IC4R012-Proteomic-2009-19201764

Project Title

• Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa

The Background of This Project

• Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos.

Plant Culture & Treatment

• Rice (Oryza sativa L. ‘Wuyunjing’ No. 7) seeds were surface-sterilized with 5% (v/v) sodium hypochlorite(NaClO) for 15 min and thoroughly washed in distilled water. Seeds were germinated on moist filter paper. Twenty seeds were randomly placed on the filter paper in 90-mm Petri dishes, and 5 mL of freshly prepared CuSO4.5H2O solution (0–200 mM) was added. Each treatment was performed in triplicate. The seeds were put to germinate for 4–8 d in the dark at 258C with renewal of the Cu solution at 2-d intervals. Seeds were considered to have germinated when the shoots were longer than 2 mm. The radicle length was measured during the exposure period. Embryos, including newly formed shoots, were isolated from the seeds after 6 d for Cu concentration and protein analyses.

Protein Extraction and 2-D PAGE

• The protein extracts were precipitated with 8 vol of ice-cold acetone containing 1 mM DTT, incubated at –208C for at least 10 h, and then centrifuged for 5 min at 12 000 g. The pellets were washed four times with ice-cold acetone containing 1 mM DTT and then dissolved in rehydration solution [8 M urea, 4% CHAPS, 65 mM DTT, 0.2% (w/v) Bio-Lyte(Bio-Rad, Hercules, CA, USA)]. Protein concentrations were assayed using a Bio-Rad RC DC Protein Assay Kit 1.For 2-DE, 1 mg of protein was loaded onto a 17-cm dry IPG strip (pH 3–10 linear gradient; Bio-Rad) using the overnight, ingel reswelling method according to the manufacturer’s instructions. The strips were placed at the top of 12.5% SDS–polyacrylamide gels and sealed with 0.5% agarose.Electrophoresis was carried out at 80 V for 30 min and then at 200 V for 5 h using a Protean Plus Dodeca cell apparatus(Bio-Rad). The 2-DE gels were stained with colloidal Coomassie brilliant blue G250 (Neuhoff et al., 1988).
• Image and data analyses of the gels were performed using PDQuest software (Version 7.2; Bio-Rad). After alignment of the gels, the spots were matched between gels automatically, and the matched spots were re-examined manually to ensure accuracy. Spot quantity normalization was conducted in the ‘total quantity of valid spots’ mode. Duplicate 2-DE gels were run for each treatment from three independent tissue extractions, and only those spots that changed reproducibly were considered to represent differentially expressed proteins. The results for Cu-treated and control samples were analysed for differences using analysis of variance (ANOVA)and Student’s t-tests. Selected protein spots were manually excised from the gels for further analysis.

Research Findings

• As compared with the control (no Cu; Fig. 1), treatment with 50 or 100 mM Cu for 4 d had no significant effect on radicle elongation. On the other hand, prolonged treatment (6 or 8 d) with Cu (�50 mM) had a negative effect, with radicle length decreasing as the concentration of Cu in the incubation medium increased.

'F I G. 1. Effect of Cu on rice radicle elongation. Rice seeds were treated with the indicated concentrations of Cu for 4, 6 or 8 d. Values shown represent means+ s.e. (n ¼ 3) for three different experiments. Means denoted by the same letter did not differ significantly (P � 0.05 according to Duncan’s multiple range test).'

• As the amount of Cu in the incubation medium increased,the Cu content in the rice embryos also increased (Fig. 2A).The maximum Cu content observed in the embryos was 0.96 mg g21 (dry weight), which occurred when the seeds germinated in the presence of 200 mM Cu.

'F I G. 2. Effect of Cu treatment on the content of (A) Cu in germinating rice embryos.'

• The soluble protein concentration and protein thiol content in the germinating rice seed embryos were determined on the sixth day after Cu treatment. At 100 or 200 mM, but not at 50 mM, Cu significantly increased the soluble protein concentration, as compared with the control (Fig. 2B). The protein thiol content was significantly higher in the embryos treated with 200 mM Cu than in the control (Fig. 2C)

'Effect of Cu treatment on the content of(B) total protein in germinating rice embryos. Rice seeds were treated with the indicated concentrations of Cu for 6 d. Values shown represent means+ s.e. (n ¼ 3) for three different experiments. Means denoted by the same letter did not differ significantly (P � 0.05 according to Duncan’s multiple range test).'

'Effect of Cu treatment on the content of(C) protein thiol (PT) in germinating rice embryos. Rice seeds were treated with the indicated concentrations of Cu for 6 d. Values shown represent means+ s.e. (n ¼ 3) for three different experiments. Means denoted by the same letter did not differ significantly (P � 0.05 according to Duncan’s multiple range test).'

• As shown in Fig. 3, SDS–PAGE of the total proteins from germinating rice embryos yielded some visible differences in the protein band patterns of the control versus Cu-treated embryos. These differences became more pronounced when the extracts were first heated at 808C for 5 min before loading. The intensities of two protein bands of about 12 kDa and 20 kDa increased as the concentration of Cu in the incubation medium increased.

'F I G. 3. SDS–PAGE of germinating rice embryo extracts from seeds treated with the indicated concentrations of Cu for 6 d. Equal amounts of protein(20 mg) were loaded in each gel lane. Arrows indicate protein bands altered by Cu treatment. Experiments were performed three times with similar results.

• The change in protein patterns was further analysed by 2-DE for better separation and characterization. Digital image analysis of a Coomassie-stained 2-DE gel revealed .520 rice embryo protein spots, of which 16 of the smaller proteins(5–25 kDa) were Cu-responsive. Compared with the control,Cu treatment up-regulated expression of 13 spots and downregulated expression of three spots (Fig. 4).

'F I G. 4. Two-dimensional PAGE of germinating rice embryo extracts from seeds treated with 0 (Con) or 200 mM Cu for 6 d. Equal amounts of protein(1 mg) were loaded on each gel. Arrows indicate the proteins identified.'

Labs working on this Project

• College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
• Asian Natural Environmental Science Center, The University of Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan

Corresponding Author

• Zhenguo Shen:zgshen@njau.edu.cn