Arabidopsis Iron Superoxide Dismutase FSD1 Protects Against Methyl Viologen-Induced Oxidative Stress in a Copper-Dependent Manner.

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear enzyme with osmoprotective and antioxidant functions. However, the current knowledge on its role in oxidative stress tolerance is ambiguous. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. In accordance with the known regulation of FSD1 expression, abundance, and activity, the findings demonstrated that the antioxidant function of FSD1 depends on the availability of Cu2+ in growth media. Arabidopsis fsd1 mutants showed lower capacity to decompose superoxide at low Cu2+ concentrations in the medium. Prolonged exposure to MV led to reduced ascorbate levels and higher protein carbonylation in fsd1 mutants and transgenic plants lacking a plastid FSD1 pool as compared to the wild type. MV induced a rapid increase in FSD1 activity, followed by a decrease after 4 h long exposure. Genetic disruption of FSD1 negatively affected the hydrogen peroxide-decomposing ascorbate peroxidase in fsd1 mutants. Chloroplastic localization of FSD1 is crucial to maintain redox homeostasis. Proteomic analysis showed that the sensitivity of fsd1 mutants to MV coincided with decreased abundances of ferredoxin and photosystem II light-harvesting complex proteins. These mutants have higher levels of chloroplastic proteases indicating an altered protein turnover in chloroplasts. Moreover, FSD1 disruption affects the abundance of proteins involved in the defense response. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

Mistletoe Eradicator - A Novel Tool for Simultaneous Mechanical and Chemical Control of Mistletoe.

Some species of mistletoes, an aerial plant hemiparasites, play a Janus-like role in ecosystems by being rich biodiversity hotspots due to their trophic and topic associations with various organisms and at the same time acting as biological pirates, exhausting the host trees by stealing water and nutrients via the haustorium. Many crop and timber trees are attacked by mistletoes, for example, dwarf mistletoes parasitize conifers, representatives of genus Tapinanthus target guava, and Phoradendron mistletoe target pecans. Recently, an increasing infestation of the European mistletoe (Viscum album subsp. album L.) in monoculture stands of common walnuts (Juglans regia L.) was recorded for the first time during 2018 in different regions of Kashmir Valley, India. The common chemical approach based on the spraying of mistletoe exophytes with the growth regulators, ethephon and glyphosate, was not efficient since V. album resprouted in the next vegetation season. Hand pruning of mistletoe was time-consuming, and a polyethylene covering resulted in the localized rotting of host tissues. The most efficient approach to eradicate mistletoe was the use of a novel tool called Mistletoe Eradicator, which allows to use mechanical and chemical means of mistletoe control simultaneously. It consists of a manipulating pole with the terminal pruning saw having a container filled with the growth regulator, from which the liquid slowly drops onto the saw blade through the nozzle. It allows working both from the ground and in the tree canopy. After the targeted application of 5%-10% ethephon or glyphosate to the base of the just pruned V. album stems (stubs), exophyte resprouting was significantly reduced 9 months after the pruning as compared to the only pruned or sprayed exophytes. An alternative variant of Mistletoe Eradicator was constructed in Ukraine filled just with a food color additive to track the liquid dropping and was successfully tested.

GR24, A Synthetic Strigolactone Analog, and Light Affect the Organization of Cortical Microtubules in Arabidopsis Hypocotyl Cells.

Strigolactones are plant hormones regulating cytoskeleton-mediated developmental events in roots, such as lateral root formation and elongation of root hairs and hypocotyls. The latter process was addressed herein by the exogenous application of a synthetic strigolactone, GR24, and an inhibitor of strigolactone biosynthesis, TIS108, on hypocotyls of wild-type Arabidopsis and a strigolactone signaling mutant max2-1 (more axillary growth 2-1). Owing to the interdependence between light and strigolactone signaling, the present work was extended to seedlings grown under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signaling using fluorescence microscopy methods with different spatiotemporal capacities, such as confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM). It was found that GR24-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, observed in living wild-type and max2-1 seedlings stably expressing genetically encoded fluorescent molecular markers for microtubules. Quantitative assessment of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signaling affected microtubule remodeling, especially under light conditions. The application of GR24 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between cytoskeletal behavior and the light-dependence of strigolactone signaling.

Parasites on parasites: hyper-, epi-, and autoparasitism among flowering plants.

All organisms engage in parasitic relations, as either parasites or hosts. Some species may even play both roles simultaneously. Among flowering plants, the most widespread form of parasitism is characterized by the development of an intrusive organ called the haustorium, which absorbs water and nutrients from the host. Despite this functionally unifying feature of parasitic plants, haustoria are not homologous structures; they have evolved 12 times independently. These plants represent ca. 1% of all extant flowering species and show a wide diversity of life histories. A great variety of plants may also serve as hosts, including other parasitic plants. This phenomenon of parasitic exploitation of another parasite, broadly known as hyper- or epiparasitism, is well described among bacteria, fungi, and animals, but remains poorly understood among plants. Here, we review empirical evidence of plant hyperparasitism, including variations of self-parasitism, discuss the diversity and ecological importance of these interactions, and suggest possible evolutionary mechanisms. Hyperparasitism may provide benefits in terms of improved nutrition and enhanced host-parasite compatibility if partners are related. Different forms of self-parasitism may facilitate nutrient sharing among and within parasitic plant individuals, while also offering potential for the evolution of hyperparasitism. Cases of hyperparasitic interactions between parasitic plants may affect the ecology of individual species and modulate their ecosystem impacts. Parasitic plant phenology and disperser feeding behavior are considered to play a major role in the occurrence of hyperparasitism, especially among mistletoes. There is also potential for hyperparasites to act as biological control agents of invasive primary parasitic host species.

In vivo light-sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection.

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue- and organ-specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light-sheet and Airyscan confocal microscopy. FSD1-GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1-GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1-GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.

Fatty acid composition in seeds of holoparasitic Orobanchaceae from the Caucasus region: Relation to species, climatic conditions and nutritional value.

The specialization of parasitic plants from the Orobanchaceae family to the heterotrophic lifestyle caused several morphological, physiological and molecular changes. One of the adaptations to the parasitic lifestyle is the production of a large number of the smallest seeds in world flora, also called "dust-seeds". Seeds of 34 holoparasitic species from the Cistanche, Orobanche, Phelipanche, and Phelypaea genera were collected in the Caucasus region (54 samples) and their fatty acid content and compositions analysed. Of these seeds, 28 were investigated for the first time, and 12 are endemic to the Caucasus (one of the most important biodiversity hotspots in the world). The influence of different hosts, populations, habitats, and climatic conditions on the fatty acid content and composition, as well as some connections of taxonomic classification are discussed. The fatty acid content in the species varied between 0.9 and 42.5%, and showed quantitative differences at generic and infrageneric levels, while displaying uniform fatty acid composition. Thirteen fatty acids were identified, of which nine were undescribed for Orobanchaceae. The fatty acid composition of the Orobanchaceae seeds represented a mixture of saturated fatty acids (SFAs) (average 7.8%) and unsaturated fatty acids (UFAs) (average 92.2%). The fatty acid content in the Orobanchaceae seeds was directly unrelated to taxonomy, while the n-6/n-3 fatty acid ratio supported the clear separation of the Orobanche and Phelipanche genera. Orobanchaceae seeds contained mainly linoleic and oleic acids, thus they could be a potential nutritional source of the unsaturated fatty acids. Additionally, the studies confirmed the hypothesis that the degree of seed oil fatty acid unsaturation increased in colder climatic conditions, especially for the Orobanche genus.

YODA-HSP90 Module Regulates Phosphorylation-Dependent Inactivation of SPEECHLESS to Control Stomatal Development under Acute Heat Stress in Arabidopsis.

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth, such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation into guard cells require a master regulatory protein phosphorylation cascade involving the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which regulate transcription factors, including SPEECHLESS (SPCH). Here, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical, and cell biology approaches, we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90s) play a crucial role in transducing heat-stress response through the YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, such as MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of the stomatal cell lineage couples stomatal development to environmental cues, providing an adaptive heat stress response mechanism in plants.

Signaling Toward Reactive Oxygen Species-Scavenging Enzymes in Plants.

Reactive oxygen species (ROS) are signaling molecules essential for plant responses to abiotic and biotic stimuli as well as for multiple developmental processes. They are produced as byproducts of aerobic metabolism and are affected by adverse environmental conditions. The ROS content is controlled on the side of their production but also by scavenging machinery. Antioxidant enzymes represent a major ROS-scavenging force and are crucial for stress tolerance in plants. Enzymatic antioxidant defense occurs as a series of redox reactions for ROS elimination. Therefore, the deregulation of the antioxidant machinery may lead to the overaccumulation of ROS in plants, with negative consequences both in terms of plant development and resistance to environmental challenges. The transcriptional activation of antioxidant enzymes accompanies the long-term exposure of plants to unfavorable environmental conditions. Fast ROS production requires the immediate mobilization of the antioxidant defense system, which may occur via retrograde signaling, redox-based modifications, and the phosphorylation of ROS detoxifying enzymes. This review aimed to summarize the current knowledge on signaling processes regulating the enzymatic antioxidant capacity of plants.

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.

Plant microtubules reorganization under the indirect UV-B exposure and during UV-B-induced programmed cell death.

The role of microtubules in cellular pathways of UV-B signaling in plants as well as in related structural cell response become into focus of few last publications. As microtubules in plant cell reorient/reorganize (become randomized, fragmented or depolymerized) in a response to direct UV-B exposure, these cytoskeletal components could be involved into UV-B signaling pathways as highly responsive players. In the current addendum, indirect UV-B-induced microtubules reorganization in cells of shielded Arabidopsis thaliana (GFP-MAP4) primary roots and the correspondence of microtubules depolymerization with the typical hallmarks of the programmed cell death in Nicotiana tabacum BY-2 (GFP-MBD) cells are discussed.

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.