IC4R008-Proteomic-2002-12203890
Contents
Project Title
- Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings
The Background of This Project
- Ozone is a destructive gaseous pollutant with serious impact on human and animal respiration as well as causing extensive damage to both natural and cultivated plant populations.
- Despite the widespread occurrence of ozone injuries, the precise mechanisms of the injury process and the plant defense systems against ozone attacks remain poorly understood. During the past few years, the effects (of ozone) and responses (in plants), especially those at the level of the gene expression are becoming clear, although there still remains an urgent need for a truly ozone-specific gene regulation pattern, or the development of a molecular probe for ozone stress. Though a variety of plant species have been examined in their response(s) to ozone, the rice plant, a monocot research model (cv. Nipponbare), remains the least investigated.
- In this project, Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, the researchers examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins.
Plant Culture & Treatment
- Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown for two weeks under white fluorescent light (wavelength 390–500 nm, 150 μM/m2/s, 12 h light period/day) at 25℃ and 70% relative humidity (RH) as previously reported. At this stage, the seedling is composed of four leaves, a shoot (leaf sheath) and roots. After completing the 12 h dark cycle, the rice seedlings were exposed continuously to 0.2 ppm ozone, or filtered pollutant free air as a control for up to a maximum of 3 d in fumigation chambers controlled at 25ºC with a RH of 70%, mean wind velocity of 0.22 m/s, and light conditions of 350 μM/ m2/s. There is a precedent in the literature for using high light intensity (400 μM/m2/s) during ozone (1 μL/L) treatment of 6-week old rice plants, after first growing the same plants under natural light conditions. After placing the seedlings in these chambers, the third and fourth leaves and leaf sheaths (see Fig. 1) were removed at appropriate times as indicated in the legends to figures, and immediately stored at –80℃ until analyzed for changes in protein profiles by SDS-PAGE and 2-DE. Ozone was prepared and its concentration monitored with an ML9810O3 analyzer (Lear Siegler Measurement Controls, Englewood, USA) as described previously.
Figure 1 Sketch of rice seedlings (cv. Nipponbare) used in this study that indicate the leaves (third and fourth) sampled during these experiments (left, 14-days old), and at 3 d after treatment (right, showing the elongating fourth leaf, 17-days old). The seedling consists of four leaves (numbered bottom to top) at two-weeks old. Arrow marks the leaf sheath region used in this study.
Protein Extraction and 2-D PAGE
- For protein extraction, 200 mg of leaf (or leaf sheath) segments were finely cut with clean scissors and homogenized with 500 μL of lysis buffer (a 10 mL solution in MilliQ water contains 4.8 g urea, 0.2 mL of NP-40, 0.2 mL carrier ampholyte (pH 3.5–10), 0.5 mL 2-mercaptoethanol, and 0.5 g PVP-40], using a glass mortar and pestle at RT. The homogenates were transferred to 1.5 mL Eppendorf tubes, and centrifuged at 18 500 x g once for 5 min and then for 10 min at 4℃, and the resulting supernatant was used as the crude protein extract. Thirty (leaf) or 50 (for leaf sheath) microliters of the supernatant was subjected to 2-DE, following O’Farrell’s method. Electrophoresis (IEF, carried out in a glass capillary tube of 13 cm length and 3 mm diameter (Nihon Eido) and SDS-PAGE in the second dimension) at a constant current of 35 mA was carried out exactly as described previously. The gels were stained with CBB, and destained. The image analysis was performed visually, and the changes observed are both qualitative and quantitative with respect to the controls. Reproducibility of 2-DE protein profiles was confirmed by first exposing three independent sets of seedlings to ozone, and running each control and ozone treated sample on 2-DE gels, and second by running each of the control/ ozone-treated samples for five times, which was subsequently used for collection of protein spots for amino acid sequencing.
Research Findings
- Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls(Fig.2 and 3).
- Out of a total of 56 identified proteins that were taken for sequencing, amino acid sequence of 14 proteins could not be determined (spots 1–3, 14, 23, 28, 29, 33, 41–43, 47, 48, 55) due to either low amounts, unclear sequence, certain modifications, and/ or blocked N-terminals, and only six proteins (spots 7, 8, 9, 15, 16, and 40) had clearly blocked N-terminals (Table 1). However, the N-terminal amino acid sequences of 36 proteins (spots 4–6, 10–13, 17–22, 24–27, 30–32, 34–39, 44–46, 49–54 and 56) were determined, and only one N-terminally blocked protein (spot 7) could be internally sequenced (Table 1).
- Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins.
- Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein.
Labs working on this Project
- Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal
- Japan Science and Technology Corporation (JST), Japan
- Food Function Laboratory, School of Agriculture, Ibaraki University, Japan
- Environmental Biology Division, National Institute for Environmental Studies (NIES), Tsukuba, Ibaraki, Japan
Corresponding Author
- rjunko@nifty.com/gkagrawal@onebox.com
