Polyamine Oxidase Triggers H2O2-Mediated Spermidine Improved Oxidative Stress Tolerance of Tomato Seedlings Subjected to Saline-Alkaline Stress

Abstract

Saline-alkaline stress is one of several major abiotic stresses in crop production. Exogenous spermidine (Spd) can effectively increase tomato saline-alkaline stress resistance by relieving membrane lipid peroxidation damage. However, the mechanism through which exogenous Spd pre-treatment triggers the tomato antioxidant system to resist saline-alkaline stress remains unclear. Whether H₂O₂ and polyamine oxidase (PAO) are involved in Spd-induced tomato saline-alkaline stress tolerance needs to be determined. Here, we investigated the role of PAO and H₂O₂ in exogenous Spd-induced tolerance of tomato to saline-alkaline stress. Results showed that Spd application increased the expression and activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and the ratio of reduced ascorbate (AsA) and glutathione (GSH) contents under saline-alkaline stress condition. Exogenous Spd treatment triggered endogenous H₂O₂ levels, SlPAO4 gene expression, as well as PAO activity under normal conditions. Inhibiting endogenous PAO activity by 1,8-diaminooctane (1,8-DO, an inhibitor of polyamine oxidase) significantly reduced H₂O₂ levels in the later stage. Moreover, inhibiting endogenous PAO or silencing the SlPAO4 gene increased the peroxidation damage of tomato leaves under saline-alkaline stress. These findings indicated that exogenous Spd treatment stimulated SlPAO4 gene expression and increased PAO activity, which mediated the elevation of H₂O₂ level under normal conditions. Consequently, the downstream antioxidant system was activated to eliminate excessive ROS accumulation and relieve membrane lipid peroxidation damage and growth inhibition under saline-alkaline stress. In conclusion, PAO triggered H₂O₂-mediated Spd-induced increase in the tolerance of tomato to saline-alkaline stress.

Keywords: Solanum lycopersicum; hydrogen peroxide; polyamine oxidase; saline-alkaline stress; spermidine.

Figure 1

Effects of Spd pre-treatment on REC, MDA content, O₂⁻ and H₂O₂ levels in tomato leaves under saline-alkaline stress. Seedlings were pretreated with 5 mL distilled water or 0.25 mM Spd and cultivated under normal conditions for 24 h, and then seedlings were irrigated with 100 mL of half strength Hoagland nutrient solution or 100 mL 300 mM saline-alkaline mixed solution. Saline-alkaline mixed solution (1:9:9:1 molar ratio of NaCl:Na₂SO₄:NaHCO₃:Na₂CO₃, Hu et al., 2014) was added to half-strength Hoagland’s nutrient solution to a final concentration of 300 mM (pH 8.6 ± 0.2). Control, pre-sprayed distilled water under normal conditions; CS, 0.25 mM Spd foliar pre-spraying under normal conditions; S, irrigation with saline-alkaline mixed solution and H₂O foliar pre-spraying; SS, 0.25 mM Spd foliar pre-spraying under salinity-alkalinity stress. The histochemical staining with NBT and DAB for detection of O₂⁻ (A) and H₂O₂ (B); MDA content (C); REC (D); O₂⁻ generation rate (E) and H₂O₂ content (F) were measured using fifth leaves of tomato seedlings after saline-alkaline stressed for 1 and 3 days. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 2

Effects of Spd pre-treatment on activities of antioxidase and the expression of related genes in tomato leaves under saline-alkaline stress. Seedlings were pretreated with 5 mL distilled water or 0.25 mM Spd and cultivated under normal conditions for 24 h, and then seedlings were irrigated with 100 mL of half strength Hoagland nutrient solution or 100 mL 300 mM saline-alkaline mixed solution. Saline-alkaline mixed solution (1:9:9:1 molar ratio of NaCl:Na₂SO₄:NaHCO₃:Na₂CO₃, Hu et al., 2014) was added to half-strength Hoagland’s nutrient solution to a final concentration of 300 mM (pH 8.6 ± 0.2). Control, pre-sprayed distilled water under normal conditions; CS, 0.25 mM Spd foliar pre-spraying under normal conditions; S, irrigation with saline-alkaline mixed solution and H₂O foliar pre-spraying; SS, 0.25 mM Spd foliar pre-spraying under salinity-alkalinity stress. The activities of SOD, CAT, APX, GR and the expression of related genes in fifth leaf of seedlings were measured after saline-alkaline stressed for 1 and 3 days. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 3

Effects of Spd pre-treatment on the redox status of glutathione and ascorbate in tomato leaves under saline-alkaline stress. Seedlings were pretreated with 5 mL distilled water or 0.25 mM Spd and cultivated under normal conditions for 24 h, then seedlings were irrigated with 100 mL a half strength Hoagland nutrient solution or 100 mL 300 mM saline-alkaline mixed solution. Saline-alkaline mixed solution (1:9:9:1 molar ratio of NaCl:Na₂SO₄:NaHCO₃:Na₂CO₃, Hu et al., 2014) was added to half-strength Hoagland’s nutrient solution to a final concentration of 300 mM (pH 8.6 ± 0.2). Control, pre-sprayed distilled water under normal conditions; CS, 0.25 mM Spd foliar pre-spraying under normal conditions; S, irrigation with saline-alkaline mixed solution and H₂O foliar pre-spraying; SS, 0.25 mM Spd foliar pre-spraying under salinity-alkalinity stress. The GSH, GSSG, AsA and DHA content in fifth leaves of seedlings were measured after saline-alkaline stressed for 1 and 3 days. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 4

The dynamic change of H₂O₂ content in Spd treated tomato leaves under normal conditions. The control and Spd-treated seedlings were sprayed with 5 mL distilled water (Control) or 0.25 mM Spd (Spd) cultivated under normal conditions for 24 h. The fifth leaves of tomato seedlings were harvested after Spd spraying for 0, 1, 3, 6, 12, 24 h. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 5

Effects of Spd pre-spraying on dynamic change of PAO activities and SlPAO4 gene expression in tomato leaves under normal conditions. Seedlings were sprayed with 5 mL distilled water (Control) or 0.25 mM Spd (Spd) cultivated under normal conditions for 24 h. The fifth leaves of tomato seedlings were harvested after Spd spraying for 0, 1, 3, 6, 12, 24 h. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 6

The H₂O₂ content of tomato seedlings treated with 5 mL distilled water (Control) or an inhibitor of PAO, 1,8-diaminooctane (1,8-DO) under normal conditions. Seedlings were sprayed with 5 mL distilled water (Control) or 1 mM 1,8-diaminooctane (1,8-DO) cultivated under normal conditions for 24 h. The fifth leaves of tomato seedlings were harvested after 1,8-diaminooctane spraying for 0, 1, 3, 6, 12, 24 h. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 7

Effects of 1,8-DO on REC (A); MDA content (B); O₂⁻ generation rate (C) and H₂O₂ content (D). Plants were foliar pretreated with 5 mL 1 mM 1,8-diaminooctane (1,8-DO, an inhibitor of polyamine oxidase). After 12 h, the leaves were sprayed with 5 mL distilled water, 0.25 mM Spd, or 5 mM H₂O₂; then seedlings were irrigated with 100 mL of half strength Hoagland nutrient solution (Control) or 100 mL 300 mM saline-alkaline mixed solution. Saline-alkaline mixed solution (molar ratio of NaCl:Na₂SO₄:NaHCO₃:Na₂CO₃ is 1:9:9:1) was added to half-strength Hoagland’s nutrient solution to a final concentration of 300 mM (pH 8.6 ± 0.2). The fifth leaves of tomato seedlings were harvested after saline-alkaline stressed for 1 day and 3 days. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.

Figure 8

Effects of SlPAO4 silencing on REC and MDA content in tomato leaves under saline-alkaline stress. pTRV2 and SlPAO4 silencing tomato seedlings were pretreated with 5 mL distilled water or 0.25 mM Spd and cultivated under normal conditions for 24 h, then seedlings were irrigated with 100 mL of half strength Hoagland nutrient solution or 100 mL 300 mM saline-alkaline mixed solution. Saline-alkaline mixed solution (1:9:9:1 molar ratio of NaCl:Na₂SO₄:NaHCO₃:Na₂CO₃, Hu et al., 2014) was added to half-strength Hoagland’s nutrient solution to a final concentration of 300 mM (pH 8.6 ± 0.2). Control, pre-sprayed distilled water under normal conditions; CS, 0.25 mM Spd foliar pre-spraying under normal conditions; S, irrigation with saline-alkaline mixed solution and H₂O foliar pre-spraying; SS, 0.25 mM Spd foliar pre-spraying under salinity-alkalinity stress. The fifth leaves of tomato seedlings were harvested after saline-alkaline stressed for 3 days. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences of p < 0.05 according to Tukey’s test.