*2.2. Heredity of Epigenetic Changes in the F1 Generation of ODN-Treated Flax Cultivated in the Field*

It has been repeatedly demonstrated that CHS gene expression may be induced in plants under stress conditions such as UV light, and bacterial or fungal infection; therefore it was decided to check during plant growth, as the environmental stress present in the field will affect the presence of induced epigenetic changes. The seeds from F0 generation plants treated with ODNs were cultivated in the experimental field. During growth of F1 plants in natural conditions, individual young plants (6-week-old plants in the middle of the vegetative growth period) were selected (and harvested) for testing from a pool of plants in order to determine the maintenance of the induced features. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 5 of 17 (6-week-old plants in the middle of the vegetative growth period) were selected (and harvested) for testing from a pool of plants in order to determine the maintenance of the induced features.

#### 2.2.1. CHS Gene Expression in the Selected Plants 2.2.1. CHS Gene Expression in the Selected Plants

Among plants cultivated in the field, the highest percentage of the flax treated ODN1 presented the primarily induced modulation of *CHS* expression (75%) Only 33% of the analyzed F1 generation of plants incubated with ODN11 possessed overexpression of the CHS gene. For ODN6, none of the analyzed plants presented expected repression of the studied gene. In Figure 1, the CHS gene expression for selected individuals is presented. Among plants cultivated in the field, the highest percentage of the flax treated ODN1 presented the primarily induced modulation of *CHS* expression (75%) Only 33% of the analyzed F1 generation of plants incubated with ODN11 possessed overexpression of the CHS gene. For ODN6, none of the analyzed plants presented expected repression of the studied gene. In Figure 1, the CHS gene expression for selected individuals is presented.

**Figure 1.** CHS gene expression in the F1 generation of plants that maintained modifications induced by short oligodeoxynucleotides (ODNs), cultivated in the experimental field. The seeds obtained from F0 plants were sown and plants were cultivated in the field. The total expression of both CHS genes, *LuCHS6* and *LuCHS7*, was determined by the real-time PCR reaction. The values are referred to the reference gene expression actin. The relative quantity (RQ) presents the transcript level in comparison to the control (set as 1, black). Plants treated with ODN6 met did not maintain in the F1 generation the initially observed level of CHS gene expression (bar presented in diagonal stripes). Data represent the mean value ± SD from at least three repeats of the experiment. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05. **Figure 1.** CHS gene expression in the F1 generation of plants that maintained modifications induced by short oligodeoxynucleotides (ODNs), cultivated in the experimental field. The seeds obtained from F0 plants were sown and plants were cultivated in the field. The total expression of both CHS genes, *LuCHS6* and *LuCHS7*, was determined by the real-time PCR reaction. The values are referred to the reference gene expression actin. The relative quantity (RQ) presents the transcript level in comparison to the control (set as 1, black). Plants treated with ODN6 met did not maintain in the F1 generation the initially observed level of CHS gene expression (bar presented in diagonal stripes). Data represent the mean value ± SD from at least three repeats of the experiment. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05.

## 2.2.2. Pattern of DNA Methylation in Variable CCGG Motifs of the CHS Gene

2.2.2. Pattern of DNA Methylation in Variable CCGG Motifs of the CHS Gene For the plants presented in Figure 1, the methylation status of the variable CCGG sites was evaluated (Figure 2 A–D). In particular F1 individuals, methylation of CCGG motifs was mostly similar to the profiles observed in our previous study [9]. The methylation status of F1 control plants For the plants presented in Figure 1, the methylation status of the variable CCGG sites was evaluated (Figure 2A–D). In particular F1 individuals, methylation of CCGG motifs was mostly similar to the profiles observed in our previous study [9]. The methylation status of F1 control plants resembled

resembled the results for F0 control plants. However, at the +1273 site, a reduced percentage of demethylation (22.5%) for the F1 generation was noted (Figure 2A), while for the F0 generation, the

F1 generation an increased methylation status (by 15.8%) was observed (Figure 2B). According to ODN6, despite the observed reversal of the tendency of change in CHS gene expression, the percentage of CCmGG at the +996 motif was stable, as observed after 48 h after incubation. Also at the +1273 site, the tendency of change was maintained and a minor increase in demethylation was observed (Figure 2C). For ODN11, significant stable methylation of internal cytosine CCmGG was (Figure 2D).

the results for F0 control plants. However, at the +1273 site, a reduced percentage of demethylation (22.5%) for the F1 generation was noted (Figure 2A), while for the F0 generation, the relevant status measured 33.9%. For ODN1, the percentage of CCmGG methylation at the +996 site was similar to the methylation level observed in the plants after treatment. The methylation profile at the +1273 site for plants modified by ODN1 was corresponding between generations; hence, in the F1 generation an increased methylation status (by 15.8%) was observed (Figure 2B). According to ODN6, despite the observed reversal of the tendency of change in CHS gene expression, the percentage of CCmGG at the +996 motif was stable, as observed after 48 h after incubation. Also at the +1273 site, the tendency of change was maintained and a minor increase in demethylation was observed (Figure 2C). For ODN11, significant stable methylation of internal cytosine CCmGG was noted in the +996 and +1273 motifs, which correlates with primarily induced ODN modifications (Figure 2D). *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 6 of 17 noted in the +996 and +1273 motifs, which correlates with primarily induced ODN modifications

**Figure 2.** Methylation in the variable CCGG motifs identified in the CHS gene sequence in the F1 generation of control plants (**A**) and individuals that maintained modifications induced by ODNs (**B**– **D**), cultivated in the experimental field. The seeds obtained from F0 plants were sown and plants were cultivated in the field. The figure presents the percentage of cytosine methylation in the variable CCGG sites (+996, +1219, +1273) located in the coding region of the CHS gene sequences. The genomic DNA was digested by restriction enzymes HpaII and MspI. The amount of non-digested DNA was determined by real-time PCR. The percentage of a particular modification is presented for studied plants and control: CCGG—lack of methylation (dark grey), CCmGG—methylation of internal cytosine (light grey) and CmCmGG—methylation of both cytosines (medium grey). The site positions were presented according to the *LuCHS6* sequence, due to the presence of all CCGG sites. Data represent the mean value ± SD from at least three independent + experimental repeats. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05. **Figure 2.** Methylation in the variable CCGG motifs identified in the CHS gene sequence in the F1 generation of control plants (**A**) and individuals that maintained modifications induced by ODNs (**B**–**D**), cultivated in the experimental field. The seeds obtained from F0 plants were sown and plants were cultivated in the field. The figure presents the percentage of cytosine methylation in the variable CCGG sites (+996, +1219, +1273) located in the coding region of the CHS gene sequences. The genomic DNA was digested by restriction enzymes HpaII and MspI. The amount of non-digested DNA was determined by real-time PCR. The percentage of a particular modification is presented for studied plants and control: CCGG—lack of methylation (dark grey), CCmGG—methylation of internal cytosine (light grey) and CmCmGG—methylation of both cytosines (medium grey). The site positions were presented according to the *LuCHS6* sequence, due to the presence of all CCGG sites. Data represent the mean value ± SD from at least three independent + experimental repeats. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05.

#### *2.3. Activity of ODNs Modified by Methylation (Met) and Thiophosphate (Pto) 2.3. Activity of ODNs Modified by Methylation (Met) and Thiophosphate (Pto)*

atoms in the phosphate backbone (pto).

Susceptibility of the oligodeoxynucleotide sequences for the nucleolytic digestion limits the lifetime of ODNs. In order to extend the time of action of short oligonucleotides, two modifications of the nucleotides included in the sequences were performed: 1. methylation of all cytosines in the Susceptibility of the oligodeoxynucleotide sequences for the nucleolytic digestion limits the lifetime of ODNs. In order to extend the time of action of short oligonucleotides, two modifications of the nucleotides included in the sequences were performed: 1. methylation of all cytosines in the

particular ODN (met) 2. substitution with a sulfur atom instead of one of the nonbridging oxygen

The impact of the unmodified, and modified 1. met and 2. pto ODNs on the gene expression of both studied isoforms is presented in Figure 3. In order to determine whether the effect obtained by modified ODNs lasts longer, the gene expression was investigated in the material harvested after at least 10 days after the incubation with oligodeoxynucleotides. The value of *CHS* expression after ODN1 and ODN11 treatments was at a similar level (RQ ~2), regardless of the sequence modification, in comparison to the control. However, the ODN6 modified by methylation and thiophosphate

the +996 site (by 8.1 %).

particular ODN (met) 2. substitution with a sulfur atom instead of one of the nonbridging oxygen atoms in the phosphate backbone (pto).
