The Vernalization Response of Arabidopsis

Vernalization refers to the acceleration of flowering that occurs in many plant species following the extended exposure of their imbibed seeds or young seedlings or buds to extended periods of cold. Vernalization is not all-or-none, but a slow, quantitative process in which increasing periods of cold cause progressively earlier flowering until a saturation point is reached. Plants that are vernalized as seeds or young seedlings do not flower immediately upon being raised to higher temperatures, but often weeks later. There is, therefore, a clear temporal separation between the perception of cold and the switch from vegetative to reproductive growth. The perception of and response to cold is localized in the meristematic cells of embryos, growing points, and buds. The changes induced in meristems by vernalization are conserved through many generations of cell division even at temperatures much higher than those required for the cold induction of flowering. In some species, this “memory” of vernalization can be maintained for up to 330 d (Lang, 1965). Vernalization is required in each generation for winter annuals and biennials and each year for perennials. Thus, meiosis appears to reset the requirement for vernalization.

There is tremendous natural variation between Arabidopsis ecotypes in the extent to which their time to flowering is shortened by vernalization. For example, a standard vernalization treatment decreased the time to floral initiation from 186 d to 49 d in a naturally late-flowering Arabidopsis accession from North Carolina, but only from 54 d to 52 d in an early-flowering accession from Köln, Germany (Nordborg and Bergelson, 1999). Both Arabidopsis ecotypes and vernalization mutants have been enormously valuable in allowing biologists to address the fundamental question of how vernalization works.

A Role for Gibberellin?

Arabidopsis grows as an inconspicuous rosette until the onset of floral initiation, whereupon bolting occurs. Gibberellic acid (GA) is the major factor underlying bolting. Vernalization not only increases GA biosynthesis in the vegetative rosette but also elevates the GA sensitivity of the shoot (Oka et al., 2001).

Although GA has been found to accelerate the time to flowering in non-vernalized Arabidopsis grown under short days (Wilson et al., 1992), the promotion of flowering by GA does not mimic vernalization precisely. For example, the time to flowering in late-flowering Arabidopsis mutants is reduced similarly by GA regardless of whether they are vernalization-sensitive (Chandler and Dean, 1994). Moreover, an Arabidopsis mutant (ga 1–3) that is severely defective in GA synthesis responds normally to vernalization (Michaels and Amasino, 1999a). Thus, although GA may promote flowering generally, it cannot substitute for vernalization, and it does not appear to have a direct role in the vernalization response in Arabidopsis.

FLOWERING LOCUS C (FLC) and FRIGIDA (FRI)

The genes FLC and FRI play central roles in vernalization in Arabidopsis. In naturally occurring late-flowering ecotypes, FRI acts to increase, and vernalization to reduce, FLC levels (Michaels and Amasino, 1999b; Sheldon et al., 1999; Johanson et al., 2000). Allelic variation at the FRI locus is a major determinant of natural variation in flowering time. Dominant alleles of FRI confer late flowering, which is reversed to earliness by vernalization. Research has shown that loss-of-function mutations at FRI have provided the basis for the evolution of many early-flowering Arabidopsis ecotypes from ancestral late-flowering types (Johanson et al., 2000).

FLC also plays a major role in vernalization. An early flowering phenotype results when FLC, which encodes for a novel MADS domain protein, is rendered dysfunctional by mutation (Michaels and Amasino, 1999b). FLC acts as a strong floral repressor by negatively regulating the genes that promote the transition to flowering. Vernalization promotes flowering by reducingFLC mRNA levels. The extent of the reduction is proportional to the duration of vernalization and is closely correlated with flowering time. Michaels and Amasino (2000) have proposed a “rheostat model” of flowering time, in which increasing levels of FLC are associated with the conversion of a species or ecotype from an annual growth habit to a biennial one.

The reduction in FLC transcript levels by vernalization is mitotically stable. FLC activity is restored in each new generation, as is the requirement for an exposure to cold for the acceleration of flowering. The level of FLC transcript determines the extent of the vernalization response in the promotion of flowering, and there is a quantitative relationship between the duration of cold treatment and the extent of down-regulation ofFLC activity. A surprising discovery, therefore, was that the complete loss of FLC function does not eliminate the effect of vernalization. Thus, vernalization is able to promote flowering via FLC-dependent and FLC-independent mechanisms (Michaels and Amasino, 2001).

Similarities to Epigenesis

Mutants have been isolated that reduce the vernalization responsiveness of late-flowering Arabidopsis mutants (Chandler et al., 1996). Some of these vernalization mutants (vrn) are unable to reduce FLC mRNA in response to cold, suggesting that they encode regulators of FLC expression (Sheldon et al., 2000). Gendall et al. (2001) have recently shown that one of the genes (VRN2) encodes a nuclear-localized Zn finger protein that is a structural homolog ofSuppressor of zeste 12, a Polycomb group (PcG) gene of enormous importance in fruit fly (Drosophila melanogaster) development. In fruit fly, PcG proteins generally act by remodeling chromatin structure and mediating the silencing of homeotic genes. VRN2 does not appear to be required for the vernalization-induced decrease inFLC mRNA, but it is essential for the stable repression of FLC later in development (Gendall et al., 2001).

The maintenance of vernalization through multiple cell divisions is reminiscent of epigenetic phenomena. Changes in epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure (Meyer, 2000;Habu et al., 2001). There is some evidence that DNA methylation may play a role in preventing early flowering in Arabidopsis ecotypes (Burn et al., 1993). Much like vernalized plants, Arabidopsis seedlings that have been treated with the demethylating compound 5-azacytidine flower more quickly. Late-flowering mutants that are insensitive to vernalization do not respond to 5-azacytidine treatments. Burn et al. (1993) found that Arabidopsis plants, either vernalized or 5-azacytidine-treated, had reduced levels of 5-methylcytosine in their DNA compared with non-vernalized plants. Moreover, normal flowering time was found to be reset in the progeny of plants induced to flower early with 5-azacytidine, paralleling the lack of inheritance of the vernalized condition. These findings led Burn et al. (1993) to propose that vernalization, through general demethylating effects, may release the block to flowering initiation, thereby allowing the plant to flower early. In support of this idea, Finnegan et al. (1998) found that Arabidopsis plants that had reduced levels of DNA methylation because of their transformation by a methyltransferase (MET1) antisense gene flowered earlier than untransformed control plants, and without the need for a cold treatment. Moreover, the promotion of flowering was directly proportional to the decrease in methylation observed in the MET1 antisense lines. Not all of the results of Finnegan et al. (1998), however, were consistent with the hypothesis that vernalization stems from a general demethylation of DNA. First, although growth at vernalizing temperatures was associated with some reduction of DNA methylation, this demethylation was transient and normal methylation levels were restored when the seedlings were transferred to warm temperatures. Second, unlike the case with vernalization, the early-flowering phenotype was inherited in sexual progeny, even when the antisense transgene was lost by segregation.

In summary, while vernalization continues to defy full explanation, plant scientists have, in this first decade of the Arabidopsis revolution, made enormous strides in identifying some of the key players in the vernalization process.