IC4R007-Proteomic-2010-19853963

Project Title

Comparative proteomic analysis of the short-term responses of rice roots and leaves to cadmium

The Background of This Project

'Fig. 1. Determination of total GSH content in rice leaf and root tissues following treatment with 100 m M CdCl 2 for 24 h. Total GSH levels in control and Cd-treated rice leaves and roots were measured.

• Cadmium (Cd) is one of the most serious pollutants, andmassive amounts are released into the environment by human activities. Contamination of the food chain by Cd is the main entry pathway for humans. Rice is known to be a leading dietary source of Cd and renal tubular dysfunction in families on rice farms has been linked with exposure to Cd. In this project, to investigate the effect of Cd in rice, the researchers conduct an systematic analysis of proteome profiles in roots and leaves, and furthermore, leaf proteins were fractionated with polyethylene glycol.

Plant Culture & Treatment

• Seeds of rice (Oryza sativa L. cv. Dongjin) were surface-sterilized in 70% ethanol for 5 min and then in 3% sodium hydrochloride for 30 min. Seeds were rinsed five times with sterilized water and imbibed in sterilized water for 1 d at room temperature. Imbibed seeds were transferred to moistened two-layer gauze with 1/2 strength MS medium (2.14 g/L MS medium including vitamins, 0.05% MES, pH 5.8) on baskets.
• The baskets were transferred to boxes containing 1/2 strength MS medium and seedlings were grown at 25 1C under a 16-h photoperiod. After 10 d, seedlings were transferred to fresh MS medium in the absence or presence of 100 m M CdCl 2 for 24 h, and roots and leaves were then collected.

Protein Extraction and 2-D PAGE

• Rice root proteins were isolated according to a modified phenol extraction method developed by Hurkman and Tanaka in 1986 and Rice leaves proteins were fractionated with PEG using the method developed by Kim in 2001. The protein concentration was determined using the 2D-Quant Kit.
• Quantified proteins (150 m g) were applied to an 18-cm IPG strip (pH 4–7, linear gradient) in a Protean IEF cell (Bio-Rad, Hercules, CA, USA) and in-gel rehydrated for 12 h. Isoelectric focusing was performed at 20 1C for 1 h at 100 V, 1 h at 1000 V, 1 h at 2500 V, 1 h at 5000 V, and 10 h at 8000 V. The strips were equilibrated with solution I (50 mM Tris–HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% DTT for 15 min, and then with solution I containing 2.5% iodoacetamide. In the second dimension, proteins were separated in 12% SDS-polyacrylamide gels and visualized by silver staining with no glutaraldehyde (Blum et al.,1987).
  

  Fig. 2. Representative 2-DE gel images of rice roots and leaves treated with Cd.
• Gel images were scanned using a GS-800 Imaging Densitometer (Bio-Rad) and analyzed with the software PDQuest version 7.2.0 (Bio-Rad). For each sample, quantitation was performed with three analytical gels originating from three independent biological replicas. The volume of each spot was normalized to a relative volume, and mean values calculated from triplicate data were compared. Quantitative analysis sets were created between each control group and Cd-treated group. Proteins showing a significant change (Po0.05) after a statistical analysis by Student’s t-test were selected. The cutoff for differentially regulated proteins was a 1.5-fold change.

Research Findings

• GSH is the major reservoir of non-protein thiols and plays an important role in defense against heavy metals and reactive oxygen species. In roots, a dramatic decrease in GSH levels was observe after Cd treatment; GSH levels were about 8 times lower in Cd-exposed roots than in control roots.In contrast, no significant difference was detected between untreated and Cd-treated leaf tissues. This result implies that GSH in roots was rapidly consumed for the chelation to Cd and incorporation into PCs following short-term Cd exposure.
  
• The expression levels of a total of 36 proteins derived from 37 gel spots were changed upon Cd treatment. In root tissues, 18 proteins from 17 gel spots exhibited changes in response to Cd, including 16 up-and 2 down-regulated proteins (Table 1). The expression of 18 proteins originated from 19 gel spots (15 up- and 3 down-regulated) in leaves was changed (Table 2).
• The identified proteins were classified into several groups: oxidative stress (antioxidant enzymes); carbohydrate metabolism; amino acid metabolism; protein metabolism (protein synthesis, degradation and folding);photosynthesis and Calvin cycle; and others. The functional distributions of up-regulated proteins are compared in Supplementary Fig. 2. The possible roles of proteins whose abundance is changed by Cd are discussed below with respect to their functional distributions.

Labs working on this Project

• Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea
• Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea
• Central Instrument Facility, Gyeongsang National University, Jinju 660-701, Republic of Korea