Molecular cloning, induction and taxonomic distribution of caffeoyl-CoA 3-O-methyltransferase, an enzyme involved in disease resistance

Abstract

Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall under conditions that trigger the disease resistance response (Pakusch, A.-E., Kneusel, R.E., and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488-494). Partial amino acid sequences of the enzyme from cultured parsley cells that had been treated with a crude elicitor were identified (Pakusch, A.-E., Matern, U., and Schiltz, E. (1991) Plant Physiol. 95, 137-143), and corresponding degenerated oligonucleotides of 29- and 30-nucleotide length were synthesized. Northern hybridizations with these probes revealed one specific RNA band, and the amount of this RNA appeared to be transiently induced upon elicitation of the cells. De novo enzyme synthesis was confirmed by Western blotting experiments using a specific antiserum. The time course of induction closely followed the pattern observed for phenylalanine ammonia-lyase and suggested the operational coordination of the methyltransferase with the general phenylpropanoid pathway in vivo. Full size cDNA of 1.258 kilobases was isolated in lambda gt11, sequenced and found to contain a remarkably long 5'-untranslated leader sequence followed by an open reading frame that codes for a 241-residue polypeptide representing the 27-kDa subunit of the native, dimeric parsley enzyme. Almost no homology was found to protein sequences filed in data banks. Southern hybridization with genomic DNA suggested that only one or two copies of the respective gene(s) are present in the parsley genome. Caffeoyl-CoA-specific methyltransferase activity was demonstrated in taxonomically widely diverse plants such as Dianthus caryophyllus (Caryophyllaceae), Carthamus tinctorius (Asteraceae) or Daucus carota, and Ammi majus (Apiaceae) where it is commonly induced by elicitor treatment. In Northern blots with RNA from Ammi or Daucus, parsley cDNA hybridized specifically to one band comparable in size to the parsley RNA, whereas Dianthus and Carthamus appear to code for slightly larger RNAs (roughly 1.45 and 1.3 kilobases, respectively). Slot-blot hybridizations revealed in all instances the rapid and transient increase of mRNA levels in response to elicitation. This emphasizes the integral role of the enzyme in disease resistance expression in plants far beyond parsley and also illustrates a new physiological context for the induction of 4-coumarate:CoA ligase.