Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed i n barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among R Nases described to date in that it contains a novel 23-amino acid insert be tween the C2 and C3 conserved sequences. Expression of the recombinant prot ein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleu rone RNase revealed that, like the pattern for cu-amylase, mRNA levels incr eased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated th at cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was requ ired to increase Lu-amylase mRNA levels to the same extent. These results a re consistent with loss of repression as an initial effect of gibberellic a cid on transcription of those genes, although the regulatory pathways for t he two genes may differ.