E. J. Finnegan

The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC)

In Arabidopsis, the MADS-box protein encoded by FLOWERING LOCUS C (FLC) is a repressor of flowering. Vernalization, which promotes flowering in the late-flowering ecotypes and many late-flowering mutants, decreases the level of FLC transcript and protein in the plant. This vernalization-induced reduction in FLC transcript levels is mitotically stable and occurs in all tissues. FLC activity is restored in each generation, as is the requirement of a low-temperature exposure for the promotion of flowering. The level of FLC 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 of FLC activity. We conclude that FLC is the central regulator of the induction of flowering by vernalization. Other vernalization-responsive late-flowering mutants, which are disrupted in genes that encode regulators of FLC, are late-flowering as a consequence of their elevated levels of FLC.

Plant DNA methyltransferases

DNA methylation is an important modification of DNA that plays a role in genome management and in regulating gene expression during development. Methylation is carried out by DNA methyltransferases which catalyse the transfer of a methyl group to bases within the DNA helix. Plants have at least three classes of cytosine methyltransferase which differ in protein structure and function. The METI family, homologues of the mouse Dnmt1 methyltransferase, most likely function as maintenance methyltransferases, but may also play a role in dexa0novo methylation. The chromomethylases, which are unique to plants, may preferentially methylate DNA in heterochromatin; the remaining class, with similarity to Dnmt3 methyltransferases of mammals, are putative dexa0novo methyltransferases. The various classes of methyltransferase may show differential activity on cytosines in different sequence contexts. Chromomethylases may preferentially methylate cytosines in CpNpG sequences while the Arabidopsis METI methyltransferase shows a preference for cytosines in CpG sequences. Additional proteins, for example DDM1, a member of the SNF2/SWI2 family of chromatin remodelling proteins, are also required for methylation of plant DNA.

Vernalization-induced flowering in cereals is associated with changes in histone methylation at the VERNALIZATION1 gene

Prolonged exposure to low temperatures (vernalization) accelerates the transition to reproductive growth in many plant species, including the model plant Arabidopsis thaliana and the economically important cereal crops, wheat and barley. Vernalization-induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone modifications at FLC chromatin. In cereals, the vernalization response is mediated by stable induction of the floral promoter VERNALIZATION1 (VRN1), which initiates reproductive development at the shoot apex. We show that in barley (Hordeum vulgare), repression of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting that vernalization promotes an active chromatin state at VRN1. Levels of these histone modifications at 2 other flowering-time genes, VERNALIZATION2 and FLOWERING LOCUS T, were not altered by vernalization. Our study suggests that maintenance of an active chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in Arabidopsis is mediated by epigenetic regulation of the floral repressor FLC, this phenomenon in cereals is mediated by epigenetic regulation of the floral activator, VRN1.

The control of flowering by vernalization

The process by which vernalization, the exposure of a germinating seed or a juvenile plant to a prolonged period of low temperature, promotes flowering in the adult plant has remained a mystery for many years. The recent isolation of one of the key genes involved in vernalization, FLOWERING LOCUS C, has now provided an insight into the molecular mechanism involved, including the role of DNA methylation.

The role of DNA methylation in the regulation of plant gene expression

The most common modification of DNA in plant cells is methylation of cytosine residues at carbon 5. In contrast to mammalian cells in which 3–8% of cytosine residues are methylated (Shapiro, 1975), in plants up to 30% of cytosine residues are modified (Adams and Burdon, 1985). There is considerable inter-species variation in the level of cytosine methylation, ranging from 4.6% in Arabidopsis thaliana (Leutwiler et al., 1984), which has a small genome with relatively little highly repeated DNA, to 33% in rye, Secale cereale (Thomas and Sherratt, 1956). The difference in the extent of methylation, between plants and animals, is due to two factors. The CG dinucleotide, which is methylated to about the same extent (70–80%) in plants and animals, occurs more frequently in plant DNA. In addition plant DNA is methylated in CNG trinucleotides where N can be any base (Gruenbaum et al., 1981). The CG dinucleotide has symmetrical cytosine residues in the two DNA strands and it has been observed that, when modified, both cytosines are methylated (Bird, 1978; Cedar et al., 1979). It is this symmetry that allows the pattern of methylation to be maintained through DNA replication. Newly replicated DNA is hemimethylated with methylation at specific sites on the parental strand. Methylation of the new strand at the unmethylated cytosine of a hemimethylated CG dinucleotide restores the original pattern of methylation; this is termed maintenance methylation (Holliday and Pugh, 1975; Riggs, 1975; Razin and Riggs, 1980). The CNG motif also has strand symmetry suggesting that methylation of this motif will probably be maintained through replication by the same mechanism.

Multiple DNA methyltransferase genes in Arabidopsis thaliana

Methylation of plant DNA occurs at cytosines in any sequence context, and as the Arabidopsis methyltransferase, METI, preferentially methylates cytosines in CG dinucleotides, it is likely that Arabidopsis has other methyltransferases with different target specificities. We have identified five additional genes encoding putative DNA methyltransferases. Three of these genes are very similar to METI throughout the coding region; these genes probably arose by a series of gene duplication events, the most recent giving rise to METIIa and METIIb. METIIa and b are expressed at low levels in vegetative and floral organs and the level of transcripts is not affected by the introduction of a METI antisense transgene, nor do the METII enzymes substitute for the reduced activity of METI in methylatingxa0CG dinucleotides. METIII is not essential as it encodes a truncated protein. Two other genes encode a second class of DNA methyltransferase with the conserved motifs characteristic of cytosine methyltransferases, but with little homology to the METI-like methyltransferases through the remainder of the protein. These two methyltransferases are characterized by the presence of a chromodomain inserted within the methyltransferase domain, suggesting that they may be associated with heterochromatin. Both these genes are transcribed at low levels in vegetative and reproductive tissues.

Structure and expression of an alcohol dehydrogenase 1 gene from Pisum sativum (cv. “Greenfeast”)

Three genomic clones for anaerobically inducible alcohol dehydrogenase (Adh) have been isolated from Pisum sativum cv. Greenfeast via cDNA cloning. One of these contains a complete gene, has exon sequences corresponding to one of the cDNA sequences and is likely to be an expressed gene. This gene has a structure similar to the Adh genes of maize, with introns in the same positions in the coding sequence but differing in their lengths and nucleotide sequences. At the nucleotide level the coding sequence is 75% homologous to both maize Adh1 and Adh2 and 80% homologous to the Adh gene from Arabidopsis, but has an extra coding triplet in exon 1 that is not found in the other plant Adh genes. The non-translated regions of all the gene transcripts are widely divergent between species. A short segment of the pea Adh promoter region (-290 to +57) was fused to a reporter gene and introduced into protoplasts of Nicotiana plumbaginifolia by electroporation. Transient expression of the introduced gene increased markedly when the transfected protoplasts were incubated under anaerobic conditions, showing that cis-acting regulatory signals necessary for anaerobic control of expression reside in the -290 to +57 segment. Sequence comparisons between this region and the corresponding regions of maize and Arabidopsis Adh genes have identified short sequences that may be involved in the anaerobic regulation of plant Adh genes.

Transposable elements can be used to study cell lineages in transgenic plants.

The beta-glucuronidase reporter gene has been used to develop a sensitive assay for the excision of transposable elements introduced into transgenic plants. The reporter gene, inactivated by the insertion of the maize transposable element Activator (Ac) into the 5-untranslated leader, was introduced into the genome of tobacco by Agrobacterium-mediated transformation. Reactivation of the beta-glucuronidase gene was detected in transgenic plants using a fluorometric or histochemical assay. Reactivation of the reporter gene was dependent on the presence of the transposase of Ac, and resulted from the excision of the Ac element. This assay, together with the improved methods for visualization, will provide a valuable and rapid method for studying the basic mechanism of transposition in plants and for developing modified transposable element systems suitable for gene tagging in transgenic plants.

Polycomb repression: It’s all in the balance

In our recent paper we suggested a molecular explanation for the long standing observation that plants need to be mitotically active to respond to a prolonged period of low temperatures by flowering early (vernalization). In Arabidopsis, vernalization is associated with the epigenetic repression of the floral repressor, FLC. FLC repression is established during the low temperature treatment and is marked by the loss of chromatin marks associated with active genes and the gain of histone H3 trimethyl-lysine 27 (K27me3) at the start of transcription/translation. After the end of the cold treatment, this repressive modification spreads across FLC chromatin to mark the entire locus. In cells not undergoing mitosis, we found that FLC is repressed by low temperatures, but that this repression is only partially maintained. We concluded that DNA replication is not required for the initial response to low temperatures, but rather for the maintenance of this response. Here we discuss the implications of our observations in terms of the plasticity of chromatin modifications in plants.