A. V. Nosov

Proline and functioning of the antioxidant system in Thellungiella salsuginea plants and cultured cells subjected to oxidative stress

The effects of proline on the functioning of antioxidant enzymes — superoxide dismutase (SOD) and ascorbate peroxidase (APO) — in Thellungiella salsuginea plants and cultured cells under normal conditions of culturing and under the influence of hydrogen peroxide (500 μM) were studied. Proline addition (0.2, 2, or 5 mM) to the medium for suspension culture or nutrient medium for plant growing resulted in the increase in the content of intracellular proline in both cultured cells and intact plant leaves and also in the activation of proline dehydrogenase, i.e., the enzyme degrading proline. Under normal conditions, treatment with proline exerted prooxidant action on both cellular and organismal levels. This was manifested in MDA accumulation and changes in APO and SOD activities. The amino acid alanine, used as a control, did not exert similar strong effect as proline. Application of 500 μM H2O2 on plant leaves resulted in the development of oxidative stress, whereas hydrogen peroxide addition into the culture medium — to the death of 50% of suspension cells. When plants and cultured cells were treated with 2 mM proline and than with H2O2, the number of dead cells in suspension was 35%, the content of MDA was decreased, APO was activated, and SOD activity was decreased in both cell culture and plant leaves. Thus, an increase in the intracellular proline concentration changed the redox balance and induced functioning of APO and SOD at both normal conditions of plant growing and cell culturing and under stress.

Variation of nuclear DNA content during somatic embryogenesis and plant regeneration of Coffea arabica L. using cytophotometry

Abstract Cytophotometric analysis of nuclear DNA was carried out in leaves of Coffea arabica L. plants grown in vitro. They were maintained for more than 1 year on MS media containing 0.53 μM NAA, and 2.32 μM kinetin, and embryogenic calli and somatic embryos were derived from them. Four suspension cultures of C. arabica differing in their embryogenic potential were also studied. In in vitro leaves used as primary explants many nuclei gave values that were hypoaneuploid, yet the somatic embryos derived from them consisted predominantly of diploid cells. As primary explants were shifted to conditioning medium (MS medium added with 0.53 μM NAA and 2.32 μM kinetin) and then to induction medium (to obtain embryogenic calli; Yasuda medium supplemented with 5 μM BAP), the frequency of hypoaneuploid values dropped. An analysis of four suspension cultures did not reveal any relationship between the cytogenetic state of cell strains and their morphogenetic potential. Of four suspensions, cultures having similar frequencies of diploid cells (60–82%), only one was capable of embryogenesis.

At the beginning of the route: ABA perception and signal transduction in plants

Nowadays, fundamentally important data toward the molecular mechanisms of phytohormone action, starting from hormonal signal perception and up to changes in hormone-regulated gene expression, became available. Signaling mechanisms for plant cell responses to ABA remained the least known. Although a substantial progress was achieved in our understanding of the role of protein kinases and protein phosphatases functioning downstream receptors, identification of ABA receptors has induced very hot debates. This review summarizes information obtained during the last decade and concerned the molecular mechanisms of perception and signal transduction of ABA signal in plants.

Can salicylic acid affect the intercellular transport of the tobacco mosaic virus by changing plasmodesmal permeability

We studied the effects of salicylic acid (SA) on the plasmodesmal permeability as evaluated by the tobacco mosaic virus (TMV) spreading in tobacco Nicotiana glutinosaleaves, where TMV induces necrotic lesions. When leaves were treated with SA simultaneously with their viral inoculation, SA retarded the development of necrotic lesions and reduced their number. When inoculated leaves were kept on the SA solution at an elevated temperature (31°C) for a short period of time, the size of the necrotic lesions, which developed after leaf transfer to room temperature, was decreased. SA stimulated the formation of “rapid” callose involved in the control of the plasmodesmal permeability, which was assessed from fluorescence after tissue staining with Aniline Blue. On the basis of these data, we suggest that SA suppressed TMV spreading in the inoculated tobacco leaves by reducing the plasmodesmal permeability.

Regulatory role of nitric oxide in plants

Research performed over the last few years identified nitric oxide (NO) as an intracellular signaling molecule involved in regulation of plant physiological processes at all stages of the life cycle. Nevertheless, some extremely important aspects of NO biology are still far from being clarified. There exist different points of view on NO formation and utilization in plants. The mechanisms of perception and transduction of the NO signal are not yet fully understood, and the origin of specificity underlying coordinated activation of responses to NO remains unresolved. It is reasonable to expect that the deep knowledge of NO functioning in animals may provide some keys to these questions. Such a comparative analysis is a way to reveal similarities and emphasize the differences in the current understanding of the NO role in plants. The present lecture highlights these aspects of NO functioning.

Opposite Effects of Synthetic Auxins, 2,4-Dichlorophenoxyacetic Acid and 1-Naphthalene Acetic Acid on Growth of True Ginseng Cell Culture and Synthesis of Ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 104 μm3 were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 104 μm3, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.

Ethylene in the proliferation of cultured plant cells: Regulating or just going along?

Ethylene, being one of five classical plant phytohormones is involved in regulation of numerous physiological processes. There are contradictory data about the effect of ethylene on the cell growth and division; although it is accepted that in culture flasks, the content of ethylene rises to a few tens of μL/L and production of ethylene is associated with the periods of active growth of the cells in vitro. We revealed a strong correlation (r = 0.96) between ethylene production and specific rate of dry weight accumulation in suspension cell cultures of Ajuga turkestanica, heterotrophic and mixotrophic strains of Arabidopsis thaliana, Beta vulgaris, Euonymus maximoviczianus, Medicago sativa, Panax ginseng, and Triticum timopheevii. In heterotrophic cell culture of A. thaliana, the peaks and general shape of the curves describing dynamics of ethylene production, the number of S-phase cells, and specific rate of increase in cell number coincided in log phase and in the phase of growth deceleration. Pretreatment of subculture inoculum with 100 μL/L ethylene caused doubling of S-phase cell number after 3-h-long culturing in fresh nutrient medium. It was found that exogenous ethylene affects the number of S-phase cells only when the level of endogenously produced ethylene is low.

Plant cell proliferation and its regulators

Plant growth, where one of the key processes is cell division, is controlled by phytohormones. In this mini-review, an analysis of the literature on the molecular mechanisms controlling plant cell proliferation by phytohormones is presented.

Phytohormone interplay controls proliferation of in vitro cultivated cells of Arabidopsis thaliana ethylene-insensitive mutants.

46 Identification of molecular mechanisms of the growth and development regulation by phytohor mones is one of the priority problems of modern plant experimental biology. Data accumulated to date indi cate that a specific hormone binding to receptors changes the functional activity of proteins which are involved in hormone signal transduction pathways. The same proteins may be involved in signal transduc tion of several hormones, thus providing conditions for their interaction. A plant growth with a cell divi sion as one of the crucial processes is regulated by sev eral hormones; consequently, it can be assumed that this regulation is a result of interaction of the hormone signal transduction pathways. The effects of auxins and cytokinins on cell proliferation have been studied in details [1], while the significance of ethylene and abscisic acid (ABA) is still a matter of debate, and there is no data on the interaction of signaling path ways of these hormones with regard to cell division. Our work on this issue resulted in understanding the necessity to establish a model system that would differ from the intact plant where dividing cells are located only in certain structures, and their proliferation is controlled from different centers making a “noise” during the study of the role of exogenous phytohor mones. Cultivated plant cells, as populations of cells on which measured effect of a substances could be made, might be a more acceptable model.

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combination with click reaction adapted for plant cells appeared, and they are help for a monitoring S-period of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.