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Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation.

Wojtasik W, Kulma A, Boba A, Szopa J - BMC Plant Biol. (2014)

Bottom Line: However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome.In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.To our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nowadays, the challenge for biotechnology is to develop tools for agriculture and industry to provide plants characterized by productivity and quality that will satisfy the growing demand for different kinds of natural products. To meet the challenge, the generation and application of genetically modified plants is justified. However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome. Four-year experiments led to the development of a technology inducing heritable epigenetic gene activation without transgenesis.

Results: The method comprises the induction of changes in methylation/demethylation of the endogenous gene by the plant's treatment with short oligodeoxynucleotides antisense to the coding region. In vitro cultured plants and F3 generation flax plants overproducing the β-1,3-glucanase gene (EMO-βGlu flax) were characterized by up-regulation of β-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the β-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.

Conclusion: To our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene. Apart from the evident effectiveness, the most important issue is that the EMO method allows generation of favourably altered plants, whose cultivation makes the plant producer independent from the complicated procedure of obtaining an agreement on GMO release into the environment and whose products might be more easily introduced to the global market.

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Expression level of genes involved in the OLIGO mechanism. The mRNA level of CMT1 (chromomethylase 1), CMT3 (chromomethylase 3), DME (Demeter), ROS1 (repressor of silencing 1), AGO1 (Argonaute 1), AGO4 (Argonaute 4), RDR2 (RNA-depended RNA polymerase 2) in flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h (light grey bars) and 48 h (dark grey bars) after exposure to OLIGOs in comparison with control, non-treated flax and in EMO-βGlu flax (EB) (black bars) in comparison with control flax was obtained from the real-time RT-PCR analysis. Actin was used as a reference gene and the transcript levels were normalized to those of the control plant (C = 1; not presented in the figure). Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P < 0.05, **P < 0.01). RQ – relative quantity.
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Fig4: Expression level of genes involved in the OLIGO mechanism. The mRNA level of CMT1 (chromomethylase 1), CMT3 (chromomethylase 3), DME (Demeter), ROS1 (repressor of silencing 1), AGO1 (Argonaute 1), AGO4 (Argonaute 4), RDR2 (RNA-depended RNA polymerase 2) in flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h (light grey bars) and 48 h (dark grey bars) after exposure to OLIGOs in comparison with control, non-treated flax and in EMO-βGlu flax (EB) (black bars) in comparison with control flax was obtained from the real-time RT-PCR analysis. Actin was used as a reference gene and the transcript levels were normalized to those of the control plant (C = 1; not presented in the figure). Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P < 0.05, **P < 0.01). RQ – relative quantity.

Mentions: To explain the possible mechanism of OLIGO action, the expression levels of genes involved in the RNAi and RNAa mechanism – RNA-dependent RNA polymerase, AGO1 and AGO4 – were investigated. Assuming that the changes could also refer to the methylase/demethylase genes, expression levels of genes encoding methylases (CMT1, CMT3) and demethylases (ROS1, DME) were measured (Figure 4).Figure 4


Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation.

Wojtasik W, Kulma A, Boba A, Szopa J - BMC Plant Biol. (2014)

Expression level of genes involved in the OLIGO mechanism. The mRNA level of CMT1 (chromomethylase 1), CMT3 (chromomethylase 3), DME (Demeter), ROS1 (repressor of silencing 1), AGO1 (Argonaute 1), AGO4 (Argonaute 4), RDR2 (RNA-depended RNA polymerase 2) in flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h (light grey bars) and 48 h (dark grey bars) after exposure to OLIGOs in comparison with control, non-treated flax and in EMO-βGlu flax (EB) (black bars) in comparison with control flax was obtained from the real-time RT-PCR analysis. Actin was used as a reference gene and the transcript levels were normalized to those of the control plant (C = 1; not presented in the figure). Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P < 0.05, **P < 0.01). RQ – relative quantity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4209061&req=5

Fig4: Expression level of genes involved in the OLIGO mechanism. The mRNA level of CMT1 (chromomethylase 1), CMT3 (chromomethylase 3), DME (Demeter), ROS1 (repressor of silencing 1), AGO1 (Argonaute 1), AGO4 (Argonaute 4), RDR2 (RNA-depended RNA polymerase 2) in flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h (light grey bars) and 48 h (dark grey bars) after exposure to OLIGOs in comparison with control, non-treated flax and in EMO-βGlu flax (EB) (black bars) in comparison with control flax was obtained from the real-time RT-PCR analysis. Actin was used as a reference gene and the transcript levels were normalized to those of the control plant (C = 1; not presented in the figure). Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P < 0.05, **P < 0.01). RQ – relative quantity.
Mentions: To explain the possible mechanism of OLIGO action, the expression levels of genes involved in the RNAi and RNAa mechanism – RNA-dependent RNA polymerase, AGO1 and AGO4 – were investigated. Assuming that the changes could also refer to the methylase/demethylase genes, expression levels of genes encoding methylases (CMT1, CMT3) and demethylases (ROS1, DME) were measured (Figure 4).Figure 4

Bottom Line: However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome.In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.To our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Nowadays, the challenge for biotechnology is to develop tools for agriculture and industry to provide plants characterized by productivity and quality that will satisfy the growing demand for different kinds of natural products. To meet the challenge, the generation and application of genetically modified plants is justified. However, the strong social resistance to genetically modified organisms and restrictive regulations in European Union countries necessitated the development of a new technology for new plant types generation which uses the knowledge resulting from analysis of genetically modified plants to generate favourably altered plants while omitting the introduction of heterologous genes to their genome. Four-year experiments led to the development of a technology inducing heritable epigenetic gene activation without transgenesis.

Results: The method comprises the induction of changes in methylation/demethylation of the endogenous gene by the plant's treatment with short oligodeoxynucleotides antisense to the coding region. In vitro cultured plants and F3 generation flax plants overproducing the β-1,3-glucanase gene (EMO-βGlu flax) were characterized by up-regulation of β-glucanase and chitinase genes, decreases in the methylation of CCGG sequences in the β-glucanase gene and in total DNA methylation and, more importantly, reasonable resistance against Fusarium infection. In addition, EMO-βGlu flax obtained by this technology showed similar features as those obtained by genetic engineering.

Conclusion: To our best knowledge, this is the first report on plant gene activation by treatment with oligodeoxynucleotides homologous to the coding region of the gene. Apart from the evident effectiveness, the most important issue is that the EMO method allows generation of favourably altered plants, whose cultivation makes the plant producer independent from the complicated procedure of obtaining an agreement on GMO release into the environment and whose products might be more easily introduced to the global market.

Show MeSH
Related in: MedlinePlus