Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two‐week‐old rice seedlings were exposed to 5°C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2‐DE and stained with CBB. Of the 250–400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress‐responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody‐affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2‐DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up‐regulated, and defense‐related proteins were down‐regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress‐related proteins are rapidly up‐regulated, while defense‐related proteins disappear, under long‐term cold stress.