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.
Sørensen
M B
MB
Department of Physiology, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Müller
M
M
Skerritt
J
J
Simpson
D
D
D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005966:Glucuronidase / Q000378:metabolism; D005983:Glutens; D001467:Hordeum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics*; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D010940:Plant Proteins; D005983:Glutens; D005966:Glucuronidase