IC4R002-Proteomic-2013-23327584
Contents
Project Title
Comparative Proteome Analyses Reveal that Nitric Oxide Is an Important Signal Molecule in the Response of Rice to Aluminum Toxicity
The Background of This Project
- Aluminum (Al) toxicity is a major contributing factor that inhibits crop production and decreases crop quality in acidic soil worldwide. 1,2 At a soil pH lower than 5, aluminum ions (Al 3+ ) aresolubilized and suppress root development in plants. Though there are various other ions in the soil, the Al ion is the major cause of phytotoxicity in acidic soils.
In this study, the researchers applied proteomic and physiological approaches to investigate the dynamic protein profiles in rice seedlings under Al stress. Our results demonstrated that NO is an important signal for modulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism as rice seedlings respond to Al toxicity.
Plant Culture & Treatment
Figure 4. Dynamic protein changes in the rice leaves responding to Al and NO stress. Treatment with Al and NO were performed as described in the legend to Figure 1 legend. After 1 day and 3 days of treatment, 1 mg of total protein was extracted from the different plants and loaded in each gel. (A) Representative BR-20 stained 2D gel of total protein from the control plants. (B) Enlarged windows (a−e) from panel A of the spot changes in the representative gels from Al- and NO-treated samples.
- Rice seeds (Oryza sativa L.) were cleaned and surface-sterilized in a solution of 2% sodium hypochlorite for 15 min, rinsed five times in sterilized water and germinated in plastic trays lined with wet paper towels for 36 h in the dark (23 °C). The seedlings were grown in Hoagland nutrient solution under controlled conditions (25 °C day/30 °C night cycle; 200 μmol photons m −2 s −1 light intensity; relative humidity of 75−80%). When the third leaf was fully expanded after two weeks of culture, they were treated for the experiments.
- For Al stress, aluminum chloride (75 μM) was added to 1/2 Hoagland nutrient solution, with the active free Al 3+ in this solution being approximately 16.5 μM calculated using Geo-Chem-EZ software. For NO treatment, the NO donor S-nitroso-N-acetylpenicillamine, dissolved into double distilled water as a stock soltion (30 mM), was added to the nutrient solution at a final concentration of 30 μM, and the treatment solution containing SNAP was changed everyday to maintain SNAP activity. For the inhibitor treatments, the rice seedlings were pretreated with the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 50 μM), the NOS inhibitor N(G)-nitro- L -arginine methyl ester (L-NAME, 10 μM), the feedback nitrate reductase inhibitor glutamine (Gln, 50 mM), or the HO (heme oxygenase, responsible for carbon monoxide generation) inhibitor zinc protoporphyrin-IX (ZnppIX, 50 μM), for 2 h before Al or NO treatment, respectively.
Protein Extraction and 2-D PAGE
- Protein extraction and 2D separation were performed according to a reported method with minor modifications. 24 Approximately 10 to 20 g of the treated leaves was ground in liquid nitrogen and the total soluble protein was extracted at 4 °C in 5 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 20 mM KCl, 13 mM DTT, 2% (v/v) NP-40, 150 mM PMSF and 1% (w/v) PVP. The homogenates were centrifuged (12000× g, 15 min, 4 °C) and the supernatants were added to five volumes of acetone containing 10% (w/v) TCA and 1% (w/v) DTT. The samples were maintained at −20 °C for 4 h and then centrifuged (25000× g, 30 min, 4 °C).
- The resulting pellets were washed with acetone containing 1% (w/v) DTT at −20 °C for 1 h and then centrifuged, and the wash step was repeated. The final pellets were vacuum-dried and then dissolved in 8 M urea, 20 mM DTT, 4% (w/v) CHAPS and 2% (w/v) ampholyte (pH 3−10). The samples in ampholyte were vortexed thoroughly for 1 h at room temperature and then centrifuged (25000× g, 20 min, 20 °C), and the supernatants were collected for 2D electrophoresis (2DE). Each experiment was repeated three times.
Research Findings
- To define the role of NO during the rice response to Al stress, the researchers applied a proteomic approach to investigate the changes in the protein profile. Approximately 1000 proteins were reproducibly resolved on each gel. A total of 256 protein spots were reproducibly detected that showed significant changes in response to the Al or NO treatments (p < 0.05) compared to the control, which received no treatment. Overall, 92 out of the 256 differential protein spots were positively identified using MALDI-TOF MS.
Figure 5(B) hierarchical clustering for the expression protein profile in rice seedlings exposed to Al or NO treatment. The hierarchical cluster analysis was conducted using the cluster 3.0 and Treeview software (Left) Cluster analysis result of the total differentially expressed proteins; (Right) enlarged results of a subset of identified differentially expressed proteins. The different colors correspond to the log-transformed values for the protein change-fold ratio shown in the bar at the bottom of the figure.
- The identified proteins were divided into 9 groups based on their biological functions (Figure 5A). The majority of these proteins were sorted to a general energy and metabolism group, followed by antioxidant enzymes, a transcriptional factor group, protein kinases and phosphatases, and cell structure and division proteins. A hierarchical cluster analysis was conducted to categorize the proteins that showed differential expression profiles during Al stress and NO treatment (Figure 5B). As would be expect for the treatments that induce oxidative stresses, we found that the proteins belonging to the antioxidant system were clustered together. In addition, the proteins involved in NO metabolism, including NR and GSNOR, were up-regulated, suggesting their critical roles during rice seedling exposure to Al stress.
Labs working on this Project
- Department of Molecular Biology, National Institute of Agrobiological Sciences; 2–1–2 Kannondai, Tsukuba, Ibaraki 305–8602, Japan.
