Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm

B- and C-hordein gene transcription is severely reduced in the endosperm of the regulatory barley mutant lys3a, and this is correlated with persistent hypermethylation of the promoters. In contrast, D-hordein is expressed at normal levels in the mutant. To confirm the connection between methylation and transcriptional activity, a genomic D-hordein clone was isolated and sequenced. The nucleotide composition of the promoter region revealed a CpG island and methylation analysis, using bisulphite treatment of genomic DNA, confirmed that the D-hordein promoter is unmethylated in endosperm and leaf tissue. Immunocytochemical studies localized D-hordein to the reticular component of protein bodies in both the wild-type Bomi and lys3a. Transient expression of GUS reporter gene constructs in barley endosperm, following transfection by particle bombardment revealed the D-hordein promotors. Comparison of transient expression in Bomi and lys3a endosperm demonstrated that the activities of the unmethylated D-hordein and the Hor1-14 C-hordein promoters were equivalent, while the activities in the mutant of the Horl-17 C-hordein and the Hor2-4 B-hordein promoters were reduced two- and tenfold, respectively. Methylation of plasmids in vitro prior to expression severely inhibited B- and D-hordein promoter activities. Based on these observations two categories of promoters for endosperm-specific expression of storage proteins are recognized and a model involving methylation and modulation of chromatin structure in the regulation by the Lys3 gene is presented.