Practical guidelines for the characterization of tobacco BY-2 cell lines

Srba, Miroslav; Černíková, Alena; Opatrný, Z.; Fischer, Lukáš

doi:10.1007/s10535-015-0573-3

Cited by 13 publications

(9 citation statements)

References 42 publications

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“…The goal of our study was to describe the precise timing and progression of the transcriptional gene silencing (TGS) in its early stages. For this purpose, we used the BY-2 cell line [48] as a model, which allowed us to monitor the process in a highly synchronised and homogeneous culture, which can be studied at a single-cell level [49]. A selected BY-2 cell line stably expressing GFP , driven by CaMV 35S promoter ( P35S ) [50], was super-transformed with an estradiol-inducible silencing construct composed of an inverted repeat prepared from a part of P35S ( IR - P35S ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Given that the cultures were exponentially growing, the observed decrease in GFP fluorescence had to result from both GFP degradation and GFP “dilution” in dividing cells. Comparing the speed of the GFP fluorescence decrease and the rate of BY-2 cell divisions (doubling time is about 20 h) [49] clearly indicated that the GFP protein was highly stable in BY-2 cells with a half-life of several days, which caused the observed delay in GFP fluorescence decline.…”

Section: Resultsmentioning

confidence: 99%

“…It clearly indicates that not only reaching a certain level of siRNAs, but also a certain duration of the exposure to siRNAs was necessary to establish dense methylation of the target region. On the other hand, once established, the dense methylation had to be very quickly introduced to newly synthetized DNA strands after replication, because we observed this dense methylation in the cells that were continually dividing approximately every 20 h [49]. Such faster methylation compared to de novo methylation of a naive locus was likely connected with the presence of the maternal highly methylated DNA strand, since Pol V is effectively recruited to loci with methylated cytosines [40].…”

Section: Discussionmentioning

confidence: 99%

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Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

Přibylová

Čermák

Tyč

et al. 2019

Epigenetics & Chromatin

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Background Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. Results We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. Conclusions Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

Přibylová

Čermák

Tyč

et al. 2019

Epigenetics & Chromatin

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“…Few years ago, we started to test an alternative model, tobacco BY-2 cell line that has been successfully used in numerous studies focused on cellular processes [ 15 ]. The BY-2 cell culture is composed of relatively homogeneous mitotically proliferating cells [ 15 , 16 ]. The absence of the gametophytic phase also prevents some types of epigenetic changes connected with this developmental stage [ 17 – 20 ].…”

Section: Introductionmentioning

confidence: 99%

“…The absence of the gametophytic phase also prevents some types of epigenetic changes connected with this developmental stage [ 17 – 20 ]. The BY-2 cell line also allows simple analyses at the level of individual cells and assessment of a large number of independent transgenic lines (in the callus form) that can be easily generated, managed and analyzed [ 16 ]. Since the behavior of individual transgenic lines of any model is affected by the T-DNA copy number and the chromosomal environment of the insertion [ 21 – 23 ], analysis of a high number of independent transformed lines, which provides a more generalized picture, is recommended.…”

Section: Introductionmentioning

confidence: 99%

Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing

Čermák

Fischer

2018

BMC Plant Biol

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BackgroundPlant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines.ResultsHere we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana.ConclusionsOur results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1482-3) contains supplementary material, which is available to authorized users.

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Analysis of Redox Relationships in the Plant Cell Cycle: Determination of Ascorbate, Glutathione, and Poly(ADPribose)polymerase (PARP) in Plant Cell Cultures

Foyer¹,

Pellny²,

Locato³

et al. 2019

Methods in Molecular Biology

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Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signaling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced reactive oxygen species accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly (ADP-ribose) polymerase during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

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Practical guidelines for the characterization of tobacco BY-2 cell lines

Cited by 13 publications

References 42 publications

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing

Analysis of Redox Relationships in the Plant Cell Cycle: Determination of Ascorbate, Glutathione, and Poly(ADPribose)polymerase (PARP) in Plant Cell Cultures

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