One of the mechanism of regulation of NO content

One of the mechanism of regulation of NO content in plant cells is it scavenging by GSH leading to formation of GSNO [59]. This reaction, depending on NO level and availability of glutatione in its reduced form, influences cellular redox potential by lowering GSH content and in consequence may impact development of root get better [60]. GSNO acts as S-transnitrosylation agent, can interact with specific sulfhydryl groups of proteins to produce high-molecular mass S-nitrosothiols, which can also transfer NO to the sulfhydryl groups of other proteins in a process of S-transnitrosation between proteins [55], [59]. GSNO is decomposed by the GSNOR to GSSG which is the substrate of the glutathione reductase that regenerates the GSH pool. We observed stimulation of GSNOR activity in roots after prolonged m-Tyr application and the effect was concentration dependent. It should be mentioned that, GSNOR activity measured using spectrophotometric method in extracts of tomato roots and determined by staining in the gel, after proteins separation by native electrophoresis did not express strictly the same pattern. Stimulation of GSNOR activity was noticed only after 72 h of culture in m-Tyr, as determined by spectrophotometric assay. In comparison, visualization of GSNOR activity in the gel seems to be more appropriate to demonstrate m-Tyr impact on NO metabolism, because significant enhancement of the signal by m-Tyr application (at both tested concentrations) was noticed just at the beginning of the experiment. Activity of GSNOR determined in our experiment in extract of roots were of the same range as presented by Kubienová et al. [43], although we were working using younger seedling and different variety of tomato. As reported in the literature, most of stressors stimulated activity of GSNOR [53], [56], [61]. Heat and cold increased GSNOR activity in pea and cucumber (Cucumis sativus L.) seedlings [42], although enhancement of enzyme activity generally in roots was less than 1.5 fold. In contrast various light conditions did not alter GSNOR activity in roots of pea plants [42], suggesting that there is no straight relationship between GSNOR activity and stress response. Zhou et al. [62], using proteomic approach, demonstrated 1.4 fold increase abundance of GSNOR in roots of tomato in response to aluminum stress. In our experiment changes in GSNOR activity in the gel correlate to the level of GSNOR protein in tomato roots. Western blot analysis indicated only slight impact of m-Tyr on this parameter. GSNOR is a constitutive protein, presence of which is common all over plant kingdom, although there are some differences in its abundance in different plant organs [28], [63]. Kubienová et al. [43] reported that in tomato GSNOR activity and gene expression differed in various organs depending on developmental stage and in old (6 weeks old) plants its activity was higher in roots than in leaves. Lack of severe correlation of protein GSNOR amount and activity is not surprising, taking also account onto GSNOR gene expression. In tomato GSNOR is encoded by a single gen, transcription of which differs depending on stress condition [42]. Gong et al. [64] showed that in tomato seedlings sodic alkaline stress induced fluctuations in transcripts of GSNOR accompanied by similar pattern of changes in protein level. In our experiment supplementation of tomato seedlings with 250 μM m-Tyr resulted in slight decline (in comparison to the control) of transcript level of GSNOR, while 50 M m-Tyr only transiently (after short term application) increased the GSNOR transcript level. In general our transcriptomic data indicate rather non-direct effect of m-Tyr on GSNOR gene transcription. Depending on literature, it is the first evidence pointing on the impact of allelopathic compound on GSNOR, as so far toxicity of allelochemicals is very rarely related to RNS formation and metabolism [30], [31], [50], [65].