Tobacco possesses two distinct classes of O-methyltransferases (OMTs; S-adenosyl-L-methionine:o-diphenol O-methyltransferases; EC 2.1.1.6). Here we report on the cloning and the expression pattern of the class I OMT that is specifically involved in lignin biosynthesis. Near-full-length cDNAs have been isolated from tobacco libraries constructed from leaf and stem poly(A)+ RNA. Sequence analysis demonstrated that the OMT I clones derived from two different mRNA species. The two types of OMT I mRNA were reverse transcribed from total RNA and the cDNAs were amplified by polymerase chain reaction and characterized by restriction analysis. The same proportion of the two transcripts was measured in stem tissue of healthy plants and in leaves reacting hypersensitively to tobacco mosaic virus, indicating a coordinate expression of the two OMT I genes. Consistently, genomic hybridization indicated the presence of two OMT I genes in the amphidiploid genome of tobacco. The pattern of expression of OMT I genes was studied by in situ mRNA hybridization. In stem, petiole, and root tissues, OMT I genes were found to be specifically expressed in vascular cells and epidermis. In healthy leaves OMT I mRNA was only detected in vascular strands, whereas, in leaves bearing tobacco mosaic virus-induced necrotic lesions, a particularly strong accumulation of the labeling was also localized in the upper and lower epidermis.

The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.