Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1.

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Arabidopsis MET1 cytosine methyltransferase mutants.

We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis.

Induced instability of two Arabidopsis constitutive pathogen-response alleles.

Paramutation is an example of a non-Mendelian-directed allelic interaction that results in the epigenetic alteration of one allele. We describe a paramutation-like interaction between two alleles, bal and cpr1-1 (constitutive expressor of PR genes 1), which map to a complex R-like gene cluster on Arabidopsis chromosome 4. Both alleles cause dwarfing and constitutive defense responses, similar to another dwarf variant, ssi1 (suppressor of SA-insensitivity 1). Previous work has demonstrated that the bal and ssi1 phenotypes are caused by overexpression of an R-like gene from the cluster, which activates an salicylic acid-dependent defense pathway. Here, we show that the cpr1-1 variant does not alter gene expression from the R-like gene cluster. The bal and cpr1-1 alleles did not complement each other in F(1) hybrids, but F(2) populations that segregated bal and cpr1-1 alleles contained plants with normal morphology at a frequency of 20%. By using molecularly marked bal and cpr1-1 lines, we found that the majority of the normal phenotypes were correlated with inheritance of an altered cpr1-1 allele. Our observation that cpr1-1 is a metastable allele suggests that cpr1-1 is an epigenetic allele. The cpr1-1 allele is the third candidate epigenetic allele originating from this R-like gene cluster, making the region a possible hotspot of epigenetic variation.

Epigenetic variation in Arabidopsis disease resistance.

Plant pathogen resistance is mediated by a large repertoire of resistance (R) genes, which are often clustered in the genome and show a high degree of genetic variation. Here, we show that an Arabidopsis thaliana R-gene cluster is also subject to epigenetic variation. We describe a heritable but metastable epigenetic variant bal that overexpresses the R-like gene At4g16890 from a gene cluster on Chromosome 4. The bal variant and Arabidopsis transgenics overexpressing the At4g16890 gene are dwarfed and constitutively activate the salicylic acid (SA)-dependent defense response pathway. Overexpression of a related R-like gene also occurs in the ssi1 (suppressor of SA insensitivity 1) background, suggesting that ssi1 is mechanistically related to bal.