Abstract
There is relatively little information concerning long-term alterations in DNA methylation following exposure of plants to environmental stress. As little is known about the ratio of non-heritable changes in DNA methylation and mitotically-inherited methylation changes, dynamics and reversibility of the DNA methylation states were investigated in grapevine plants (Vitis vinifera) stressed by in vitro cultivation. It was observed that significant part of induced epigenetic changes could be repeatedly established by exposure to particular planting and stress conditions. However, once stress conditions were discontinued, many methylation changes gradually reverted and plants returned to epigenetic states similar to those of maternal plants. In fact, in the period of one to three years after in vitro cultivation it was difficult to distinguish the epigenetic states of somaclones and maternal plants. Forty percent of the observed epigenetic changes disappeared within a year subsequent to termination of stress conditions ending and these probably reflect changes caused by transient and reversible stress-responsive acclimation mechanisms. However, sixty percent of DNA methylation diversity remained after 1 year and probably represents mitotically-inherited epimutations. Sequencing of regions remaining variable between maternal and regenerant plants revealed that 29.3% of sequences corresponded to non-coding regions of grapevine genome. Eight sequences (19.5%) corresponded to previously identified genes and the remaining ones (51.2%) were annotated as “hypothetical proteins” based on their similarity to genes described in other species, including genes likely to undergo methylation changes following exposure to stress (V. vinifera gypsy-type retrotransposon Gret1, auxin-responsive transcription factor 6-like, SAM-dependent carboxyl methyltransferase).
Highlights
Very early in the history of plant tissue culture it was observed that clonally propagated plants often exhibit some level of variability, termed somaclonal variation [1]
Among variants derived from grapevine cultivar Müller Thurgau there were evaluated 1854 MSAP bands and among variants derived from Riesling the amount was 2007 MSAP bands
In this work we have shown the dynamics and reversibility of DNA methylation landscape found in plants stressed by in vitro cultivation
Summary
Very early in the history of plant tissue culture it was observed that clonally propagated plants often exhibit some level of variability, termed somaclonal variation [1]. The occurrence of phenotypic or genetic changes after in vitro cultivation depends on a wide range of factors, including the original role of the cultured tissue and the plant’s regeneration systems [2,3]; the strength and duration of stressful conditions may play a role [4,5,6]. How a single progenitor plant can produce a variety of phenotypic outcomes under the same in vitro culture conditions is still far from completely understood, but it is likely to result from various causes. Genetic changes observed in regenerated plants include alterations in chromosome number, point mutations and new insertions of transposable elements [7,8]. Changes in phenotype may reflect effects occurring in vitro, such as chimerical segregation or loss of a pathogen [9]
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