STRUCTURE OF THE PARSLEY CAFFEOYL-COA O-METHYLTRANSFERASE GENE, HARBORING A NOVEL ELICITOR RESPONSIVE CIS-ACTING ELEMENT

The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methy ltransferase (CCoAOMT, EC 2.1.1.104) gene, including the 5'-flanking r egion of 5 kb, was determined from parsley (Petroselinum crispum) plan ts. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in leng th. The genomic sequence matches the ORF of the cDNA previously report ed from elicited parsley cell cultures, showing only three base change s that do not affect the enzyme polypeptide sequence. S1 nuclease prot ection assays and primer extension analyses with genomic and cDNA temp lates revealed the transcription start site 67 bp upstream of the tran slation start codon, indicating a shorter 5'-UTR than reported previou sly for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, and an S V 40-like enhancer element is located 347 bp upstream. Most notably, t hree putative cis-regulatory elements were recognized by sequence alig nments, which represent motifs recurring in the promoters of several g enes of the stress-inducible phenylpropanoid pathway (boxes P, A and L ). Transient expression assays with a set of 5'-truncated promoter-GUS fusions show that significant promoter activity is retained in a 354 bp promoter fragment. In vitro DNase 1 footprint experiments and elect rophoretic mobilty shift assays (EMSA) identified in this fragment a u nique sequence motif with elicitor-inducible trans-factor binding acti vity, which was unrelated to boxes P, A, or L. This novel cis-regulato ry element, designated box E, appears to be conserved in the TATA-prox imal regions of other stress-inducible phenylpropanoid genes, and in v itro binding of nuclear protein was confirmed in EMSA assays for such an element from the PAL-1 promoter (-54 to -45). Moreover, the deletio n of box E reduced the activity and erased the elicitor-responsiveness of the CCoAOMT promoter in transient expression assays. The results c orroborate the proposed physiological function of CCoAOMT in elicited plant cells and may shed new light on the sequential action of trans-a ctive factors in the regulation of phenylpropanoid genes.