cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme

Abstract

Four caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) cDNA clones were isolated from RNA extracted from TMV-infected tobacco leaves using an heterologous DNA probe. The cDNAs were 84-93% identical in their nucleotide sequences, indicating that they are the products of four closely related genes. A comparison of the CCoAOMT cDNAs with database sequences and Southern blot analysis indicated that they are encoded by a new CCoAOMT family of tobacco. Overall expression of this gene family in tobacco tissues was investigated by RNA blot analysis. The expression of each individual gene was studied by RT-PCR coupled with RFLP analysis of PCR products, taking advantage of the presence of specific restriction sites in each cloned cDNA. Two members of the CCoAOMT gene family appeared to be constitutively expressed in various plant organs and tissues whereas the two others were preferentially expressed in flower organs, after tobacco mosaic virus (TMV) infection or elicitor treatment of leaves. The CCoAOMT enzymatic protein expressed in bacteria was purified and shown to be specific for the caffeoyl-CoA and 5-hydroxyferuloyl-CoA esters and to have no activity against free caffeic acid and 5-hydroxyferulic acid. The pattern of CCoAOMT transcript accumulation during development of tobacco stem was found closely related to that of COMT I genes which have been shown to be specifically involved in lignin biosynthesis. Moreover, the inhibition of COMT I gene expression in transgenic tobacco was also shown to decrease CCoAOMT gene expression, particularly in the most lignified tissues. Thus, the expression pattern and the substrate specificity of tobacco CCoAOMT sustain a preferential role in lignin biosynthesis.