Identification of Cu-binding proteins in embryos of germinating rice in response to Cu toxicity

Seed germination, an early and important process for the growth and development of plants, is hypersensitive to environmental changes. Copper (Cu) is a necessary micronutrient for plants; however, an excessive dose of Cu had an extremely negative effect at the cellular level as a result of inevitable binding to proteins. In contrast, some structural motifs of proteins can bind free Cu ions and relieve Cu toxicity. This study aimed to understand the expression characteristics of Cu-binding proteins induced by excess Cu during rice seed germination. We investigated Cu-binding proteins in germinating rice embryos treated with 200 µM Cu using a Sephadex G-50 column or immobilized Cu affinity chromatography combined with two-dimensional gel electrophoresis. Proteomics analysis indicated that 12 protein spots exhibited a > 2.0-fold increase in intensity in response to Cu toxicity as compared with controls. Among nine proteins in ten spots identified as Cu-binding proteins, three proteins (from four spots) were involved in antioxidative defense: copper, zinc superoxide dismutase, glutathione S-transferase and protein disulfide isomerase. These results show that reactive oxygen species may be involved in the expression regulation of Cu-binding proteins in germinating rice in response to Cu stress.