Abstract
Copper (Cu) is an essential micronutrient for plants, but excess Cu can inactivate and disturb the protein function due to unavoidable binding to proteins at the cellular level. As a redox-active metal, Cu toxicity is mediated by the formation of reactive oxygen species (ROS). Cu-binding structural motifs may alleviate Cu-induced damage by decreasing free Cu2+ activity in cytoplasm or scavenging ROS. The identification of Cu-binding proteins involved in the response of plants to Cu or ROS toxicity may increase our understanding the mechanisms of metal toxicity and tolerance in plants. This study investigated change of Cu-binding proteins in radicles of germinating rice seeds under excess Cu and oxidative stress using immobilized Cu2+ affinity chromatography, two-dimensional electrophoresis, and mass spectra analysis. Quantitative image analysis revealed that 26 protein spots showed more than a 1.5-fold difference in abundances under Cu or H2O2 treatment compared to the control. The identified Cu-binding proteins were involved in anti-oxidative defense, stress response and detoxification, protein synthesis, protein modification, and metabolism regulation. The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to low concentration Cu (20 μM Cu) and H2O2 stress, and 5 out of 24 were increased under low and high concentration Cu (100 μM Cu) but unaffected under H2O2 stress, which hint Cu ions can regulate Cu-binding proteins accumulation by H2O2 or no H2O2 pathway to cope with excess Cu in cell. The change pattern of these Cu-binding proteins and their function analysis warrant to further study the roles of Cu ions in these Cu-binding proteins of plant cells.
Highlights
Copper (Cu), an essential micronutrient required for growth and development in all plants, is a structural and catalytic cofactor of several proteins and enzymes involved in electron transfer and redox reactions
Our results showed that exposure to excess Cu for 12 h caused an evident accumulation of H2O2 in the radicles (Figure 1A)
The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to 20 μM Cu and H2O2 stress in rice radicles
Summary
Copper (Cu), an essential micronutrient required for growth and development in all plants, is a structural and catalytic cofactor of several proteins and enzymes involved in electron transfer and redox reactions. More than 100 proteins comprising two groups are estimated to have the ability to complex with Cu in Arabidopsis: Cu-binding proteins/chaperones and enzymes (Häensch and Mendel, 2009). Excess Cu can inactivate and disturb the protein structure via unavoidable protein binding (Yruela, 2009). To control metal homeostasis and redox status, plants have several mechanisms of metal tolerance, including exclusion, compartmentalization, and binding to organic ligands such as organic acids, amino acids, peptides, and proteins (Hall, 2002; Yruela, 2009). The identification of metal-binding proteins involved in the responses of plants to metal toxicity may improve our understanding regarding the mechanisms of metal toxicity and tolerance in plants
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