A GCC ELEMENT AND A G-BOX MOTIF PARTICIPATE IN ETHYLENE-INDUCED EXPRESSION OF THE PRB-1B GENE

The PRB-1b gene codes for a basic-type pathogenesis-related protein an d is activated at the transcriptional level by the plant hormone ethyl ene. To identify cis-acting DNA elements essential for ethylene induct ion, deleted and mutant forms of the PRB-1b promoter, fused to the bet a-glucuronidase (GUS) coding region, were introduced in transgenic tob acco plants. A 73 bp fragment (X1 region) of the PRB-2b promoter, loca ted between positions -213 and -141, was sufficient to confer ethylene responsiveness to the reporter gene. The X1 region contains a TAAGAGC CGCC motif (GCC-box) well conserved in several ethylene-inducible gene s. A substitution mutation in this sequence, in the context of a 213 b p PRB-1b promoter, completely abolished ethylene induction in transgen ic tobacco, defining this conserved motif as part of a cis-acting elem ent responsive to ethylene. Three other mutations in the X1 region cau sed a pronounced decrease in the PRB-1b promoter activity in transgeni c plants, but did not affect ethylene inducibility. One of them, local ized in a G-box like motif (CACGTG), disrupted the binding site for a nuclear factor, as observed in gel-shift analysis. Interestingly, the mobility of the complex formed on the G-box element was dependent on i ts phosphorylation state. These results suggest that a cis-acting elem ent involved in the perception of the ethylene signal resides in a GCC motif and acts in concert with additional elements in the regulation of ethylene-induced PRB-1b expression.