Proteomic analysis of rice leaves during drought stress and recovery

Three‐week old plants of rice (Oryza sativa L. cv CT9993 and cv IR62266) developed gradual water stress over 23 days of transpiration without watering, during which period the mid‐day leaf water potential declined to ∼–2.4 MPa, compared with ∼–1.0 MPa in well‐watered controls. More than 1000 protein spots that were detected in leaf extracts by proteomic analysis showed reproducible abundance within replications. Of these proteins, 42 spots showed a significant change in abundance under stress, with 27 of them exhibiting a different response pattern in the two cultivars. However, only one protein (chloroplast Cu‐Zn superoxide dismutase) changed significantly in opposite directions in the two cultivars in response to drought. The most common difference was for proteins to be up‐regulated by drought in CT9993 and unaffected in IR62266; or down‐regulated by drought in IR62266 and unaffected in CT9993. By 10 days after rewatering, all proteins had returned completely or largely to the abundance of the well‐watered control. Mass spectrometry helped to identify 16 of the drought‐responsive proteins, including an actin depolymerizing factor, which was one of three proteins detectable under stress in both cultivars but undetectable in well‐watered plants or in plants 10 days after rewatering. The most abundant protein up‐regulated by drought in CT9993 and IR62266 was identified only after cloning of the corresponding cDNA. It was found to be an S‐like RNase homologue but it lacked the two active site histidines required for RNase activity. Four novel drought‐responsive mechanisms were revealed by this work: up‐regulation of S‐like RNase homologue, actin depolymerizing factor and rubisco activase, and down‐regulation of isoflavone reductase‐like protein.