Kinetin-induced caffeic acid O-methyltransferases in cell suspension cultures of Vanilla planifolia Andr. and isolation of caffeic acid O-methyltransferase cDNAs

Abstract

Two different S-adenosyl-L-methionine:caffeic acid methyltransferases are induced in kinetin-treated cell cultures of Vanilla planifolia. Upon addition of kinetin to the culture, maximum caffeic acid 4-O-methyltransferase and caffeic acid 3-O-methyltransferase activity is obtained after around 2 and 40 h, respectively. The former enzyme is involved in the biosynthesis of vanillic acid and the latter enzyme is co-induced with other enzymes of the general phenylpropanoid pathway and is involved in the biosynthesis of lignin precursors. A number of cDNA clones encoding S-adenosyl-L-methionine:caffeic acid-methyltransferases were isolated by heterologous probe screening of a λZapII cDNA library constructed from mRNA of a kinetin-treated cell suspension culture of V. planifolia. A full-length cDNA clone, Vpomt35, contains a 1 089-bp open reading frame coding for 363 amino acid residues, a 25-bp 5′-end sequence and a 214-bp 3′-end non-coding sequence. The deduced amino acid sequence of Vpomt35 revealed 56 to 80 % sequence identity when compared to those of other plant caffeic acid O-methyltransferases. Detection of methyltransferase activity in extracts of Escherichia coli XL-1 Blue cells transformed with a pBluescript phagemid construct containing Vpomt35 confirmed that this cDNA clone encodes a caffeic acid 3-O-methyltransferase. The enzyme also exhibits a low caffeic acid 4-O-methyltransferase activity (2 % of the caffeic acid 3-O-methyltransferase activity). A number of other cDNA clones encoding caffeic acid O-methyltransferases was also sequenced. Comparison of nucleotide and deduced amino acid sequences of these cDNAs indicated two types of caffeic acid O-methyltransferase cDNAs which showed 94 % sequence identity in the coding region, but only 55 % homology with several gaps in the 3′ untranslated end. Results from Southern blot analysis suggest that the caffeic acid O-methyltransferase gene is organized as a small family with at least two genes, which are expressed in Vanilla cell suspension cultures.