Genome-wide identification and evolution of the tubulin gene family in Camelina sativa.

BACKGROUND: Tubulins play crucial roles in numerous fundamental processes of plant development. In flowering plants, tubulins are grouped into α-, ß- and γ-subfamilies, while α- and ß-tubulins possess a large isotype diversity and gene number variations among different species. This circumstance leads to insufficient recognition of orthologous isotypes and significantly complicates extrapolation of obtained experimental results, and brings difficulties for the identification of particular tubulin isotype function. The aim of this research is to identify and characterize tubulins of an emerging biofuel crop Camelina sativa. RESULTS: We report comprehensive identification and characterization of tubulin gene family in C. sativa, including analyses of exon-intron organization, duplicated genes comparison, proper isotype designation, phylogenetic analysis, and expression patterns in different tissues. 17 α-, 34 ß- and 6 γ-tubulin genes were identified and assigned to a particular isotype. Recognition of orthologous tubulin isotypes was cross-referred, involving data of phylogeny, synteny analyses and genes allocation on reconstructed genomic blocks of Ancestral Crucifer Karyotype. An investigation of expression patterns of tubulin homeologs revealed the predominant role of N6 (A) and N7 (B) subgenomes in tubulin expression at various developmental stages, contrarily to general the dominance of transcripts of H7 (C) subgenome. CONCLUSIONS: For the first time a complete set of tubulin gene family members was identified and characterized for allohexaploid C. sativa species. The study demonstrates the comprehensive approach of precise inferring gene orthology. The applied technique allowed not only identifying C. sativa tubulin orthologs in model Arabidopsis species and tracking tubulin gene evolution, but also uncovered that A. thaliana is missing orthologs for several particular isotypes of α- and ß-tubulins.

Involvement of ROS and calcium ions in developing heat resistance and inducing antioxidant system of wheat seedlings under melatonin's effects.

The stress-protective effect of melatonin (N-acetyl-5-methoxytryptamine) on plant cells is mediated by key signaling mediators, in particular calcium ions and reactive oxygen species (ROS). However, the links between changes in calcium and redox homeostasis and the formation of adaptive responses of cultivated cereals (including wheat) to the action of high temperatures have not yet been studied. In the present study, we investigated the possible involvement of ROS and calcium ions as signaling mediators in developing heat resistance in wheat (Triticum aestivum L.) seedlings and activating their antioxidant system. Treatment of 3-day-old etiolated seedlings with melatonin solutions at concentrations 0.01-10 µM increased their survival after exposure to 45 °C for 10 min. The most significant stress-protective effect was exerted by melatonin treatment at 1 µM concentration. Under the influence of melatonin, a transient enhancement of superoxide anion radical (O2•-) generation and an increase in hydrogen peroxide content were observed in roots, with a maximum at 1 h. Four hours after treatment with melatonin, the activity of catalase and guaiacol peroxidase increased in roots, while the activity of superoxide dismutase did not change significantly. After exposure to 45 °C, the activity of catalase and guaiacol peroxidase was higher in the roots of melatonin-treated wheat seedlings, and the indices of ROS generation, content of the lipid peroxidation product malonic dialdehyde, and cell membrane damage were lower than in control seedlings. Melatonin-induced changes in root ROS generation and antioxidant enzyme activities were eliminated by pretreatment with the hydrogen peroxide scavenger dimethylthiourea (DMTU), NADPH oxidase inhibitor imidazole, and calcium antagonists (the extracellular calcium chelator EGTA and phospholipase C inhibitor neomycin). Treatment with DMTU, imidazole, EGTA, and neomycin also abolished the melatonin-induced increase in survival of wheat seedlings after heat stress. The role of calcium ions and ROS, generated with the participation of NADPH oxidase, as signaling mediators in the melatonin-induced antioxidant system and heat stress resistance of wheat seedlings have been demonstrated.

Chromosome-level assembly and analysis of Camelina neglecta: a novel diploid model for Camelina biotechnology research.

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa. Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and ß) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa, we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa. Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD, FAD2, FAD3, FAE1, ABI3, WRI1 and FUS3 displaying high expression levels in C. neglecta seeds. The high representability of C. neglecta as a model species for Camelina-based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta, leading to overexpression of CvLPAT2, CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research.

Overcoming genetic paucity of Camelina sativa: possibilities for interspecific hybridization conditioned by the genus evolution pathway.

Camelina or false flax (Camelina sativa) is an emerging oilseed crop and a feedstock for biofuel production. This species is believed to originate from Western Asian and Eastern European regions, where the center of diversity of the Camelina genus is located. Cultivated Camelina species arose via a series of polyploidization events, serving as bottlenecks narrowing genetic diversity of the species. The genetic paucity of C. sativa is foreseen as the most crucial limitation for successful breeding and improvement of this crop. A potential solution to this challenge could be gene introgression from Camelina wild species or from resynthesized allohexaploid C. sativa. However, both approaches would require a complete comprehension of the evolutionary trajectories that led to the C. sativa origin. Although there are some studies discussing the origin and evolution of Camelina hexaploid species, final conclusions have not been made yet. Here, we propose the most complete integrated evolutionary model for the Camelina genus based on the most recently described findings, which enables efficient improvement of C. sativa via the interspecific hybridization with its wild relatives. We also discuss issues of interspecific and intergeneric hybridization, aimed on improving C. sativa and overcoming the genetic paucity of this crop. The proposed comprehensive evolutionary model of Camelina species indicates that a newly described species Camelina neglecta has a key role in origin of tetra- and hexaploids, all of which have two C. neglecta-based subgenomes. Understanding of species evolution within the Camelina genus provides insights into further research on C. sativa improvements via gene introgression from wild species, and a potential resynthesis of this emerging oilseed crop.

The role of nitric oxide and hydrogen sulfide in regulation of redox homeostasis at extreme temperatures in plants.

Nitric oxide and hydrogen sulfide, as important signaling molecules (gasotransmitters), are involved in many functions of plant organism, including adaptation to stress factors of various natures. As redox-active molecules, NO and H2S are involved in redox regulation of functional activity of many proteins. They are also involved in maintaining cell redox homeostasis due to their ability to interact directly and indirectly (functionally) with ROS, thiols, and other molecules. The review considers the involvement of nitric oxide and hydrogen sulfide in plant responses to low and high temperatures. Particular attention is paid to the role of gasotransmitters interaction with other signaling mediators (in particular, with Ca2+ ions and ROS) in the formation of adaptive responses to extreme temperatures. Pathways of stress-induced enhancement of NO and H2S synthesis in plants are considered. Mechanisms of the NO and H2S effect on the activity of some proteins of the signaling system, as well as on the state of antioxidant and osmoprotective systems during adaptation to stress temperatures, were analyzed. Possibilities of practical use of nitric oxide and hydrogen sulfide donors as inductors of plant adaptive responses are discussed.

Brassinosteroids application induces phosphatidic acid production and modify antioxidant enzymes activity in tobacco in calcium-dependent manner.

Brassinosteroids (BRs) are steroid hormones regulating various aspects of plant metabolism, including growth, development and stress responses. However, little is known about the mechanism of their impact on antioxidant systems and phospholipid turnover. Using tobacco plants overexpressing H+/Ca2+vacuolar Arabidopsis antiporter CAX1, we showed the role of Ca2+ ion balance in the reactive oxygen species production and rapid phosphatidic acid accumulation induced by exogenous BR. Combination of our experimental results with public transcriptomic and proteomic data revealed a particular role of Ca2+-dependent phospholipid metabolizing enzymes in BR signaling. Here we provide novel insights into the role of calcium balance and lipid-derived second messengers in plant responses to exogenous BRs and propose a complex model integrating BR-mediated metabolic changes with phospholipid turnover.

A journey through a plant cytoskeleton: Hot spots in signaling and functioning.

This survey paper contains a brief analysis of publications included in the special issue of the scientific journal Cell Biology International titled "Plant Cytoskeleton Structure, Dynamics and Functions". The manuscripts in this special issue reflect some new aspects of plant cytoskeleton organization, signaling and functioning, and results from different Ukrainian research groups, and focuses on bringing together scientists working across different instrumental scales.

RNAi-Based Biocontrol of Wheat Nematodes Using Natural Poly-Component Biostimulants.

With the growing global demands on sustainable food production, one of the biggest challenges to agriculture is associated with crop losses due to parasitic nematodes. While chemical pesticides have been quite successful in crop protection and mitigation of damage from parasites, their potential harm to humans and environment, as well as the emergence of nematode resistance, have necessitated the development of viable alternatives to chemical pesticides. One of the most promising and targeted approaches to biocontrol of parasitic nematodes in crops is that of RNA interference (RNAi). In this study we explore the possibility of using biostimulants obtained from metabolites of soil streptomycetes to protect wheat (Triticum aestivum L.) against the cereal cyst nematode Heterodera avenae by means of inducing RNAi in wheat plants. Theoretical models of uptake of organic compounds by plants, and within-plant RNAi dynamics, have provided us with useful insights regarding the choice of routes for delivery of RNAi-inducing biostimulants into plants. We then conducted in planta experiments with several streptomycete-derived biostimulants, which have demonstrated the efficiency of these biostimulants at improving plant growth and development, as well as in providing resistance against the cereal cyst nematode. Using dot blot hybridization we demonstrate that biostimulants trigger a significant increase of the production in plant cells of si/miRNA complementary with plant and nematode mRNA. Wheat germ cell-free experiments show that these si/miRNAs are indeed very effective at silencing the translation of nematode mRNA having complementary sequences, thus reducing the level of nematode infestation and improving plant resistance to nematodes. Thus, we conclude that natural biostimulants produced from metabolites of soil streptomycetes provide an effective tool for biocontrol of wheat nematode.

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.

Structural and functional features of lysine acetylation of plant and animal tubulins.

The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3).

Protein phosphatases potentially associated with regulation of microtubules, their spatial structure reconstruction and analysis.

According to the sequence and profile comparison with known catalytic domains, where identified protein phosphatases potentially involved in regulation of microtubule dynamics and structure from Arabidopsis thaliana, Nicotiana tabacum, Medicago sativa, Oryza sativa subsp. japonica, Zea mays, and Triticum aestivum. Selected proteins were related to classical non-receptor, serine/threonine-specific and dual protein phosphatases. By application of template structures of human protein phosphatases, it was performed homology modelling of the catalytic domains of 17 plant protein phosphatases. Based on the results of the structural alignment, molecular dynamics, and conservatism in positions of functionally importance, it was confirmed homology of selected plant proteins and known protein phosphatases regulating structure and dynamics of microtubules.

3D-modeling of carboxyl-terminal phosphorylation of plant αß-tubulin and its role in kinesin-8/microtubule interaction.

The results of computer modeling of plant kinesin-8/αß-tubulin complexes with such αß-tubulins' modified amino acid residues as phosphorylated Tyr262 and Tyr107 are reported in this paper. The molecular dynamics of these modified complexes in comparison with the dynamics of non-modified ones suggests that the phosphorylation of both α- and ß-tubulins reveals stabilizing effect on the protein structure around the modified residue. It was found also that the phosphorylation of Tyr107 in ß-tubulin molecule favors to more advantageous kinesin-8 binding with the phosphorylated microtubule surface in terms of energy.

MAST-like protein kinase IREH1 from Arabidopsis thaliana co-localizes with the centrosome when expressed in animal cells.

MAIN CONCLUSION: The similarity of IREH1 (Incomplete Root Hair Elongation 1) and animal MAST kinases was confirmed; IREH1cDNA was cloned while expressing in cultured animal cells co-localized with the centrosome. In mammals and fruit flies, microtubule-associated serine/threonine-protein kinases (MAST) are strongly involved in the regulation of the microtubule system. Higher plants also possess protein kinases homologous to MASTs, but their function and interaction with the cytoskeleton remain unclear. Here, we confirmed the sequence and structural similarity of MAST-related putative protein kinase IREH1 (At3g17850) and known animal MAST kinases. We report the first cloning of full-length cDNA of the IREH1 from Arabidopsis thaliana. Recombinant GFP-IREH1 protein was expressed in different cultured animal cells. It revealed co-localization with the centrosome without influencing cell morphology and microtubule arrangement. Structural N-terminal region of the IREH1 molecule co-localized with centrosome as well.

Involvement of Inositol Biosynthesis and Nitric Oxide in the Mediation of UV-B Induced Oxidative Stress.

The involvement of NO-signaling in ultraviolet B (UV-B) induced oxidative stress (OS) in plants is an open question. Inositol biosynthesis contributes to numerous cellular functions, including the regulation of plants tolerance to stress. This work reveals the involvement of inositol-3-phosphate synthase 1 (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives, in the response to NO-dependent OS in Arabidopsis. Homozygous mutants deficient for IPS1 (atips1) and wild-type plants were transformed with a reduction- grx1-rogfp2 and used for the dynamic measurement of UV-B-induced and SNP (sodium nitroprusside)-mediated oxidative stresses by confocal microscopy. atips1 mutants displayed greater tissue-specific resistance to the action of UV-B than the wild type. SNP can act both as an oxidant or repairer depending on the applied concentration, but mutant plants were more tolerant than the wild type to nitrosative effects of high concentration of SNP. Additionally, pretreatment with low concentrations of SNP (10, 100 µM) before UV-B irradiation resulted in a tissue-specific protective effect that was enhanced in atips1. We conclude that the interplay between nitric oxide and inositol signaling can be involved in the mediation of UV-B-initiated oxidative stress in the plant cell.

Extracellular Synthesis of Luminescent CdS Quantum Dots Using Plant Cell Culture.

The present study describes a novel method for preparation of water-soluble CdS quantum dots, using bright yellow-2 (BY-2) cell suspension culture. Acting as a stabilizing and capping agent, the suspension cell culture mediates the formation of CdS nanoparticles. These semiconductor nanoparticles were determined by means of an UV-visible spectrophotometer, photoluminescence, high-resolution transmission electron microscopy (HRTEM), and XRD. Followed by the electron diffraction analysis of a selected area, transmission electron microscopy indicated the formation of spherical, crystalline CdS ranging in diameter from 3 to 7 nm and showed wurtzite CdS quantum dots. In the present work, the toxic effect of synthesized CdS quantum dots on Nicotiana tabacum protoplasts as a very sensitive model was under study. The results of this research revealed that biologically synthesized CdS nanoparticles in low concentrations did not induce any toxic effects.

DMAEM-based cationic polymers as novel carriers for DNA delivery into cells.

Different transformation systems and vectors have been improved to increase the effectiveness of transformation and achieve stable expression of target genes. Because classical direct and indirect transformation processes commonly suffer from instability of a gene in the environment, gene deletion, transgene silencing, and poor gene transfer efficiency. Nowadays, gene transformation technologies are based on the use of new carriers (nanoparticles, carbon nanotubes, whiskers, and polymers) characterized by better efficiency and reproducibility for the direct DNA delivery into cells. In this review, we have focused on the novel DMAEM-based direct DNA delivery system and its possible applications for cell transformation.

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.

Biosynthesis of luminescent CdS quantum dots using plant hairy root culture.

CdS nanoparticles have a great potential for application in chemical research, bioscience and medicine. The aim of this study was to develop an efficient and environmentally-friendly method of plant-based biosynthesis of CdS quantum dots using hairy root culture of Linaria maroccana L. By incubating Linaria root extract with inorganic cadmium sulfate and sodium sulfide we synthesized stable luminescent CdS nanocrystals with absorption peaks for UV-visible spectrometry at 362 nm, 398 nm and 464 nm, and luminescent peaks at 425, 462, 500 nm. Transmission electron microscopy of produced quantum dots revealed their spherical shape with a size predominantly from 5 to 7 nm. Electron diffraction pattern confirmed the wurtzite crystalline structure of synthesized cadmium sulfide quantum dots. These results describe the first successful attempt of quantum dots synthesis using plant extract. PACS: 81.07.Ta; 81.16.-c; 81.16.Rf.

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.