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 encode 4-coumarate:CoA ligase (4CL), were cloned from a library of Lithospermum erythrorhizon cell suspension cultures by the use of heterologous probe of potato 4CL. These cDNAs are 2.1 kb and 2.2 kb in length, respectively. LE4CL-1 encodes 636 amino acids, whose homologies to the 4CL protein sequences known to potato, parsley, pine and rice, were found to be 68%, 66%, 56% and 50% (identities on amino acid level), respectively, whereas those of the predicted translation product of LE4CL-2 (594 amino acids) to the above 4CL proteins were 49 approximately 54%. The similarity of the deduced amino acid sequences between the two 4CLs from Lithospermum cell cultures was 49% in identity. Northern analyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2 were much higher under illumination than in the dark, as reported for the 4CL genes of such plants as parsley. In comparison of mRNA levels of LE4CL-1 and LE4CL-2, the former was demonstrated to be generally higher than the latter by means of an application of RT-PCR. The genomic southern blot experiments suggested that there are probably three copies of LE4CL-1 in the Lithospermum genome DNA, whereas only one copy was detected for LE4CL-2.