When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence.

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

A cDNA encoding a DNA methyltransferase, with a predicted polypeptide of 1556 amino acid residues containing all motifs conserved in this enzyme family, was isolated from tobacco plants, and the corresponding gene was designated as NtMET1. RNA blot analysis indicated NtMET1 transcripts to accumulate in dividing tissues of tobacco plants, and they could be detected during the S phase in synchronized dividing BY2 cells. In situ hybridization revealed the transcripts to be localized exclusively in actively proliferating tissues around axillary apical meristem. In order to ascertain physiological roles, transgenic tobacco plants that had the antisense construct were made and examined for phenotypes. Methylation levels of genomic DNA from transgenic plants significantly decreased in comparison with wild-type levels, and distinct phenotypic changes including small leaves, short internodes and abnormal flower morphology were noted. Microscopic observation revealed that leaf structure differed between transgenic and wild-type plants. These results suggest that NtMET1 functions during DNA replication, and that DNA methylation plays an important role in plant morphogenesis.

No abstract

The wheat gene WPK4 encodes a 56-kDa protein kinase that belongs to group 3 of the SNF1-related protein kinase family (SnRK3), and is up-regulated by light and cytokinins and down-regulated by sucrose. In order to determine whether or not this particular regulation pattern is general among plant species, we isolated and characterized homologous genes from rice and maize. Two rice genes, OsPK4 and OsPK7, encode proteins comprising 508 and 520 amino acids, and show, respectively, 75% and 76% sequence similarity to WPK4. OsPK4 and OsPK7 proteins produced in Escherichia coli were able to phosphorylate themselves and myelin basic proteins, the reaction requiring magnesium and/or manganese ions. Transcripts of OsPK4 were detected in all tissues tested, and amounts were increased upon illumination, nutrient deprivation and treatment with cytokinins. In contrast, transcripts of OsPK7 were not found in any tissues except in mature leaves at low levels, and did not accumulate under any of the stress conditions examined. A maize gene, ZmPK4, encodes a protein with 518 amino acids that shows 74% similarity to WPK4. Its transcripts were constitutively expressed in all tissues, regardless of light, nutrient and cytokinin status, but were increased upon exposure to low temperature. These results indicate that, despite the sequence similarity between their products, genes for SnRK3 proteins are differentially regulated in response to environmental stimuli.