Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency
Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu<sup>2+</sup> ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathi...
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| author | Wioletta Monika Dynkowska Renata Orłowska Piotr Waligórski Piotr Tomasz Bednarek |
| author_facet | Wioletta Monika Dynkowska Renata Orłowska Piotr Waligórski Piotr Tomasz Bednarek |
| author_sort | Wioletta Monika Dynkowska |
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| description | Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu<sup>2+</sup> ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu<sup>2+</sup> and Zn<sup>2+</sup> that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu<sup>2+</sup> or Zn<sup>2+</sup> presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu<sup>2+</sup> and those treated with Zn<sup>2+</sup> formed distinct groups based on DNA sequence and methylation markers. Zn<sup>2+</sup> treated and control samples formed separate groups. Also, Cu<sup>2+</sup> discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu<sup>2+</sup> or Zn<sup>2+</sup> ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye. |
| format | Article |
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| spelling | doaj-art-673992d73106412f95537d789c8c48212025-08-20T04:00:54ZengMDPI AGCells2073-44092025-07-011415116710.3390/cells14151167Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration EfficiencyWioletta Monika Dynkowska0Renata Orłowska1Piotr Waligórski2Piotr Tomasz Bednarek3Plant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870 Błonie, PolandPlant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870 Błonie, PolandThe Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Kraków, PolandPlant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870 Błonie, PolandRye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu<sup>2+</sup> ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu<sup>2+</sup> and Zn<sup>2+</sup> that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu<sup>2+</sup> or Zn<sup>2+</sup> presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu<sup>2+</sup> and those treated with Zn<sup>2+</sup> formed distinct groups based on DNA sequence and methylation markers. Zn<sup>2+</sup> treated and control samples formed separate groups. Also, Cu<sup>2+</sup> discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu<sup>2+</sup> or Zn<sup>2+</sup> ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye.https://www.mdpi.com/2073-4409/14/15/1167rye anther cultureDNA sequence contextalbino plantsGPREcopperzinc |
| spellingShingle | Wioletta Monika Dynkowska Renata Orłowska Piotr Waligórski Piotr Tomasz Bednarek Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency Cells rye anther culture DNA sequence context albino plants GPRE copper zinc |
| title | Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency |
| title_full | Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency |
| title_fullStr | Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency |
| title_full_unstemmed | Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency |
| title_short | Cu<sup>2+</sup> and Zn<sup>2+</sup> Ions Affecting Biochemical Paths and DNA Methylation of Rye (<i>Secale cereale</i> L.) Anther Culture Influencing Plant Regeneration Efficiency |
| title_sort | cu sup 2 sup and zn sup 2 sup ions affecting biochemical paths and dna methylation of rye i secale cereale i l anther culture influencing plant regeneration efficiency |
| topic | rye anther culture DNA sequence context albino plants GPRE copper zinc |
| url | https://www.mdpi.com/2073-4409/14/15/1167 |
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