Identification of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-targeted proteomics approach.
In plants, environmental stresses cause an increase in the intracellular level of reactive oxygen species (ROS), leading to tissue injury. To obtain biochemical insights into this damage process, we investigated the protein carbonyls formed by ROS or by the lipid peroxide-derived α,β-unsaturated aldehydes and ketones (i.e. reactive carbonyl species, or RCS) in the leaves of Arabidopsis thaliana under salt stress. A. thaliana Col-0 plants that we treated with 300 mM NaCl for 72 h under continuous illumination suffered irreversible leaf damage. Several RCS such as 4-hydroxy-(E)-2-nonenal (HNE) were increased within 12 h of this salt treatment. Immunoblotting using distinct antibodies against five different RCS, i.e. HNE, 4-hydroxy-(E)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde, revealed that RCS-modified proteins accumulated in leaves with the progress of the salt stress treatment. The band pattern of Western blotting suggested that these different RCS targeted a common set of proteins. To identify the RCS targets, we collected HNE-modified proteins via an anti-HNE antiserum affinity trap and performed an isobaric tag for relative and absolute quantitation, as a quantitative proteomics approach. Seventeen types of protein, modified by 2-fold more in the stressed plants than in the non-stressed plants, were identified as sensitive RCS targets. With aldehyde-reactive probe-based affinity trapping, we collected the oxidized proteins and identified 22 additional types of protein as sensitive ROS targets. These RCS and ROS target proteins were distributed in the cytosol and apoplast, as well as in the ROS-generating organelles the peroxisome, chloroplast and mitochondrion, suggesting the participation of plasma membrane oxidation in the cellular injury. Possible mechanisms by which these modified targets cause cell death are discussed.
Science Research Center, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8515 JapanGraduate School of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8515 Japan mano@yamaguchi-u.ac.jp.
Jun'ichi
J
Mano
Graduate School of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8515 Japan.
Mitsuaki
M
Nagata
Graduate School of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8515 Japan.
Shoutarou
S
Okamura
Faculty of Agriculture, Niigata University, Ikarashi-Ninocho 8050, Nishi-ku, Niigata, 950-2181 JapanNiigata Crop Research Center, Niigata Agricultural Research Institute, Nagakura-cho 857, Nagaoka, 940-0826 Japan.
Takeshi
T
Shiraya
Faculty of Agriculture, Niigata University, Ikarashi-Ninocho 8050, Nishi-ku, Niigata, 950-2181 Japan.
Toshiaki
T
Mitsui
D000447:Aldehydes / Q000378:metabolism; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000378:metabolism*; D008027:Light; D015227:Lipid Peroxidation; D018384:Oxidative Stress; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D050050:Protein Carbonylation; D020543:Proteome; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*; D013312:Stress, Physiological
D000447:Aldehydes; D029681:Arabidopsis Proteins; D020543:Proteome; D017382:Reactive Oxygen Species; C013166:2-nonenal; D012965:Sodium Chloride
4-Hydroxyl-(E)-2-nonenal (HNE); Acrolein; Lipid peroxide; Oxidative stress; Reactive carbonyl species; Reactive electrophiles