On the mechanism of the cell cycle control of suspension-cultured tobacco cells after exposure to static magnetic field

doi:10.1016/j.plantsci.2018.08.004

Plant Science

Volume 277, December 2018, Pages 139-144

https://doi.org/10.1016/j.plantsci.2018.08.004 Get rights and content

Highlights

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H₂O₂ and NO contents of tobacco cells increased by SMF.
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SMF increased the expression Rbp and p21, as tumor suppressor genes.
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SMF delayed the CDKA and CycD gene expression.
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G1/S transition was delayed in SMF treated tobacco cells.

Abstract

One of the main sites of the magnetic fields influence on living cells is the cell cycle. The intensity of this influence however, varies depending on the cell type and the duration of the treatment. Suspension of cultured tobacco cells (Nicotiana tabacum cv. Barley 21) were synchronized via sucrose starvation at their stationary growth phase. The cells were then exposed to 0.2 m T SMF up to 24 h. The progression of different cell cycle phases was monitored through flow cytometry in a time course manner. Expression of cell cycle controlling genes and amounts of certain signaling molecules were measured as well. Exposure to SMF delayed G1.S transition which was accompanied by decrease of cyclin-dependent kinases A (CDK A) and D-type cyclin, but an increase in the adenylyl cyclase (AC), transcription factor E2F, retinoblastoma protein (Rbp), and CDK-inhibitor protein 21 (p21) transcript accumulation. Exposure to SMF also increased the contents of nitric oxide (NO), hydrogen peroxide (H₂O₂), and salicylic acid (SA), compared to the control group. The results suggest a signaling pathway triggered by SMF starting from accumulation of NO and H₂O₂ followed by downstream events including the increase of cyclic nucleotides and subsequent decrease of both CDKA and CycD.

Introduction

Static magnetic fields (SMF) are inevitable factors in the plants environment and their effects are constantly increasing due to the widespread use of industrial and medical devices. There is a considerable literature denoting physiological effects of SMF on plant growth and development, photosynthesis, seed germination, nutrients uptake, enzyme activity, and redox state [1], although the exact underlying mechanisms are still a matter of debate. Increase in the activity, concentration, and lifetime of free radicals, alteration in internal calcium concentration (Ca²⁺ signaling), and alteration in the activity and contents of antioxidant enzymes, and alteration of tertiary structure of certain iron-containing antioxidant proteins have been recently introduced as possible mechanisms of SMF effects on plants [2,3]. Effects of SMF on the plants varies depending on the species, field strength, treatment duration, etc. Exposure of Arabidopsis thaliana magnetic field around 65 μT for 2 days altered the internal calcium ion concentration, compared to the controls [4]. The effects of SMF on the cell growth and death of suspension-cultured tobacco cells were investigated after exposure to 10 and 30 m T SMF for 5 days, 5 h each [5]. It was observed that both fields, particularly 30 m T, adversely affected the growth and promoted cell death which was accompanied by a harmonized increase in the activity of peroxidase and increase of lignification of cell walls. Overproduction of reactive oxygen species, increase of catalase gene expression and activity and altered mitogen activated protein kinase (MAPK) gene expression were observed in Vicia faba seedlings after exposure to a SMF of 30 m T for 8 days, 8 h each. The authors suggested that adverse effects of SMF, it at least in part, is raised from oxidative express mediated by a MAPK cascade [6].

One of the main sites of the magnetic fields influence is the cell cycle progression and proliferation [7,8]. Cell cycle machinery is regulated through various cyclin-dependent kinases (CDK). They phosphorylate and block a tumor suppressor, retinoblastoma protein (Rbp) which binds cellular transcription factor E2F and inhibits the expression of genes whose products are necessary for G1.S transition [9]. The CDK proteins by themselves are blocked by CDK inhibitors (CKI) e.g., p21, p27 and p16, in response to signals from the unfavorable conditions or from damaged DNA [10]. The G1phase is particularly important in the cell cycle because it determines whether a cell commits to division or to leave the cell cycle.

Extensive researches have been carried out on the effects of SMF on cell cycle machinery of human cells. For instance, it has been reported that the level of p21 increased at 6 h after exposure of human amniotic fluid cells to the MF of 1 m T for 30 h, whereas the expression of cyclin D1 was enhanced after 6 h and 18 h [11]. A temporary G2 arrest was observed in human p53 mutant Jurkat cells after 6 m T SMF exposure for 36 h [12].

Few studies of this kind however have been conducted on plant cells and, as mentioned above, the available literature has mainly concentrated on the growth retarding effects of magnetic fields on the plant cells and activation of oxidative stress. To the best of our knowledge, molecular mechanisms of plant cell cycle progression after exposure to SMF have not been comprehensively investigated. Underlying molecular mechanisms of SMF effects on the cell cycle machinery of plant cells were monitored in synchronized suspension-cultured tobacco cells, which provide us with the most reliable and productive system resembling stem cells.

Section snippets

Plant cell culture and synchronization

Suspension cultures of a rapidly growing tobacco (Nicotiana tabacum L. cv. Burley 21) cell line were used in this study. The cells were grown in a modified LS medium composed of 3% sucrose, (NH₄)NO₃, 20.61 mM; KH₂PO₄, 1.25 mM; CaCl₂, 2.99 mM; MgSO₄, 1.50 mM; MnSO₄, 0.1 mM; Fe-EDTA, 0.1 mM; H₃BO₃, 0.1 mM; CoCl₂, 0.11 mM; CuSO₄, 0.1 mM; Na₂MoO₄, 1.03 mM; ZnSO₄, 29.91 mM, and KI, 5 mM. All chemicals were purchased from Merck (Germany) and used as received without any further purification. The pH

Growth rate and Cell cycle progression

The cell cycle progression was monitored by flow cytometry of purified nuclei of both control and the SMF-exposed cells at appropriate intervals (Supplementary data 4). Sucrose depletion at day 11 efficiently synchronized them so that 74% of the cells were arrested in G1 (Table 2). Exposure of synchronized tobacco cells to SMF remarkably reduced the cell growth and their dry weight were 20% lower than the controls at 24 h (Fig. 1).

Monitoring of cell cycle progression showed that most of the

Discussion

Adverse effects of SMF on the viability and growth of plant cells have been previously shown in maize, sunflower, sweet basil, and suspension-cultured tobacco cells [20,21]. Exposure of tobacco cells to 0.2 m T SMF in the present study significantly decreased the dry weight of the cells. Similar to typical response of plant cells to other abiotic stresses [14], a delay of G1. S transition occurred after treatment of the tobacco cells with SMF (10 h vs. 6 h for the control cells).

There are many

Acknowledgments

The authors wouldlike to thank Tarbiat Modares University for providing the laboratory facilities and financial support for this project. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Research ArticleOn the mechanism of the cell cycle control of suspension-cultured tobacco cells after exposure to static magnetic field

Highlights

Abstract

Introduction

Section snippets

Plant cell culture and synchronization

Growth rate and Cell cycle progression

Discussion

Acknowledgments

J. Plant Physiol.

J. Plant Physiol.

Adv. Space Res.

Micron

Mol. Cells

Food Chem.

Anal. Biochem.

Mol. Plant

Magnetic field effects on plant growth, development, and evolution

Front. Plant Sci.

Antioxidant capacity of parsley cells (Petroselinum crispum L.) in relation to iron-induced ferritin levels and static magnetic field

Electromagn. Biol. Med.