Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species.
The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.
Sakamoto
Atsushi
A
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Tsukamoto
Shigefumi
S
Yamamoto
Hiroshi
H
Ueda-Hashimoto
Manami
M
Takahashi
Misa
M
Suzuki
Hitomi
H
Morikawa
Hiromichi
H
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D002736:Chloroplasts / Q000201:enzymology*; D004926:Escherichia coli; D016166:Free Radical Scavengers / Q000378:metabolism; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D009569:Nitric Oxide / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D030421:Peroxynitrous Acid / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013997:Time Factors
D029681:Arabidopsis Proteins; D016166:Free Radical Scavengers; D026361:Reactive Nitrogen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D010544:Peroxidases; C108214:BAS1 protein, Arabidopsis; D054464:Peroxiredoxins