Thus one of the aim of the work was

Thus, one of the aim of the work was to link the phytotoxic effect of m-Tyr on root growth of tomato seedlings to modification of RNS level and tissue distribution accompanied by determination of 3-NT content. Due to the role of GSNOR, which is suggested to be a key element in the interplay between the ROS and RNS metabolisms [34], we pointed our interest at its activity, protein level and gene expression. Even if (as reviewed by Petřivalský et al. [28]) GSNOR activity and expression varied depending on environmental conditions, no simple correlation between application of oxidative agents or ROS (H2O2 or paraquat) and GSNOR activity, gene expression or protein level are confirmed [35], [36]. The experimental model, we are using for investigation of regulation of elongation growth of roots [30], [31], [37], [38] seems to be very convenient to build up a general idea of mode of action of m-Tyr, with a special attention pointed at its putative, secondary effect related to RNS metabolism.
Materials and methods
Results
Discussion According to the idea of “root-brain” or “plant intelligence” root is the most important organ of the plant body responsible for signal perception [47]. Ability of plants to combine all environmental information depends on root function, but on the other hand involves modification in root growth depending on the stress factor. A detailed explanation of plant susceptibility implies the existence of short-life and membrane permeable signaling molecules such as RNS or ROS. Both of them are considered also as important morphogens. m- Tyr as natural chemical of a strong phytotoxic potential is in a center of attention of allelopathic/environmental toxicology studies due to is potential as environmental friendly herbicide, but also focuses interest of other researchers as a useful marker of oxidative stress. Supplementation of tomato seedlings with m-Tyr inhibited elongation growth of roots, accompanied with slight decline in cell viability. This data are in agreement to previous reports [2], [7], [48], describing m-Tyr dose dependent restriction of seedlings' growth of various plant species. In addition to inhibitory effect on root elongation we demonstrated also alteration in the number of border notch inhibitor in plants exposed to this NPAA. After 24 h of m-Tyr supplementation number of border cells was reduced, while in contrast after 72 h of culture it doubled in comparison to the control, but it was not accompanied by decline in cell viability. Enrichment in border cells number was noticed in roots of tomato exposed to canavanine [31], while after application of other allelochemicals e.g. cyanamide to maize (Zea mays L.) [38] or caffeine, naringenin, liquiritigenin to pea (Pisum sativum L.) [49] the number of border cells was significantly reduced. Correlation of chemical toxicity to amount and viability of border cells are not clear and most likely depend on plant material and tested chemical. Roots shortening of tomato seedlings treated with m-Tyr was linked also to other changes in morphology. They were visibly thicker, of higher diameter but significantly reduced surface area, suggesting malformations in cytoskeleton arrangement and/or cell cycle or differentiation. Similar to m-Tyr effect on root morphology was noticed in roots of Arabidopsis thaliana subjected to farnesene (a volatile sesquiterpene commonly found in the essential oils of some plants), and despite disruptions of microtubule formation were linked to modification in ROS and RNS production [50]. m-Tyr led also to formation of more lateral root primordia, a commonly observed effect of NO in plant developmental processes (see Ref. [52] for review). In tomato plants exposed to m-Tyr enhanced fluorescence signal corresponding to NO and ONOO− was characteristic only for border cells and epidermis of root tips and was accompanied by high level of both RNS detected in roots of plants cultured in 50 μM NPAA for 24 h. Prolonged treatment with m-Tyr or higher concentration of phytotoxin resulted in reduction of fluorescence of DAF-FM derivatives and have a slight impact on fluorescence of APF derivatives. Although accumulation of ROS seems to be a common and typical secondary stress induced by various biotic and abiotic stressors, there is no rule pointing at the direction of changes in RNS concentration. Some stress factors (e.g. heavy metals) in various plant material provoke NO or ONOO− generation, while in another lead to their reduction (see Ref. [33] for review). Nevertheless, alterations in NO level may be one of the reason of disturbances in root growth induced by m-Tyr application as fundamental role of this signaling molecule in regulation of root growth was confirmed e.g. in seedlings of NO-deficient Arabidopsis mutants. In noa1, nia1, and nia2 reduced length of primary root and shortened meristem were typical [52].