Two cis-acting elements necessary and sufficient for gibberellin-upregulated proteinase expression in rice seeds

In germinating rice seeds, a cysteine proteinase (REP-1), synthesized in aleurone-layer cells, is a key enzyme in the degradation of the major storage protein, glutelin. The expression of the gene for REP-1 (Rep1) is induced by gibberellins (GAs) and repressed by abscisic acid (ABA). To identify GA-responsive elements in the Rep1 promoter, we developed a transient expression system in rice aleurone cells. Deletion and point-mutation analyses indicated that the GA-response complex was composed of TAACAGA, TAACGTA, and two copies of CAACTC. The two former sequences were identical to GAREs conserved in the promoter of genes for alpha-amylase and proteinases in cereals. The latter, termed as CAACTC regulatory elements (CAREs), were novel GAREs. Gain-of-function experiments revealed that two pairs of GARE and CARE were necessary and sufficient to confer GA inducibility. The sequences were also required for effective transactivation by the transcription factor OsGAMyb. Four copies of either GARE or CARE showed transactivation neither by OsGAMyb nor by GA induction. CARE and GARE were also found in the promoters of a rice alpha-amylase gene, RAmy1A, and a barley proteinase gene, EPB1, which are expressed in germinating seeds. Mutations of CARE in their promoters caused a loss of GA inducibility and GAMyb transactivation, suggesting that CARE is the regulatory element for GA-inducible expression of hydrolase genes in the germinating seeds.