Cloning and sequencing of a full-length cDNA coding for phenylalanine ammonia-lyase from tobacco cell culture.

PAL (EC 4.3.1.5) plays a key role in linking primary metabolism to phenylpropanoid metabolism by catalyzing the deamination of L-Phe to produce trans-cinnamic acid. This reaction is considered a key step in phenylpropanoid metabolism. In bean (Edwards et al., 1985) and parsley (Lois et al., 1989), PAL genes are transcriptionally activated by elicitor treatment, UV light, and wounding. We reported that the addition of kinetin to tobacco (Nicotiana tabacum L. Bright Yellow T-13) cell cultures increased PAL activity (about 2fold) and resulted in high-leve1 accumulations of scopoletin and scopolin (Hino et al., 1982). The increase in PAL activity was shown to result from a stimulation of de novo synthesis of the PAL enzyme, based on immunochemical studies (Nagai et al., 1988). However, the regulatory mechanism is not clearly understood. To clarify the mechanism of this kinetin-mediated PAL induction, we have cloned a fulllength PAL cDNA that corresponds to the PAL induced by kinetin . Total cellular RNA was isolated from kinetin-treated cells (final concentration of 1 pg kinetin/mL) by a modification of the method of Chirgwin et al. (1979). Poly(A)+ RNA was isolated by adsorption and elution from an oligo(dT)-cellulose column and used for the construction of the cDNA library. Double-stranded cDNAs were synthesized and cloned into the EcoRI site of Xgtll. The cDNA library was screened with a 32P-labeled PAL cDNA isolated from bean (a 1520-bp open reading frame and a 223-bp 3‘ noncoding region) (Edwards et al., 1985). In this paper, we present the sequence of the full-length cDNA encoding tobacco PAL. It contains a 2136-bp open reading frame capable of coding for a polypeptide with 712 amino acids, a 204-bp 5’ noncoding region, and a 206-bp 3’ noncoding region containing a 19-bp poly(A) (Table I). The molecular mass of the polypeptide was calculated to be 77,400 D from the deduced amino acid sequence. We compared the nucleotide sequence of the coding region and the deduced amino acid sequence of tobacco PAL cDNA with the partial sequences of PAL cDNA from the other plants. The homology between the nucleotide sequences was 74 to 83% for potato (Joos and Hahlbrock, 1992), tomato (Lee et al., 1992), sweet potato (Tanaka et al., 1989), parsley (Lois et al., 1989), poplar (Subramaniam et al., 1993), soybean