Vernalisation Pathway

The vernalisation pathway (Figure 1.1) promotes flowering in plants that have been exposed to prolonged low temperatures. Winter annual accessions of Arabidopsis respond to vernalisation and contain the alleles of FLC and FRI, which play a central role in the vernalisation response (see also section 1.4.1; Lee et al., 1993; Clarke and Dean, 1994; Sheldon et al., 1999; Sheldon et al., 2000) . The correct function of these genes is normally lacking in early flowering accessions, many of which carry loss-of-function fri alleles; Ler also has a transposon inserted in the first intron of FLC (Gazzani et al., 2003).

The FRI gene

FRI is an upstream positive regulator of FLC. FRI does not repress flowering in the absence of FLC activity (Johanson et al., 2000; Michaels and Amasino, 2001) . FRI encodes an unknown protein, which is predicted to contain coiled-coil domains in two positions (Johanson et al., 2000).

The repressor of flowering FLC Protein

The MADS box protein FLC is a repressor of flowering (Michaels and Amasino, 1999; Sheldon et al., 1999) . The expression of FLC is high in winter-annual accessions or in autonomous pathway mutants and is mainly expressed in the vegetative apex and roots (Michaels and Amasino, 2000) . The expression levels of FLC remain high throughout vegetative growth, but are low during reproductive growth, this suggests that developmental processes may override the repression of flowering caused by FLC expression (Michaels and Amasino, 1999; Sheldon et al., 1999) . FLC expression is progressively reduced during vernalisation treatment and reaches trough levels after approximately six weeks vernalisation. Repression of FLC expression is maintained when vernalised plants are returned to warmer growth conditions (Michaels and Amasino, 2001) , but is reset through meiosis so that the progeny of vernalised plants again show high FLC expression.

Although FLC plays a central role in the vernalisation process, flc null mutants still respond to vernalisation (Michaels and Amasino, 2001; Reeves and Coupland, 2001) . This suggests that vernalisation can also promote flowering in an FLC independent manner.

Vernalization Pathway Mutants

Genes involved in the vernalization pathway were identified by screening for mutants that are late flowering even after a vernalization treatment. Mutants were identified on the basis that they did not respond to vernalization of the late flowering autonomous pathway mutant fca1, and two mutants were identified called vernalization 1 and vernalization 2 (vrn1; vrn2) (Chandler et al., 1996; Gendall et al., 2001; Levy et al., 2002). In vrn1 fca-1 or vrn2 fca-1 plants FLC expression is reduced by vernalization but is not maintained at low levels when plants are returned to a warm environment. Therefore vrn1 and vrn2 are involved in the maintenance but not in the initiation of FLC repression (Gendall et al., 2001; Levy et al., 2002) .

Both VRN genes have been cloned. VRN1 encodes a nuclear protein with B3 domains related to those in plant-specific transcription factors and able to bind DNA (Levy et al., 2002) . VRN2 encodes a nuclear-localised zinc-finger protein, which shows a similarity to Polycomb Group (PcG) proteins of plants and animals (Gendall et al., 2001) . In animals these proteins have been implicated in chromatin remodelling through histone modification (van Lohuizen, 1998; Birve et al., 2001) .

Consistent with this function for VRN genes, vernalisation treatments cause changes in histone methylation in discrete domains of the FLC gene, the changes seen in the H3 K9 domains of FLC do not occur in vrn mutants, whilst dimethylation of the H3 K27 domain is lost in vrn2 mutants; this is consistent with VRN1 functioning downstream of VRN2 (Bastow et al., 2004) .

Vernalization Insensitive Genes

The expression levels of VRN1 and VRN2 are not changed by vernalisation. However, a third gene involved in the repression of FLC by vernalisation is VERNALIZATION INSENSITIVE 3 (VIN3), which encodes a plant homeodomain (PHD) finger containing protein (Sung and Amasino, 2004b) . This group of proteins often play a role in chromatin remodelling complexes (Aasland et al., 1995) . In contrast to vrn1 and vrn2 mutants, in vin3 mutants repression of FLC does not occur. VIN3 is expressed in the shoot and root apical meristems, where cold temperatures are perceived. The expression of VIN3 increases during vernalization, and a subsequent reduction of FLC is observed. When plants are returned to warm conditions, there is a decrease in the expression of VIN3. VIN3 is therefore essential for the initial repression of FLC during the vernalization response.

The current model of vernalization proposes that VIN3 represses FLC expression by reducing acetylation of histones in domains of the FLC gene. Subsequent histone methylation by VRN1 and VRN2 then ensures that FLC expression remains in a stable and repressed state after the plants are returned to warm temperatures (Sung and Amasino, 2004a, b), and until the chromatin is reset during meiosis.

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