title: Nitration of plant apoplastic proteins from cell suspension cultures.
abstract: Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases.
  
  
  CONCLUSIONS
  High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long-term adapted to stressful conditions, and results showing that such conditions did not always cause an increase in nitrotyrosine content. These findings may be interpreted as characteristic features of apoplastic protein nitration.
authors: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland; Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik Poland. Electronic address: agnieszkalapa@wp.pl.
authors: Agnieszka
authors: A
authors: Szuba
authors: Department of Molecular and Cellular Biology, Adam Mickiewicz University, Umultowska 89, 61-613 Poznań, Poland.
authors: Anna
authors: A
authors: Kasprowicz-Maluśki
authors: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland; Department of Molecular and Cellular Biology, Adam Mickiewicz University, Umultowska 89, 61-613 Poznań, Poland.
authors: Przemysław
authors: P
authors: Wojtaszek
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D002478:Cells, Cultured; D009569:Nitric Oxide / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D001059:Apoproteins; D051017:Apoptosis Regulatory Proteins; D010940:Plant Proteins; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D009584:Nitrogen
keywords: Apoplast; Arabidopsis thaliana; Nicotina tabacum; Nitrotyrosines; Proteomics; Suspension cultures