TY - JOUR A1 - Staszek, Pawel A1 - Krasuska, Urszula A1 - Otulak-Koziel, Katarzyna A1 - Fettke, Jörg A1 - Gniazdowska, Agnieszka T1 - Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots JF - Frontiers in plant science N2 - Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 mu M) for 24-72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-mu M CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-mu M CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-mu M CAN, while 10-mu M CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration. KW - canavanine KW - cellular antioxidant system KW - GSNOR-GSNO reductase KW - nitrated proteins KW - nitric oxide-NO KW - nonproteinogenic amino acid KW - NOS-like activity KW - reactive nitrogen species (RNS) Y1 - 2019 U6 - https://doi.org/10.3389/fpls.2019.01077 SN - 1664-462X VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Krasuska, Urszula A1 - Ciacka, Katarzyna A1 - Orzechowski, Slawomir A1 - Fettke, Jörg A1 - Bogatek, Renata A1 - Gniazdowska, Agnieszka T1 - Modification of the endogenous NO level influences apple embryos dormancy by alterations of nitrated and biotinylated protein patterns JF - Planta N2 - NO donors and Arg remove dormancy of apple embryos and stimulate germination. Compounds lowering NO level (cPTIO, L -NAME, CAN) strengthen dormancy. Embryo transition from dormancy state to germination is linked to increased nitric oxide synthase (NOS)-like activity. Germination of embryos is associated with declined level of biotin containing proteins and nitrated proteins in soluble protein fraction of root axis. Pattern of nitrated proteins suggest that storage proteins are putative targets of nitration. Nitric oxide (NO) acts as a key regulatory factor in removal of seed dormancy and is a signal necessary for seed transition from dormant state into germination. Modulation of NO concentration in apple (Malus domestica Borkh.) embryos by NO fumigation, treatment with NO donor (S-nitroso-N-acetyl-d,l-penicillamine, SNAP), application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), N (omega)-nitro-l-arginine methyl ester (l-NAME), canavanine (CAN) or arginine (Arg) allowed us to investigate the NO impact on seed dormancy status. Arg analogs and NO scavenger strengthened embryo dormancy by lowering reactive nitrogen species level in embryonic axes. This effect was accompanied by strong inhibition of NOS-like activity, without significant influence on tissue NO2 (-) concentration. Germination sensu stricto of apple embryos initiated by dormancy breakage via short term NO treatment or Arg supplementation were linked to a reduced level of biotinylated proteins in root axis. Decrease of total soluble nitrated proteins was observed at the termination of germination sensu stricto. Also modulation of NO tissue status leads to modification in nitrated protein pattern. Among protein bands that correspond to molecular mass of approximately 95 kDa, storage proteins (legumin A-like and seed biotin-containing protein) were identified, and can be considered as good markers for seed dormancy status. Moreover, pattern of nitrated proteins suggest that biotin containing proteins are also targets of nitration. KW - Apple KW - Nitro-tyrosine KW - Nitric oxide synthase-like activity KW - Reactive nitrogen species KW - Seed germination Y1 - 2016 U6 - https://doi.org/10.1007/s00425-016-2553-z SN - 0032-0935 SN - 1432-2048 VL - 244 SP - 877 EP - 891 PB - Springer CY - New York ER -