| Phenylalanine ammonia lyase (PAL) (EC 4.3.1.5) is a key branchpoint enzyme leading to the phenylpropanoid metabolic pathways in plants. It plays a role in such physiological disorders as russet spotting in lettuce, as well as plant pathogen interactions, including with such unusual plant pathogens as Agrobacterium tumefaciens. In tomato (Lycopersicon esculentum, Mill.), PAL is encoded by a seven to twelve gene family. One of these genes, designated LesPAL1, was cloned from a genomic library and characterized. Its sequence showed strong homology (ca. 77% identity) with the structural regions of other plant pal genes, including identical placement of the intron. Approximately 35% identity was observed with pal clones from yeast, and about 20% identity with the histidine ammonia lyase from bacteria and mammals. Comparison of the predicted amino acid sequence of 13 pal genes and 3 hal genes revealed a putative active site (190-197) and substrate binding domain (383-481) for these enzymes. One striking difference is the presence of a UAA stop in LesPAL1, rather than a CAA codon, found in other pal genes, at amino acid position 469. The sequence of the mRNA revealed a consensus CAA at this position, however, indicative of RNA editing which may be involved in regulation of the 53 kD and 77 kD isozymes of PAL monomer. The LUX reporter system was evaluated for use in fusion constructs, but abandoned due to insufficient reducing potential in planta. The promoter of the tomato pal gene was subcloned in front of a gus reporter and proved to be active in transient expression assays and developmentally regulated in stable transgenic tobacco. This pal gene was also cloned from a tomato cDNA library of fruit, but not root tissue, suggesting differential regulation. The sequence of the promoter region contains 7 boxes of homology to known regulatory elements which have been identified in other plant defense response genes, but it was not induced in response to fungal elicitor. A putative regulatory element with the sequence GGnAGCCATTT was found in the protein coding region and appears to be required for pathogen induction. |
