Svetlana S. Makarova

Biosynthesis of stable iron oxide nanoparticles in aqueous extracts of Hordeum vulgare and Rumex acetosa plants.

We report the synthesis and characterization of amorphous iron oxide nanoparticles from iron salts in aqueous extracts of monocotyledonous (Hordeum vulgare) and dicotyledonous (Rumex acetosa) plants. The nanoparticles were characterized by TEM, absorbance spectroscopy, SAED, EELS, XPS, and DLS methods and were shown to contain mainly iron oxide and iron oxohydroxide. H. vulgare extracts produced amorphous iron oxide nanoparticles with diameters of up to 30 nm. These iron nanoparticles are intrinsically unstable and prone to aggregation; however, we rendered them stable in the long term by addition of 40 mM citrate buffer pH 3.0. In contrast, amorphous iron oxide nanoparticles (diameters of 10-40 nm) produced using R. acetosa extracts are highly stable. The total protein content and antioxidant capacity are similar for both extracts, but pH values differ (H. vulgare pH 5.8 vs R. acetosa pH 3.7). We suggest that the presence of organic acids (such oxalic or citric acids) plays an important role in the stabilization of iron nanoparticles, and that plants containing such constituents may be more efficacious for the green synthesis of iron nanoparticles.

Exogenous proline modifies differential expression of superoxide dismutase genes in UV-B-irradiated Salvia officinalis plants

Grown in water culture 6-week-old Salvia officinalis plants with 4–5 true leaves were exposed to irradiation with UV-B (10 min, 12.3 kJ/m2), subjected to 5 mM exogenous proline in the nutrient solution, and treated with a combination of both factors. The plants responded to short UV-B irradiation by the appearance of oxidative stress, which was manifested in elevated content of malondialdehyde in leaves. Exogenous proline added 24 h before the irradiation inhibited lipid peroxidation. The total activity of superoxide dismutase (SOD) was analyzed in plant leaves, and three SOD isoforms—Mn-SOD, Fe-SOD, and Cu/Zn-SOD—were identified. Activities of these isoforms were measured over time, and the expression of their respective genes was analyzed by reverse transcription polymerase chain reaction (RT-PCR). It is shown that the addition of proline, UV-B irradiation, or combination of both treatments regulated in a differential manner the activities of SOD isoforms localized in various cell compartments. The activity of the cytosolic Cu/Zn-SOD isoform was limited by the presence of its mRNA, the content of which was regulated by mRNA synthesis or decay rate. By contrast, the activity of plastidic Fe-SOD isoform was regulated on the substrate (allosteric) level, not on the level of FSD gene expression. The activity of mitochondrial Mn-SOD isoform was insensitive to UV-B irradiation, addition of proline, or combination of both treatments, even though the level of MSD gene transcripts increased significantly after UV-B irradiation. The results indicate that MSD gene transcripts induced by UV-B were not completely processed to produce mature mRNA or mature mRNA was not capable of translation. It cannot be excluded that the synthesized macromolecule, the Mn-SOD precursor did not undergo posttranslational maturation to produce biologically active enzyme molecules. It appears that proline is involved in the differentially regulated complex expression of various SOD isoforms. This regulation is largely based on various extents of oxidative stress in different cell compartments.

Possible role of the Nt-4/1 protein in macromolecular transport in vascular tissue.

The Arabidopsis thaliana 4/1 (At-4/1) protein has a highly α-helical structure with potential to interact both with itself and other protein ligands, including the movement proteins of some plant viruses; the Nicotiana tabacum ortholog (Nt-4/1) has similar structure. Here we describe localization of GUS expression in transgenic N. tabacum seedlings under control of the Nt-4/1 promoter, which indicates that transcription is associated with the veins at certain developmental stages, and especially in the hypocotyl. Viroid accumulation and movement was altered in plants in which 4/1 expression was reduced by virus-induced gene silencing. These localization studies support a role of 4/1 in signaling in the vasculature, including mobility of pathogen-related and cellular RNAs.

Subcellular localization and self-interaction of plant-specific Nt-4/1 protein

The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.

Coilin, the signature protein of Cajal bodies, differentially modulates the interactions of plants with viruses in widely different taxa

Cajal bodies (CBs) are distinct nuclear bodies physically and functionally associated with the nucleolus. In addition to their traditional function in coordinating maturation of certain nuclear RNAs, CBs participate in cell cycle regulation, development, and regulation of stress responses. A key “signature” component of CBs is coilin, the scaffolding protein essential for CB formation and function. Using an RNA silencing (loss-of-function) approach, we describe here new phenomena whereby coilin also affects, directly or indirectly, a variety of interactions between host plants and viruses that have RNA or DNA genomes. Moreover, the effects of coilin on these interactions are manifested differently: coilin contributes to plant defense against tobacco rattle virus (tobravirus), tomato black ring virus (nepovirus), barley stripe mosaic virus (hordeivirus), and tomato golden mosaic virus (begomovirus). In contrast, with potato virus Y (potyvirus) and turnip vein clearing virus (tobamovirus), coilin serves to increase virus pathogenicity. These findings show that interactions with coilin (or CBs) may involve diverse mechanisms with different viruses and that these mechanisms act at different phases of virus infection. Thus, coilin (CBs) has novel, unexpected natural functions that may be recruited or subverted by plant viruses for their own needs or, in contrast, are involved in plant defense mechanisms that suppress host susceptibility to the viruses.

Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling

Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled–coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein–protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.

RNA-binding properties of the plant protein Nt-4/1.

The tobacco α-helical protein Nt-4/1 with unknown function forms ribonucleoprotein (RNP) complexes in vitro. Results obtained by retardation of RNP complexes in agarose gel were confirmed by Western-Northern hybridization. Several deletion and point mutants of Nt-4/1 were constructed, and the RNA-binding site was mapped in a positively charged region of the C-terminal domain of the protein. The results of this study and those described earlier support our hypothesis of the participation of Nt-4/1 protein in spreading RNA-containing pathogens in the plant.

In vitro properties of hordeivirus TGB1 protein forming ribonucleoprotein complexes.

Hordeivirus movement protein encoded by the first gene of the triple gene block (TGB1 protein, TGBp1) interacts in vivo with viral genomic and subgenomic RNAs to form ribonucleoprotein (RNP) particles that are considered to be a form of viral genome (non-virion transport form) capable of cell-to-cell and long-distance transport in infected plants. The structures of these RNPs have not been elucidated. The poa semilatent virus (PSLV) TGBp1 contains a structured C-terminal NTPase/helicase domain and an N-terminal extension region consisting of two domains - a completely intrinsically disordered extreme N-terminal domain and an internal domain (ID) with structure resembling a partially disordered molten globule. Here, we characterized the structures assembled in vitro by the full-length PSLV TGBp1 alone or in the presence of viral RNA. The PSLV TGBp1 was capable of multimerization and self-assembly into extended high-molecular-mass complexes. These complexes disassembled to apparent monomers upon incubation with ATP. Upon incubation with viral RNA, the PSLV TGBp1 in vitro formed RNP structures that appeared as filamentous particles resembling virions of helical filamentous plant viruses in morphology and dimensions. By comparing the biophysical characteristics of PSLV TGBp1 and its domains in the presence and absence of RNA, we show that the ID plays the main structural role in the self-interactions and RNA interactions of TGBp1 leading to the assembly of virus-like RNP particles.

The Multiple Functions of the Nucleolus in Plant Development, Disease and Stress Responses

The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribose)polymerase (PARP), which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses.

Virus resistance in potato: Current state and prospects

The potato (Solanum tuberosum), one of the world’s most important food crops, is infected by numerous viruses, nine of which are of major economic importance as they cause significant yield losses and reduce the quality of the crop. To mitigate the consequences of viral infections, countries with highly developed agriculture are improving and promoting sanitary measures, which include constantly monitoring viruses and certification of potato seed material based on virus diagnostics and improved potato cultivars. However, the development of virus-resistant potato cultivars seems to be a preferable choice in the long term. By employing conventional breeding techniques and genetic engineering approaches using natural virus resistance genes, normally obtained from the wild growing Solanum species, or virus-specific sequences, a number of potato cultivars/lines, which are resistant to the majority of known potato viruses, have already been obtained. However, the above-mentioned techniques have certain limitations, which are determined, in particular, by the high virus specificity of the acquired resistance (against certain viruses only), its short duration, and the ability of the virus to overcome it, as well as the regularly declared bans on the use of genetically modified plants. In the modern era, the novel technologies allowing us to edit genomes for the purposes of gene design have created the possibility to engineer a new generation of resistance genes. The most promising approaches are thought to be the directed mutagenesis of resistance genes, which allows broadening the spectrum of the effects of the gene and the use of nonhost resistance (NHR) (nonspecific resistance) genes, allowing engineering plants resistant to unrelated viruses and in certain cases, to other pathogens and even abiotic stresses. The identification of genes involved in the mechanisms of NHR has only just begun. The nucleus is now being considered as a new source of still unknown factors involved in diverse signal pathways controlling the plant’s defensive response against virus infection. The review describes the approaches which are used to generate virus-resistant potato plants and the challenges faced in this path.