Nitric oxide synthase inhibitor L-NAME affects Arabidopsis root growth, morphology, and microtubule organization.

The presence of evolutionarily conserved NOS or NOS-like enzymes in land plants different than those in animals is still unclear, despite their activity has been revealed in cytosol and some organelles. At the same time, the emerging evidence for the importance of L-arginine-dependent pathways of NO synthesis in plant cells is still accumulating. The aim of our study was to reveal physiological effects on growth and differentiation processes, and microtubular cytoskeleton organization of the competitive mammalian NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME). Thus, the treatment of Arabidopsis with L-NAME (50-1 mM) caused dose- and time-dependent inhibition of primary roots growth. Moreover, the morphology of primary roots under the influence of L-NAME also underwent changes. L-NAME (>100 µM) induced the formation of novel over-elongated root hairs in shortened elongation zone, while in higher concentrations (500 µM) it caused a slight swelling of epidermal cells in differentiation zone. L-NAME also provoked microtubule reorganization in epidermal cells of different root growth zones. Thus, L-NAME at concentrations of 50-1 mM induced cortical microtubules randomization and/or depolymerization in epidermal cells of the root apex, meristem, transition, elongation, and differentiation zones after 2 h of treatment. Disordered microtubules in trichoblasts could initiate the formation of actively elongating root hairs that reveals longitudinal microtubules ensuring their active growth at 24 h of treatment. Therefore, L-NAME inhibits primary root growth, induces the differentiation processes in roots, reorganizes cortical microtubules in epidermal root cells suggesting the importance of L-arginine-dependent pathways of NO synthesis in plants.

Nitric oxide modulates actin filament organization in Arabidopsis thaliana primary root cells at low temperatures.

Cytoskeleton is gaining the increasing recognition as one of nitric oxide (NO)-downstream targets because of its involvement in plenty of NO-controlled processes in plants throughout the entire life cycle starting from seed germination to pollination as well as (a)biotic stress tolerance. It has been revealed that low temperature (+0.5°C) has an inhibitory effect on A. thaliana primary root growth and causes an anisotropic increase of epidermal cells diameter in elongation zone. Furthermore, actin filaments' organization of epidermal cells in different zones of primary roots is modulated by NO content. Thus, the exogenous NO donor (SNP) favors to actin filaments network reorganization, while both cold and NO scavenger (c-PTIO) increase its randomization. According to the data obtained, it can be assumed that not only actin filaments act as NO sensors, but NO is also involved into plant cell response on low temperatures by the signaling via such important cytoskeleton machinery as actin network.

Cytoskeleton in the Parasitic Plant Cuscuta During Germination and Prehaustorium Formation.

Although cytoskeleton is a driving force for cell division and growth in higher plants, there is little evidence about its components in parasitic angiosperms. Microtubules and actin filaments in cells of shoot apical meristem and root-like structure of stem holoparasites European (C. europaea L.) and Eastern (C. monogyna Vahl.) dodders, as well as in prehaustorium, the specific organ adapted to parasitism, were visualized for the first time by immunolabeling and fluorescence microscopy. The significance of cytoskeletal elements during germination and prehaustorium formation was addressed by treatments with taxol, oryzalin, latrunculin B, cytochalasin B/D, jasplakinolide, and 2,3-butanedione monoxime. In shoot apical meristem many dividing cells were observed, in contrast to root-like structure, devoid of cell divisions. Cortical microtubules were oriented transversely and/or obliquely, while actin filaments were randomly distributed in cells of both organs. Furthermore, longitudinal cortical microtubules were present in digitate cells of prehaustorium, and transverse arrays were found in its file cells. Long and short random actin filaments were also observed in prehaustorium cells. Thus, it was shown that the cytoskeleton in dodder shoot cells is organized in a similar way to non-parasitic dicots, while cytoskeletal organization has some peculiarities in quickly senescing root-like structure and prehaustorium.

Plant-based biopharming of recombinant human lactoferrin.

Recombinant proteins are currently recognized as pharmaceuticals, enzymes, food constituents, nutritional additives, antibodies and other valuable products for industry, healthcare, research, and everyday life. Lactoferrin (Lf), one of the promising human milk proteins, occupies the expanding biotechnological food market niche due to its important versatile properties. Lf shows antiviral, antimicrobial, antiprotozoal and antioxidant activities, modulates cell growth rate, binds glycosaminoglycans and lipopolysaccharides, and also inputs into the innate/specific immune responses. Development of highly efficient human recombinant Lf expression systems employing yeasts, filamentous fungi and undoubtedly higher plants as bioreactors for the large-scale Lf production is a biotechnological challenge. This review highlights the advantages and disadvantages of the existing non-animal Lf expression systems from the standpoint of protein yield and its biological activity. Special emphasis is put on the benefits of monocot plant system for Lf expression and the biosafety aspects of the transgenic Lf-expressing plants.

Tubulin tyrosine nitration regulates microtubule organization in plant cells.

During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics.

Nitric oxide as a critical factor for perception of UV-B irradiation by microtubules in Arabidopsis.

Influence of ultraviolet-B (UV-B) as an abiotic stress factor on plant microtubules (MTs) and involvement of nitric oxide (NO) as a secondary messenger mediating plant cell response to environmental stimuli were investigated in this study. Taking into account that endogenous NO content in plant cells has been shown to be increased under a broad range of abiotic stress factors, the effects of UV-B irradiation and also the combined action of UV-B and NO donor sodium nitroprusside (SNP) or NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) on the MTs organization in different root cells of Arabidopsis thaliana were tested. Subsequently, realization of the MT-mediated processes such as root growth and development was studied under these conditions. Arabidopsis thaliana seedlings expressing the chimeric gene gfp-map4 were exposed to the enhanced UV-B with or without SNP or c-PTIO pretreatment. The UV-B irradiation alone led to a dose-dependent root growth inhibition and to morphological alterations of the primary root manifested in their swelling and excessive root hair formation. Moreover, dose-dependent randomization and depolymerization of MTs in both epidermal and cortical cells under the enhanced UV-B were found. However, SNP pretreatment of the UV-B irradiated A. thaliana seedlings recovered the UV-B inhibited root growth as compared to c-PTIO pretreatment. It has been shown that in 24 h after UV-B irradiation the organization of MTs in root epidermal cells of SNP-pretreated A. thaliana seedlings was partially recovered, whereas in c-PTIO-pretreated ones the organization of MTs has not been distinctly improved. Therefore, we suppose that the enhanced NO levels in plant cells can protect MTs organization as well as MT-related processes of root growth and development against disrupting effects of UV-B.

Nitric oxide signalling via cytoskeleton in plants.

Nitric oxide (NO) in plant cell mediates processes of growth and development starting from seed germination to pollination, as well as biotic and abiotic stress tolerance. However, proper understanding of the molecular mechanisms of NO signalling in plants has just begun to emerge. Accumulated evidence suggests that in eukaryotic cells NO regulates functions of proteins by their post-translational modifications, namely tyrosine nitration and S-nitrosylation. Among the candidates for NO-downstream effectors are cytoskeletal proteins because of their involvement in many processes regulated by NO. This review discusses new insights in plant NO signalling focused mainly on the involvement of cytoskeleton components into NO-cascades. Herein, examples of NO-related post-translational modifications of cytoskeletal proteins, and also indirect NO impact, are discussed. Special attention is paid to plant α-tubulin tyrosine nitration as an emerging topic in plant NO research.