The Dynamic Changes of the Plasma Membrane Proteins and the Protective Roles of Nitric Oxide in Rice Subjected to Heavy Metal Cadmium Stress

Abstract

The heavy metal cadmium is a common environmental contaminant in soils and has adverse effects on crop growth and development. The signaling processes in plants that initiate cellular responses to environmental stress have been shown to be located in the plasma membrane (PM). A better understanding of the PM proteome in response to environmental stress might provide new insights for improving stress-tolerant crops. Nitric oxide (NO) is reported to be involved in the plant response to cadmium (Cd) stress. To further investigate how NO modulates protein changes in the plasma membrane during Cd stress, a quantitative proteomics approach based on isobaric tags for relative and absolute quantification (iTRAQ) was used to identify differentially regulated proteins from the rice plasma membrane after Cd or Cd and NO treatment. Sixty-six differentially expressed proteins were identified, of which, many function as transporters, ATPases, kinases, metabolic enzymes, phosphatases, and phospholipases. Among these, the abundance of phospholipase D (PLD) was altered substantially after the treatment of Cd or Cd and NO. Transient expression of the PLD fused with green fluorescent peptide (GFP) in rice protoplasts showed that the Cd and NO treatment promoted the accumulation of PLD in the plasma membrane. Addition of NO also enhanced Cd-induced PLD activity and the accumulation of phosphatidic acid (PA) produced through PLD activity. Meanwhile, NO elevated the activities of antioxidant enzymes and caused the accumulation of glutathione, both which function to reduce Cd-induced H2O2 accumulation. Taken together, we suggest that NO signaling is associated with the accumulation of antioxidant enzymes, glutathione and PA which increases cadmium tolerance in rice via the antioxidant defense system.

Keywords: cadmium pollution; lipid hydrolysis; nitric oxide; quantitative proteomics; reactive oxygen species; rice.

Figure 1

Effects of cadmium, SNAP, and different chemicals on rice growth and on photosynthetic capability. These experiments were repeated three times with similar results. (A) The rice seedlings at the third leaf stage were treated with 10 μM cadmium (Cd) with or without 30 μM SNAP, 1 μM PA (16:0-18:2), 0.1% 1-Bu, or 30 μM cPTIO for 7 days, and phenotypes from one experiment were noted. (B) The effect of cadmium treatment on total chlorophyll content after 7 days of treatment (n = 30/experiment). (C) The effect of cadmium treatment on stem and root length after 7 days of treatment (n = 30/experiment). (D) The effects of cadmium treatment on the Fv/Fm ratio and ion leakage in roots after 7 days of treatment. Values reflect means ± SEs of at least three independent experiments (n = 30/experiment). Different symbols above the bars indicate significant differences (Tukey's test, p < 0.05).

Figure 2

Effects of cadmium, SNAP, and different chemicals on rice biomass (A) and phytochelatins (B). The rice seedlings growth and treatment was the same as in Figure 1, but the biomass and phytochelatin content were measured after 7 day of treatment. Values reflect means ± SEs of three independent experiments (n = 30/experiment). Different symbols above the bars indicate significant differences (Tukey's test, p < 0.05).

Figure 3

Effects of cadmium, SNAP, and different chemicals on NO accumulation. Third-leaf-stage rice seedlings were treated with 10 μM cadmium with or without 30 μM SNAP, 1 μM PA (16:0-18:2), 0.1% 1-Bu, or 30 μM cPTIO. NO fluorescence was detected by DAF-FMDA after 6 h of exposure (A). This experiment was repeated three times with similar results and one replicate is represented. Flu: NO fluorescence; BF: Bright Field. NO content were also measured (B). Values reflect means ± SEs of at least three independent experiments (n = 10/experiment). Different symbols above the bars indicate significant differences (Tukey's test, p < 0.05).

Figure 4

Protein expression patterns in the total plasma membranes of rice seedling roots exposed to cadmium or cadmium and SNAP. The total plasma membrane proteins from third-leaf-stage rice seedling roots were treated with 10 μM cadmium with or without 30 μM SNAP for 12 h and 1 day, and the differential expression protein were identified by the iTRAQ method. The number of up-regulated and down-regulated proteins were listed (A), and hierarchical clustering of cadmium- and SNAP-responsive proteins were also presented (B). The colors correspond to the log-transformed values of the protein change-fold ratios, as depicted in the bottom-right bar.

Figure 5

NO promotes cadmium-induced PLD protein migration and its enzyme activity. (A) GFP fluorescence in rice protoplasts transiently expressing PLDa-GFP fusion protein. Control, untreated; Cd, treated with 10 μM cadmium for 12 h; Cd+SNAP, treated with 10 μM cadmium plus 30 μM SNAP for 12 h; Cd+SNAP+cPTIO, treated with 10 μM cadmium plus 30 μM SNAP and 30 μM cPTIO for 12 h. From left to right: Flu, GFP fluorescence; BF:bright field; Merge (Flu+BF): merged image of FLU and BF. The GFP fluorescence intensity in the nucleus and membrane were quantified by Image J software (http://rsb.info.nih.gov/ij/download.html). (B,C) Western blotting and an anti-PLD antibody assay to detect PLDa accumulation in the enriched plasma membrane protein (B) and the total proteins (C). Third-leaf-stage rice seedlings were treated with 10 μM cadmium with or without 30 μM SNAP or 30 μM cPTIO for 1 day. The enriched plasma membranes proteins and total proteins were isolated for PLD content analysis. (D,E) The rice seedling at three leaf stage were treated with 10 μM cadmium, the time course of PLD activity with (closed circle) or without Cd stress (open circle) were measured (D), and the effect of different chemicals on PDL activity were measured after 1 day of treatment (E). Values reflect means ± SEs of three independent experiments (n = 10/experiment). Different symbols above the bars indicate significant differences (Tukey's test, p < 0.05).

Figure 6

Effects of cadmium, SNAP, and different chemicals on PA accumulation. Third-leaf-stage rice seedlings were treated with 10 μM cadmium or other chemicals as above. The time course of total PA content with (closed circle) or without Cd stress (open circle) were measured (A), and the effect of different chemicals on total PA content (B). The different PA molecular species content was measured at the indicated times (C) and different PA molecular species content (D) were also measured after 12 h of treatment. The asterisk in (C,D) indicates that the mean value is significantly different from that of the control without any treatment (P < 0.05). Values reflect means ± SEs of six independent experiments (n = 10/experiment).

Figure 7

NO and PA reduce cadmium-induced H₂O₂ accumulation and increase antioxidant accumulation. (A) The effects of cadmium and different treatments on H₂O₂ and glutathione accumulation in rice seedlings. Rice was treated with cadmium, Cd and SNAP, Cd+SNAP+cPTIO, Cd+PA, and Cd+1-Bu. The content of H₂O₂ and glutathione were measured after 3 days of treatment. (B) The effects of cadmium, Cd and SNAP, Cd+SNAP+cPTIO, Cd+PA, and Cd+1-Bu on SOD, APX, and GR antioxidant enzymes activities. The asterisk in (A,B) indicates that the mean value is significantly different from that of the control without any treatment (P < 0.05). Values reflect means ± SEs of three independent experiments (n = 5/experiment). (C) The effects of cadmium, Cd and SNAP, Cd+SNAP+cPTIO, Cd+PA, and Cd+1-Bu on enzyme protein accumulations. The antioxidant enzyme activities and protein accumulation were measured after 3 days of treatments. Anti-actin antibodies were used as the loading control.

Figure 8

Proposed model for the roles of NO and PA in facilitating rice seedling tolerance to cadmium stress. Once subjected to Cd stress, NO is quickly generated in rice. NO increases the accumulation of PLD around the plasma membrane allowing PA to be generated. The PA induces the accumulation of antioxidants and reduces H₂O₂ accumulation. The accumulation of antioxidants and reduction in H₂O₂ increases the plant tolerance to cadmium stress.