Proteomic analysis of reactive oxygen species (ROS)-related proteins in rice roots

To investigate the rice root proteome, we applied the PEG fractionation technique combined with two-dimensional gel electrophoresis which rendered more well-separated protein spots. Out of the 295 chosen proteins, 93 were identified by MALDI-TOF mass spectrometry. The proteins were classified as relating to metabolism (38.7%), reactive oxygen species (ROS)-related proteins (22.5%), protein processing/degradation (8.6%), stress/defense (7.5%), energy (6.5%) and signal transduction (5.4%). The high percentage of ROS-related proteins found in rice root brings us to assess the roles of ROS on rice root growth. Treatment with ROS quenching chemicals such as reduced glutathione (GSH), diphenyleneiodonium (DPI) and ascorbate inhibited root growth dose-dependently. Forty-nine proteins identified were either up- or down-regulated by GSH treatment, of which 14 were ROS-related proteins, such noticeably modulated ones as glutathione-S-transferase (GST), superoxide dismutases (SOD) and L-ascorbate peroxidases. The protein levels of four GSTs (NS4, 8, 56 and 57), three APXs (NS46, 49 and 50) and MnSOD (NS45) were strongly reduced by GSH treatment but slightly reduced by ascorbate and DPI. Ascorbate and DPI strongly inhibited expression levels of a catalase A (NP23) and an APX (NS65) but did not affect APXs (NS46, 49 and 50) protein levels. Northern analysis demonstrated that changes in transcript levels of five genes--GST (NS4), GST (NS43), Mn-SOD (NS45), APX (NS50) and APX (NS46/49) in response to ROS quenching chemicals were coherent with patterns shown in two-dimensional electrophoresis analyses. Taken together, we suggest that these proteins may take part in an important role in maintaining cellular redox homeostasis during rice root growth.