# Cu2+ and Zn2+ Ions Affecting Biochemical Paths and DNA Methylation of Rye (Secale cereale L.) Anther Culture Influencing Plant Regeneration Efficiency

## TL;DR

This study shows how Cu2+ and Zn2+ ions influence DNA methylation and biochemical pathways in rye anther cultures, affecting the efficiency of plant regeneration and the occurrence of albino plants.

## Contribution

The study identifies specific DNA methylation and biochemical markers influenced by Cu2+ and Zn2+ ions, offering new insights into improving rye plant regeneration.

## Key findings

- Cu2+ and Zn2+ ions significantly alter DNA methylation patterns and biochemical profiles in rye regenerants.
- Demethylation in the CG context explains up to 89% of the variance across genotypes.
- Changes in cadaverine and SAM levels most strongly affect green plant regeneration efficiency.

## Abstract

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu2+ 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 Cu2+ and Zn2+ 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 Cu2+ or Zn2+ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu2+ and those treated with Zn2+ formed distinct groups based on DNA sequence and methylation markers. Zn2+ treated and control samples formed separate groups. Also, Cu2+ 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 Cu2+ or Zn2+ 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.