Structure of the tobacco caffeic acid O-methyltransferase (COMT) II gene: identification of promoter sequences involved in gene inducibility by various stimuli.

Abstract

The tobacco gene encoding caffeic acid-O-methyltransferase of class II (COMT II) was isolated, including a 1.7 kb 5'-flanking region. Sequence motifs were identified in COMT II gene promoter which are present in many genes of the phenylpropanoid pathway or in stress-inducible pathogenesis-related (PR) genes. A 1215 bp COMT II promoter fragment was transcriptionally fused to the GUS coding region and its activity pattern studied by stable expression of the fusion gene in tobacco. Transgenic lines were analysed for GUS and OMT activities upon infection, UV irradiation, wounding and treatment by various signalling compounds. The promoter proved responsive to various biotic and abiotic elicitors and to infection by avirulent and virulent pathogens. During the course of the hypersensitive reaction of tobacco to TMV two peaks were detected, an early one induced by the inoculation process and a second one at the onset of lesion formation. Parallel changes were observed between GUS activity that reflected the activity of the COMT II promoter fragment and COMT II activity that mirrored expression of the endogenous COMT II gene, indicating that COMT II pattern of expression is established at the transcriptional level. Various promoter fragments were fused to the GUS gene and revealed that gene induction by MeJA or UV and by TMV or wounding requires different sequences included in a 74 bp fragment. When the 74 bp sequence was multimerized and inserted ahead of the CaMV 35S RNA minimal promoter, one construct was shown to be capable of driving expression of the reporter gene around the TMV-infected sites in transgenic tobacco plants.