Dynamics and Reversibility of the DNA Methylation Landscape of Grapevine Plants (Vitis vinifera) Stressed by In Vitro Cultivation and Thermotherapy

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).

Fig 1. Schema of the preparation of individual variants.

Fig 2. Dendrograms of epigenetic similarity between individuals with different period after stress.

(A) Degree of epigenetic changes within variants derived from Müller Thurgau show strong influence of the time which elapsed from the plants's exposure to stress to its testing. Once stress conditions were discontinued, many methylation changes gradually reverted and plants returned to epigenetic states similar to those of maternal plants. Furthermore, it is apparent that epigenetic states in variants after in vitro thermotherapy were regularly more different from states in maternal plants than epigenetic states in variants after standard in vitro cultivation. (B) Degree and development of epigenetic changes within variants derived from Riesling cultivar. As visible, it is possible to observe the same regularities as for group of Müller Thurgau variants in the Fig 2A. The system used to identify analysed accessions is described in Table 1, biological repeats are recognisable by letters “A” or “B” at the end of the names of respective accessions.