Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner

Abstract

Chalcone synthase (CHS) has been recognized as an essential enzyme in the phenylpropanoid biosynthesis pathway. Apart from the leading role in the production of phenolic compounds with many valuable biological activities beneficial to biomedicine, CHS is well appreciated in science. Genetic engineering greatly facilitates expanding knowledge on the function and genetics of CHS in plants. The CHS gene is one of the most intensively studied genes in flax. In our study, we investigated engineering of the CHS gene through genetic and epigenetic approaches. Considering the numerous restrictions concerning the application of genetically modified (GM) crops, the main purpose of this research was optimization of the plant's modulation via epigenetics. In our study, plants modified through two methods were compared: a widely popular agrotransformation and a relatively recent oligodeoxynucleotide (ODN) strategy. It was recently highlighted that the ODN technique can be a rapid and time-serving antecedent in quick analysis of gene function before taking vector-mediated transformation. In order to understand the molecular background of epigenetic variation in more detail and evaluate the use of ODNs as a tool for predictable and stable gene engineering, we concentrated on the integration of gene expression and gene-body methylation. The treatment of flax with a series of short oligonucleotides homologous to a different part of CHS gene isoforms revealed that those directed to regulatory gene regions (5′- and 3′-UTR) activated gene expression, directed to non-coding region (introns) caused gen activity reduction, while those homologous to a coding region may have a variable influence on its activity. Gene expression changes were accompanied by changes in its methylation status. However, only certain (CCGG) motifs along the gene sequence were affected. The analyzed DNA motifs of the CHS flax gene are more accessible for methylation when located within a CpG island. The methylation motifs also led to rearrangement of the nucleosome location. The obtained results suggest high specificity of ODN action and establish a potential valuable alternative for improvement of crops.

3-Hydroxybutyrate Is Active Compound in Flax that Upregulates Genes Involved in DNA Methylation

“…The assumption is supported by the finding that DNA methylation regulates the structure of chromatin, leading to modulation of gene expression via changes of nucleosome positioning [42]. In addition, our own observation that the nucleosome of the chalcone synthase (CHS) gene methylated at variable motifs in the CpG island revealed higher energy and shift towards the 3'-end of the DNA also supports this suggestion [43].…”

“…Notably, in transgenic flax (line H1 and H5) in particular chalcone synthase expression was significantly increased. We have shown recently that expression of this gene is regulated by methylation [43]. Of other phenylpropanoid pathways, the genes involved in the lignin synthesis were highly activated.…”

Nucleotide‐Selective Templated Self‐Assembly of Nanoreactors under Dissipative Conditions

“…Akey characteristic of biological systems is that reaction pathways are transiently up-or downregulated caused by the temporary availability of atrigger. [52,53] We therefore studied nucleotide-selective transient reaction activation under dissipative conditions by following formation of product NBD-SC 8 .UPLC studies revealed that in the absence of the enzyme AP the product concentration increased gradually in time to reach am aximum value after around 70 minutes for both systems (A•GMP and B•TMP) (Figure 7a). However,i nt he presence of enzyme (3 UmL À1 for A•GMP and 6UmL À1 for B•TMP) the product concentration ceased to increase much earlier after addition of GMP or TMP,r espectively,a nd reached al ower final concentration (Figure 7a).…”

Nucleotide‐Selective Templated Self‐Assembly of Nanoreactors under Dissipative Conditions

“…Akey characteristic of biological systems is that reaction pathways are transiently up-or downregulated caused by the temporary availability of atrigger. [52,53] We therefore studied nucleotide-selective transient reaction activation under dissipative conditions by following formation of product NBD-SC 8 .UPLC studies revealed that in the absence of the enzyme AP the product concentration increased gradually in time to reach am aximum value after around 70 minutes for both systems (A•GMP and B•TMP) (Figure 7a). However,i nt he presence of enzyme (3 UmL À1 for A•GMP and 6UmL À1 for B•TMP) the product concentration ceased to increase much earlier after addition of GMP or TMP,r espectively,a nd reached alower final concentration (Figure 7a).…”