Limits...
Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide.

Zhou L, Bokhari SA, Dong CJ, Liu JY - PLoS ONE (2011)

Bottom Line: Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins.Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs.Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, School of Life Sciences, Tsinghua University, Beijing, People's Republic of China.

ABSTRACT

Background: Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H(2)O(2)) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H(2)O(2)-responsive proteins in the apoplast of rice seedling roots.

Methodology/principal findings: 14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H(2)O(2) for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H(2)O(2) were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H(2)O(2) conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H(2)O(2) stress.

Conclusions/significance: The present study is the first apoplast proteome investigation of plant seedlings in response to H(2)O(2) and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.

Show MeSH

Related in: MedlinePlus

Effects of H2O2 treatments in rice seedlings, protein yields and intracellular contamination ratio.(A) Effects of H2O2 treatments on REL in rice seedlings. (B) Effects of H2O2 treatments on MDA concentrations in rice seedling roots. (C) Accumulation of apoplastic H2O2 in rice seedling roots. (D) Protein yields from vacuum infiltrates. (E) Intracellular contamination of vacuum infiltrates. Two-week-old seedlings were treated with H2O2 at different concentrations for 6 h. Values are means of independent replicates±SE, n = 3. Levels of significance of T-test are shown by * and ** for p<0.05 and 0.01, compared to the control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037377&req=5

pone-0016723-g001: Effects of H2O2 treatments in rice seedlings, protein yields and intracellular contamination ratio.(A) Effects of H2O2 treatments on REL in rice seedlings. (B) Effects of H2O2 treatments on MDA concentrations in rice seedling roots. (C) Accumulation of apoplastic H2O2 in rice seedling roots. (D) Protein yields from vacuum infiltrates. (E) Intracellular contamination of vacuum infiltrates. Two-week-old seedlings were treated with H2O2 at different concentrations for 6 h. Values are means of independent replicates±SE, n = 3. Levels of significance of T-test are shown by * and ** for p<0.05 and 0.01, compared to the control.

Mentions: To minimize the contamination of apoplast proteins with intracellular proteins, it is absolutely critical to adopt appropriate H2O2 concentrations for the treatments of rice seedling roots. Electrolyte leakage is an important indicator of the cell membrane damage under adverse conditions including oxidative stress [18]. To evaluate the effect of H2O2 stress on the cell membrane, changes in relative electrolyte leakage (REL) were measured in rice seedling roots treated with different concentrations of H2O2. As shown in Figure 1A, REL increased slightly from 24.76% (control) to 34.82% (300 µM) or 40.83% (600 µM) as the concentration of H2O2 increased from 0 to 600 µM, indicating that these treatments with H2O2 indeed triggered some root responses. When the concentrations of H2O2 were increased to 900 µM or higher, REL jumped to more than 50%, suggesting some oxidative damage to the cell membrane. It has been reported that, if the seedling REL of the chilling-sensitive rice cultivar TN.1 reaches 50% or higher under chilling (5°C) treatment, the survival ratio is less than 70%, demonstrating severe oxidative damage to the cell membrane [19]. These data suggest that treatments with higher than 600 µM H2O2 may be inappropriate for this study. The effect of H2O2 concentrations on rice seedling roots was further evaluated by an assay for malondialdehyde (MDA), which is a breakdown product of membrane lipid peroxidation and can also be used as a marker to indicate the degree of damage of the cell membrane under oxidative stress [20]. As presented in Figure 1B, there were almost no changes in the MDA concentration between the control (12.1±1.22 nmol/g FW) and samples treated with 300 µM H2O2 (12.3±2.03 nmol/g FW). Even under the 600 µM H2O2 treatment, the MDA concentration increased only slightly to 18.2±0.6 nmol/g FW (0.01<p<0.05, compared to the control) (Figure 1B). Based on the results of the REL and MDA assays, it is clear that 300 µM H2O2 led to some responses in the cell membrane without obvious lipid peroxidation change. However, 600 µM H2O2 caused slight oxidative damage to the cell membrane with increased lipid peroxidation and electrolyte leakage. Additionally, the levels of apoplastic H2O2 in roots treated with exogenous H2O2 of 300 and 600 µM H2O2 concentrations were increased by 30.8% and 76.8% over control, respectively, showing a dose-dependent pattern of accumulation (Figure 1C). Furthermore, 600 µM H2O2 treatment for 6 h caused the seedling leaves to roll inward, and the net photosynthetic rate was declined by about 20% over control [3]. Taken together, these results suggest that these two concentrations represent different oxidative conditions and rice seedlings could tolerate these concentrations without serious destruction to the cell membrane, therefore, 300 µM and 600 µM H2O2 were adopted for the treatments in this study.


Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide.

Zhou L, Bokhari SA, Dong CJ, Liu JY - PLoS ONE (2011)

Effects of H2O2 treatments in rice seedlings, protein yields and intracellular contamination ratio.(A) Effects of H2O2 treatments on REL in rice seedlings. (B) Effects of H2O2 treatments on MDA concentrations in rice seedling roots. (C) Accumulation of apoplastic H2O2 in rice seedling roots. (D) Protein yields from vacuum infiltrates. (E) Intracellular contamination of vacuum infiltrates. Two-week-old seedlings were treated with H2O2 at different concentrations for 6 h. Values are means of independent replicates±SE, n = 3. Levels of significance of T-test are shown by * and ** for p<0.05 and 0.01, compared to the control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037377&req=5

pone-0016723-g001: Effects of H2O2 treatments in rice seedlings, protein yields and intracellular contamination ratio.(A) Effects of H2O2 treatments on REL in rice seedlings. (B) Effects of H2O2 treatments on MDA concentrations in rice seedling roots. (C) Accumulation of apoplastic H2O2 in rice seedling roots. (D) Protein yields from vacuum infiltrates. (E) Intracellular contamination of vacuum infiltrates. Two-week-old seedlings were treated with H2O2 at different concentrations for 6 h. Values are means of independent replicates±SE, n = 3. Levels of significance of T-test are shown by * and ** for p<0.05 and 0.01, compared to the control.
Mentions: To minimize the contamination of apoplast proteins with intracellular proteins, it is absolutely critical to adopt appropriate H2O2 concentrations for the treatments of rice seedling roots. Electrolyte leakage is an important indicator of the cell membrane damage under adverse conditions including oxidative stress [18]. To evaluate the effect of H2O2 stress on the cell membrane, changes in relative electrolyte leakage (REL) were measured in rice seedling roots treated with different concentrations of H2O2. As shown in Figure 1A, REL increased slightly from 24.76% (control) to 34.82% (300 µM) or 40.83% (600 µM) as the concentration of H2O2 increased from 0 to 600 µM, indicating that these treatments with H2O2 indeed triggered some root responses. When the concentrations of H2O2 were increased to 900 µM or higher, REL jumped to more than 50%, suggesting some oxidative damage to the cell membrane. It has been reported that, if the seedling REL of the chilling-sensitive rice cultivar TN.1 reaches 50% or higher under chilling (5°C) treatment, the survival ratio is less than 70%, demonstrating severe oxidative damage to the cell membrane [19]. These data suggest that treatments with higher than 600 µM H2O2 may be inappropriate for this study. The effect of H2O2 concentrations on rice seedling roots was further evaluated by an assay for malondialdehyde (MDA), which is a breakdown product of membrane lipid peroxidation and can also be used as a marker to indicate the degree of damage of the cell membrane under oxidative stress [20]. As presented in Figure 1B, there were almost no changes in the MDA concentration between the control (12.1±1.22 nmol/g FW) and samples treated with 300 µM H2O2 (12.3±2.03 nmol/g FW). Even under the 600 µM H2O2 treatment, the MDA concentration increased only slightly to 18.2±0.6 nmol/g FW (0.01<p<0.05, compared to the control) (Figure 1B). Based on the results of the REL and MDA assays, it is clear that 300 µM H2O2 led to some responses in the cell membrane without obvious lipid peroxidation change. However, 600 µM H2O2 caused slight oxidative damage to the cell membrane with increased lipid peroxidation and electrolyte leakage. Additionally, the levels of apoplastic H2O2 in roots treated with exogenous H2O2 of 300 and 600 µM H2O2 concentrations were increased by 30.8% and 76.8% over control, respectively, showing a dose-dependent pattern of accumulation (Figure 1C). Furthermore, 600 µM H2O2 treatment for 6 h caused the seedling leaves to roll inward, and the net photosynthetic rate was declined by about 20% over control [3]. Taken together, these results suggest that these two concentrations represent different oxidative conditions and rice seedlings could tolerate these concentrations without serious destruction to the cell membrane, therefore, 300 µM and 600 µM H2O2 were adopted for the treatments in this study.

Bottom Line: Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins.Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs.Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, School of Life Sciences, Tsinghua University, Beijing, People's Republic of China.

ABSTRACT

Background: Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H(2)O(2)) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H(2)O(2)-responsive proteins in the apoplast of rice seedling roots.

Methodology/principal findings: 14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H(2)O(2) for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H(2)O(2) were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H(2)O(2) conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H(2)O(2) stress.

Conclusions/significance: The present study is the first apoplast proteome investigation of plant seedlings in response to H(2)O(2) and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.

Show MeSH
Related in: MedlinePlus