Krasylenko Y. The role of microtubules in mediation of nitric oxide action on plant cell

The current thesis is dedicated to the investigation of the effects of both eхogenous and endogenous NO content modulators (NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (c-PTIO), NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NАМE) and 3-nitrotyrosine as a marker of NO activity on the growth and morphology of Arabidopsis thaliana (GFP-MAP4) primary roots as well as on microtubules (MTs) organization in root cells. It was revealed that MTs in epidermal cells of transition and elongation zones were the most sensitive to the fluctuations of intracellular NO content. NO donors SNP and SNAP promoted A. thaliana primary root growth and provoked MTs reorganization in epidermal cells of the differentiation zone that, in turn, initiated root hair's primordia formation. At the same time, NO scavenger c-PTIO acts as a negative regulator of primary root growth and root hairs formation, since it caused MTs depolymerization in trichoblasts along with swelling of A. thaliana primary root cells. It was also shown that the processes of growth and differentiation of A. thaliana primary roots as well as MTs organization in root cells were altered after the treatment of seedlings with the competitive NO-synthase inhibitor L-NAME. This supports the importance of L-arginin-dependent pathway of endogenous NO synthesis for the maintenance of MTs organization. In turn, 3-nitrotyrosine led to the inhibition of A. thaliana primary root growth and to the randomization of endoplasmic MTs in meristematic cells as well as cortical microtubules in epidermal cells of transition and elongation zones that might have caused the root growth inhibition. The presence of alplha-tubulin nitrotyrosination in the untreated BY-2 cells was demonstrated for the first time by Western-blot analysis. Nitrotyrosination of alpha-tubulin under physiological conditions may explain its regulatory potential. Using the method of immunofluorescent microscopy with specific antibodies against alpha-tubulin and nitrotyrosine, the colocalization of alpha-tubulin and nitrotyrosine on preprophase bands, mitotic spindles and fragmoplasts of the untreated cells of Bright Yellow-2 (ВY-2) Nicotiana tabacum suspension culture were revealed. This proves the presence of nitrotyrosinated alpha-tubulin in the polymerized MTs. It was assumed that the functional role of this posttranslational modification can be the regulation of MTs dynamic properties, since SNP treatment caused dose-dependent decrease of general number of mitotic MTs without any changes of their organization. The influence of the UV-B (280-315 nm) as one of the key abiotic stress factors becomes more evident due to the unswerving ozone layer depletion that impairs the morphogenesis and productivity of plants. A. thaliana primary root growth inhibition along with morphological alterations acompanied by MTs randomization or depolymerization was revealed after the seedlings exposure to the enhanced UV-B (6.8-68 kJ/m2). Is was also found that SNP pretreatment of A. thaliana seedlings with further UV-B exposure reestablished the root growth activity and initial primary roots morphology as well as MTs organization in root cells. The obtained results indicate functional interrelation between MTs organization and fluctuations of intracellular NO content under the UV-B influence. Moreover, the enhanced UV-B exposure of the aboveground part of A. thaliana seedlings caused the indirect inhibition of primary root growth, intense root hairs formation and MTs reorganization in epidermal cells of the transition zone. This possibly indicates the long-distance signal transduction by various secondary messengers and NO fits this role as well.