The control of expression of storage protein genes in pisum sativum L

Abstract

Pea cotyledon and leaf genomic DNA were found to be methylated in a series of defined methylation states. ͫ CG and ͫ C-X-G methylations were detected and the latter was more prevalent in leaf DNA. Pea rDNA was also found to be highly methylated but was relatively undermethylated in the developing cotyledon. The significance of the relative hypo-' methylation of cotyledon genomic DNA (and rDNA) is discussed with respect to the endoreduplication phase of seed development. Two post-expression demethylation events associated with the legumin gene family were detected using a cDNA probe. The methylation of specific CCGG sequences in and around two legumin genes was also investigated. The extent of the methylation of the genes was found to increase in a 5' to 3' direction and one gene was found to have an unmethylated site about 500bp upstream from the transcription start site. Minor changes in the extent of methylation of two sites in the protein coding regions of the two genes were detected and these are thought to represent 'fine-tuning' of gene expression, rather than major gene switching events. One or two post-expression demethylation events associated with the vicilin gene family, were detected using cDNA probes. In addition, there was evidence that some cytosines associated with the vicilin genes became hypermethylated during cotyledon development. A normal pattern of 50,000-M vicilin gene demethylation and hypermethylation was detected in the cotyledon DNA of a mutant pea line, which produces reduced levels of 50,000-M vicilin polypeptide and message.Analysis of the sequence data of two legumin genes indicated that in general the CG dinucleotide was suppressed although one exon was found to have a cluster of CG dinucleotides and an increased usage of the CG-containing arginine codons. The mutability of 5-methylcytosine is discussed in relation to possible legumin protein coding requirements.