Cloning of cytochrome P450 cDNAs from cultured Glycyrrhiza echinata L cells and their transcriptional activation by elicitor-treatment

From a cDNA library of cultured Glycyrrhiza echinata L. (Fabaceae) cells treated with yeast extract (YE) elicitor, eight cytochrome P450 (P450) fragments (Ge-1 to 8) were isolated using the PCR method based on conserved sequences of P450s. Comparison of amino acid sequences revealed that two of the fragments (Ge-1 and 2) belong to the CYP73 family encoding trans-cinnamic acid 4-hydroxylase. Ge-4 had 63% identity with the sequence included in CYP93, which has recently been reported to be induced by methyl jasmonate in soybean cells (Suzuki et al., FEES Lett., 383 (1996) 83-86), and Ge-6 and 7 were highly homologous to the partial sequence of CYP84 encoding ferulic acid 5-hydroxylase of Arabidopsis thaliana (Meyer et al., Proc. Natl. Acad. Sci. USA: 93 (1996) 6869-6874). Others (Ge-3, 5 and 8) displayed homology less than 43% with known plant P450s. A full-length cDNA (CYP73A14) containing Ge-l sequence was isolated from the cDNA library, and its amino acid sequence showed 91-93% identity with CYP73 of other leguminous plant species. Northern blot analysis of mRNAs of G. echinata cells indicated that CYP73 (Ge-l and 2) genes were transcribed at the early stages post-elicitation (< 10 h). The transcription level of the Ge-1 gene was much higher than that of Ge-2. Transcription of Ge-3, 5, 6 and 7 was also activated by elicitation, but no clear signal from Ge-4 and 8 was observed. When total RNAs of cultured cells of Medicago sativa were analyzed by Northern blotting, transcripts hybridized with G. echinata cDNA clones containing Ge-1, 3, 5 and 6 sequences could be detected. Possible involvement of cloned P450s in elicitor-induced phenylpropanoid/flavonoid biosynthesis is discussed. (C) 1997 Elsevier Science Ireland Ltd.