (Cu) stress. Soybeans were grown in a greenhouse in soil supplemented with 164 and 244 mg kg^-1 Cu and treated with a free or nanoencapsulated NO donor at 1 mM, as well as with nanoparticles without NO. There were also soybean plants treated with distilled water and maintained in soil without Cu addition (control), and with Cu addition (water). The exogenous application of the nanoencapsulated and free S-nitroso-MSA improved the growth and promoted the maintenance of the photosynthetic activity in Cu-stressed plants. However, only the nanoencapsulated S-nitroso-MSA increased the bioavailability of NO in the roots, providing a more significant induction of the antioxidant activity, the attenuation of oxidative damage, and a greater capacity to mitigate the root nutritional imbalance triggered by Cu stress. The results suggest that the nanoencapsulation of the NO donors enables a more efficient delivery of NO for the protection of soybean plants under Cu stress.

Keywords: S-nitrosothiol; biopolymer; metal stress; nanotechnology.

Figure 1

Representative transmission electron microscopy image of the chitosan nanoparticles loaded with mercaptosuccinic acid (MSA_NP) (a) and the corresponding size histogram of MSA_NP (b).

Figure 2

Visual aspect of the roots from soybean plants cultivated in soil with natural copper (Cu) content (a) and Cu supplementation to 164 mg kg⁻¹ (b) in experiment 1. Visual aspect of the roots from the soybean plants cultivated in soil with a natural Cu content (c) and Cu supplementation of 244 mg kg⁻¹ (d) in experiment 2. Control = treatment with distilled water; water = treatment with distilled water after the Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM.

Figure 3

Potential activity of photosystem II (F_v/F₀) (a,b) and the relative rate of the linear electron transport of photosystem II (rETR) (c,d) of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after the Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 8). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 4

Net photosynthetic rate (A) (a,b) and stomatal conductance (g_s) (c,d) of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after the Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 8). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 5

Instantaneous carboxylation efficiency (k) (a,b) of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 8). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 6

Root S-nitrosothiol content (a,b) of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 4). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 7

Root H₂O₂ (a,b) and conjugated dienes (CD) (c,d) contents of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 4). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 8

Root superoxide dismutase (SOD) (a,b) and peroxidase (POD) (c,d) activities of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 4). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 9

Root ascorbate peroxidase (APX) (a,b) and catalase (CAT) (c,d) activities of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 4). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 10

Root Cu content (a,b) of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg⁻¹; moderate Cu stress: 164 mg kg⁻¹; severe Cu stress: 244 mg kg⁻¹). Control = treatment with distilled water; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM. Data are the mean ± standard error (n = 4). The same uppercase and lowercase letters on the column (referring to experiments 1 and 2, respectively) indicate values that do not differ by ANOVA, followed by the Tukey test (p < 0.05).

Figure 11

Corrplot depicting the correlation coefficient of the essential nutrients in the roots of soybean plants treated and cultivated in soil with natural Cu content (Control) and 164 mg kg^₋1 (a) and natural Cu content (Control) and 244 mg kg^₋1 (b). The areas of the circles show the value of the corresponding Pearson correlation coefficients. The significant (p < 0.05; p < 0.10) correlation values are presented in the upper panel in circle; The non-significant (p > 0.10) correlation values are present in the upper panel with a blank square; the positive correlations are displayed in blue and the negative correlations in red. Data are the average of the four replicates per treatment.

Figure 12

Principal component analysis (a), and heatmap hierarchical clustering analysis (b) of the essential nutrients in the roots of soybean plants cultivated in soil, containing different copper (Cu) levels (natural content: 11 mg kg^₋1; moderate Cu stress: 164 mg kg^₋1). Control = treatment with distilled water and soil with natural copper content; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM and soil with copper supplementation; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM and soil with copper supplementation; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM and soil with copper supplementation. Data are the average of the four replicates per treatment.

Figure 13

Principal component analysis (a), and heatmap hierarchical clustering analysis (b) of the essential nutrients in the roots of soybean plants cultivated in soil containing different copper (Cu) levels (natural content: 11 mg kg^₋1; severe Cu stress: 244 mg kg^₋1). Control = treatment with distilled water and soil with natural copper content; water = treatment with distilled water after Cu addition; S-nitroso-MSA_NP = treatment with a nanoencapsulated NO donor (S-nitroso-mercaptosuccinic acid) at 1 mM and soil with copper supplementation; S-nitroso-MSA = treatment with a NO donor (S-nitroso-mercaptosuccinic acid) in the free form at 1 mM and soil with copper supplementation; MSA_NP = treatment with nanoparticles containing the non-nitrosated MSA at 1 mM and soil with copper supplementation. Data are the average of the four replicates per treatment.