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.

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