Figure 4.

Protein carbonyl patterns in Arabidopsis seeds following CDT or natural aging. Protein extracts were prepared as described in “Materials and Methods” from the dry mature seeds and analyzed by 2D PAGE. Carbonylated proteins were characterized in nondeteriorated seeds (0 d, control seeds; A), 7-d deteriorated seeds (B), freshly harvested seeds (C), and 11-year-old dry mature seeds (D). Proteins were separated by 2D gel electrophoresis as shown in Figure 2A. Following transfer to nitrocellulose, the appearance of carbonyl groups in proteins was analyzed by immunodetection of protein-bound DNP after derivatization with the corresponding hydrazine, as described (Job et al., 2005). Proteins undergoing carbonylation are labeled with black arrows; they are listed in Table III. These findings show that both CDT and natural aging induce an oxidative stress on specific proteins, presumably through the generation of ROS.