DNA methylation and the promotion of flowering by vernalization

Abstract

We have tested the hypothesis that the promotion of flowering by prolonged exposure to low temperatures (vernalization) is mediated by DNA demethylation [Burn, J. E., Bagnall, D. J., Metzger, J. M., Dennis, E. S. & Peacock, W. J. (1993) Proc. Natl. Acad. Sci. USA 90, 287-291]. Arabidopsis plants that have reduced levels of DNA methylation because of the presence of a methyltransferase (METI) antisense gene flowered earlier than untransformed control plants, without the need for a cold treatment. Decreased DNA methylation mutants (ddm1) also flowered earlier than the wild-type progenitor under conditions where they respond to vernalization. We conclude that demethylation of DNA is sufficient to cause early flowering, and we have found that the promotion of flowering is directly proportional to the decrease in methylation in METI antisense lines. The early-flowering phenotype was inherited in sexual progeny, even when the antisense transgene had been lost by segregation. Methyltransferase antisense plants with low DNA methylation levels responded to a low-temperature treatment by flowering even earlier than their untreated siblings indicating that the promotion of flowering by cold and by demethylation was additive when neither treatment saturated the early-flowering response. As in untransformed control plants, the cold-induced early-flowering signal was reset in progeny of METI antisense plants. These observations suggest that the demethylation brought about by a METI antisense can account for some properties of vernalization, but not for the need for revernalization in each generation.

Figure 1

The early-flowering phenotype segregates with low levels of DNA methylation but not with the METI antisense. The T3 progeny of T2 plant 10.1, which was hemizygous for the METI antisense transgene, flowered early even when they had lost the transgene by segregation. (A) Flowering times of unvernalized C24. (B) Flowering times of progeny that inherited the transgene; flowering times of progeny that did not inherit the transgene are shown in C. Methylation levels of a subset of plants from B were estimated, and in general, the level of methylation (35–45% of normal) correlated with the flowering time. The plant that flowered after 78 days (B) had a higher level of methylation (55% of normal), which may account for the later flowering time.

Figure 2

Flowering time of progeny from vernalized and unvernalized C24 or antisense plants. The progeny of vernalized and unvernalized plants flowered at the same time, indicating the cold-induced signal for early flowering was not inherited in progeny of the vernalized antisense plants. All plants in the progeny generation responded to a cold treatment by flowering earlier than untreated sibs, confirming that the vernalization signal had been reset. (A) Progeny of vernalized C24 plants. (B) Progeny of unvernalized C24 plants. (C) Progeny of vernalized T3 10.5 antisense plants. (D) Progeny of unvernalized T3 10.5 antisense plants. For A–D, the left graph shows flowering time of plants vernalized in this generation, and the right graph shows flowering time of plants not vernalized in this generation.

Figure 3

Proposed pathways to flowering in Arabidopsis. Some of the genes in the pathways (11, 21, 22) are indicated, but no order of function is intended from the order in which the genes are listed. There may be some functional interaction and/or overlap of these pathways that is not indicated in this simplified model. The step in the vernalization pathway that is blocked, in the absence of a cold treatment, is indicated by the box. The block represented by the shaded part of the box can be alleviated by demethylation, but the unshaded portion is only alleviated by cold. The methylation block may lie within the vernalization pathway and/or in a parallel pathway, as indicated.