The endogenous nitric oxide mediates selenium-induced phytotoxicity by promoting ROS generation in Brassica rapa

Abstract

Selenium (Se) is suggested as an emerging pollutant in agricultural environment because of the increasing anthropogenic release of Se, which in turn results in phytotoxicity. The most common consequence of Se-induced toxicity in plants is oxidative injury, but how Se induces reactive oxygen species (ROS) burst remains unclear. In this work, histofluorescent staining was applied to monitor the dynamics of ROS and nitric oxide (NO) in the root of Brassica rapa under Se(IV) stress. Se(IV)-induced faster accumulation of NO than ROS. Both NO and ROS accumulation were positively correlated with Se(IV)-induced inhibition of root growth. The NO accumulation was nitrate reductase (NR)- and nitric oxide synthase (NOS)-dependent while ROS accumulation was NADPH oxidase-dependent. The removal of NO by NR inhibitor, NOS inhibitor, and NO scavenger could alleviate Se(IV)-induced expression of Br_Rbohs coding for NADPH oxidase and the following ROS accumulation in roots, which further resulted in the amelioration of Se(IV)-induced oxidative injury and growth inhibition. Thus, we proposed that the endogenous NO played a toxic role in B. rapa under Se(IV) stress by triggering ROS burst. Such findings can be used to evaluate the toxic effects of Se contamination on crop plants.

Figure 1. Effect of Se(IV) on the growth of B. rapa root.

(A–B) The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM of Se(IV) solution for 72 h. After that, the root length were measured (A). The images of roots were photographed (B). (C–D) The root length and root image were obtained when the roots of seedlings were exposed to 0.06 mM of Se(IV) solution for 12, 24, 36, 48, 60, and 72 h, respectively. Asterisk indicates that mean values of three replicates are significantly different between the treatments of Se(IV) and the control group (CK) (P<0.05).

Figure 2. Effect of Se(IV) on endogenous total ROS (A–D), endogenous O₂ ^{• −} (E), lipid peroxidation (F), and the loss of plasma membrane integrity (G) in the roots of B. rapa.

The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM of Se(IV) solution for 48 h. Afterwards, the roots were loaded with DCFH-DA for 15 min and immediately photographed (A). The relative DCF fluorescent density in roots was estimated (B). (C–D) The image and density of DCF fluorescence were obtained when the roots of seedlings were exposed to 0.06 mM of Se(IV) solution for 0, 1, 3, 6, 12, and 24 h, respectively. The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM of Se(IV) solution for 48 h. Afterwards, the roots were stained with NBT (E), Schiff's reagent (F), and Evan blue (G), respectively. Asterisk indicates that mean values of three replicates are significantly different between the treatments of Se(IV) and the control group (CK) (P<0.05).

Figure 3. Effect of NADPH oxidase inhibitors (DPI, PY, and IMZ) on endogenous O₂ ^{• −} (A), endogenous total ROS (B–C), lipid peroxidation (D), the loss of plasma membrane integrity (E), and the growth of roots under Se(IV) stress.

The roots of seedlings were exposed to Se(IV) (0.06 mM), Se(IV) (0.06 mM)+DPI (10 µM), Se(IV) (0.06 mM)+PY (5 mM), and Se(IV) (0.06 mM)+IMZ (0.5 mM) for 48 h. Afterwards, the roots were stained with NBT (A), DCFH-DA (B), Schiff's reagent (D), and Evan blue (E), respectively. The density of DCF fluorescence were estimated (C). The roots of seedlings were exposed to the above treatment solutions for 72 h. Afterwards, the root length were measured (F). Asterisk indicates that mean values of three replicates are significantly different between the different treatments (P<0.05).

Figure 4. Effect of Se(IV) on the transcripts of Br_RbohA-J.

The roots of seedlings were exposed to 0.06 mM of Se(IV) solution for 6, 12, 24, 48, and 72 h, respectively. The total RNA was extracted from roots for qRT-PCR analysis. Actin was used for cDNA normalization. The data were presented as the relative fold-change in transcript abundance of the target genes. Red column means up-regulation. Black column means down-regulation.

Figure 5. Effect of Se(IV) on the generation of endogenous NO in the roots of B. rapa.

The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM of Se(IV) solution for 48 h. Afterwards, the roots were loaded with DAF-FM DA for 15 min and immediately photographed (A). The relative DAF-FM fluorescent density in roots was estimated (B). (C–D) The image and density of DAF-FM fluorescence were obtained when the roots of seedlings were exposed to 0.06 mM of Se(IV) solution for 0, 1, 3, 6, 12, and 24 h, respectively.

Figure 6. Identification of the sources of Se(IV)-induced NO generation in the roots of B. rapa.

(A–B) The image and density of DAF-FM fluorescence were obtained when the roots of seedlings were exposed to Se(IV) (0.06 mM), Se(IV) (0.06 mM)+_L-NMMA (200 µM), Se(IV) (0.06 mM)+Tungstate (30 µM), and Se(IV) (0.06 mM)+cPTIO (0.1 mM) for 48 h. Asterisk in (B) indicates that mean values of three replicates are significantly different between different treatments (P<0.05). The roots of seedlings were exposed to 0, 0.03, 0.06, 0.12, 0.23, and 0.46 mM of Se(IV) solution for 48 h. After that, NR activity (C) and NOS activity (D) in roots were measured, respectively. Asterisk in (C) and (D) indicates that mean values of three replicates are significantly different between Se(IV) treatment and the control (P<0.05).

Figure 7. Effects of NO synthesis inhibitors (_L-NMMA and tungstate) and NO scavenger (cPTIO) on the generation of endogenous ROS (A–B), O₂ ^{• −} (C), and the expression of several Br_Rbohs (D) in Se(IV)-treated roots of B. rapa.

The roots of seedlings were exposed to Se(IV) (0.06 mM), Se(IV) (0.06 mM)+_L-NMMA (200 µM), Se(IV) (0.06 mM)+Tungstate (30 µM), and Se(IV) (0.06 mM)+cPTIO (0.1 mM) for 72 h. The roots were loaded with DCFH-DA for 15 min and immediately photographed (A). The relative DCF fluorescent density in roots was estimated (B). Asterisk indicates that mean values of three replicates are significantly different between the different treatments (P<0.05). The roots were stained with NBT for the detection of O₂ ^{• −} (C). The total RNA was extracted from roots under the above treatments for 24 h for qRT-PCR analysis of the transcripts of Br_RbohD, F, G1, G2, and I. Actin was used for cDNA normalization (D).

Figure 8. Effects of NO synthesis inhibitors (_L-NMMA and tungstate) and NO scavenger (cPTIO) on lipid peroxidation (A), the loss of plasma membrane integrity (B), and root elongation under Se(IV) stress.

The roots of seedlings were exposed to Se(IV) (0.06 mM), Se(IV) (0.06 mM)+_L-NMMA (200 µM), Se(IV) (0.06 mM)+Tungstate (30 µM), and Se(IV) (0.06 mM)+cPTIO (0.1 mM) for 72 h. Afterwards, the roots were stained with Schiff's reagent (A) and Evan blue (B), respectively. The root length was measured as well (C). Asterisk indicates that mean values of three replicates are significantly different between the different treatments (P<0.05).

Figure 9. Effect of SNP on the root growth under Se(IV) stress.

The roots of seedlings were exposed to Se(IV) (0.06 mM), SNP (250 mM), and Se(IV) (0.06 mM)+SNP (250 mM) for 72 h. After that, the length of roots were measured. The mean values of three replicates followed by the same letter were not significantly different at P<0.05.