A MAJOR CYSTEINE PROTEINASE, EPB, IN GERMINATING BARLEY-SEEDS - STRUCTURE OF 2 INTRONLESS GENES AND REGULATION OF EXPRESSION

The barley cysteine proteinase B (EPB) is the main protease responsibl e for the degradation of endosperm storage proteins providing nitrogen ous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormon e, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in t he scutellar epithelium within 24 h of seed germination, but the aleur one tissue surrounding the starchy endosperm eventually becomes the ma in tissue expressing this enzyme. The EPB gene family of barley consis ts of two very similar genes, EPB1 and EPB2, both of which have been m apped to chromosome 3. The sequences of EPB1 and EPB2 match with the t wo previously published cDNA clones indicating that both genes are exp ressed. Interestingly, neither of these genes contain any introns, a r are phenomenon in which all members of a small gene family are active intronless genes. Sequence comparison indicates that the barley EPB fa mily can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage p rotein degradation, The promoters of EPB1 and EPB2 have been linked to the coding sequence of a reporter gene, GUS, encoding beta-glucuronid ase, and introduced into barley aleurone cells using the particle bomb ardment method. Transient expression studies indicate that EPB promote rs are sufficient to confer the hormonal regulation of these genes.