Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)