Does DNA Methylation Pattern Mark Generative Development in Winter Rape?

Abstract

In this paper we report on changes in DNA methylation pattern in rape apices and leaves during transition from vegetative to reproductive stage due to grafting and/or vernalization. Grafted plants of winter rape (Brassica napus L., var. "Gó rczań ski") (stock from vernalized, scion from non-vernalized plants) were used together with vernalized non-grafted plants. In addition, methylation status was determined also in spring rape (var. "Młochowski") grown under normal and low temperature. The methylation-sensitive amplification polymorphism (MSAP) method with EcoRI/MspI and EcoRII/HpaII restriction enzymes was employed. The majority (ca. 68%) of analyzed loci (566 in winter and 551 in spring rape) were monomorphic, i.e. did not undergo methylation. Both cultivars showed a similar degree of methylation. 188 loci in winter and 176 in spring cultivars expressed changes in the methylation pattern. All differentially amplified fragments resulted from either full methylation of an internal cytosine or from hemi-methylation of an external cytosine. A pair-wise comparison showed that a similar number of loci underwent development-related methylation changes in apices of the winter and spring rape. The majority (80%) of changes were demethylation events in generative (vernalized) apices of the winter cultivar. However, an increased number of demethylated loci was detected in vernalized apices in comparison with generative, nonvernalized ones. In apices of vegetative and generative grafted plants the same number of demethylation events was observed. Overall, 10 MSAP loci were detected that expressed methylation changes in vernalized apices only; among them 7 loci underwent demethylation after vernalization and remained methylated in both vegetative and generative non-vernalized stage. Only 1 locus was demethylated in generative non-vernalized apices. Thus, most of demethylation events can be ascribed to vernalization and not to the generative stage. In leaves of winter rape methylation and demethylation events occurred with similar frequency, while in the spring cultivar more demethylation events were detected. The results show that during vernalization and transition to the generative stage different sets of genes are activated.

Vernalization and photoperiod-related changes in the DNA methylation state in winter and spring rapeseed

“…These results confirmed the authors' previous analysis, which suggested the presence of MSAP loci with changes in the methylation state specific for vernalization and a significant prevalence of demethylation events during the cold treatment in the winter rape (Filek et al 2006b). The importance of the first weeks of a prolonged exposure to cold was also suggested.…”

“…Seeds of two rapeseed cultivars, a winter ('Górczański', G) and spring type ('Młochowski', M), were germinated as described in Filek et al (2006b). Plants were grown in a growth chamber at 17/15°C (day/night, 16 h day) until the stage of a 5-leaf rosette and then transferred for 8 weeks (56 days) into 5/2°C (day/night, 16 h day) for vernalization.…”

“…The restriction, ligation, PCR preamplification, selective amplification and the visualization of products on a sequencer (Model 4300, LI-COR, Lincoln, NE) were as described in Filek et al (2006b). The adapters for the EcoRI and MspI/HpaII recognition sites were as proposed by Vos et al (1995) and Xu et al (2000), respectively.…”

“…The presence/absence of the amplification product in a single MSAP locus was scored in both restriction systems. Owing to the differential sensitivity of MspI and HpaII to the methylation state at 5 0 -CCGG-3 0 sites, it was possible to define whether a demethylation or methylation event had occurred in a single MSAP locus in the samples compared (for details, Filek et al 2006b). Briefly, when two DNA samples are subjected to MSAP analysis, there are 13 possible band pattern combinations in a single MSAP locus between two samples compared.…”

“…It was also shown that the methylation state of DNA is intimately linked to chromatin structure and vice versa (Lindroth et al 2004). Therefore, it was suggested that vernalization might also result in DNA demethylation and the active state of specific genes involved in the induction of flowering (Lizal and Relichova 2001;Filek et al 2006b). …”