Cloning and sequence of a cDNA encoding phenylalanine ammonia-lyase from the tropical forage legume Stylosanthes humilis.

PAL (EC 4.3.1.5) catalyzes the deamination of Phe to trans-cinnamic acid in the first step of the phenylpropanoid pathway, a key process for the synthesis of phenolic compounds in plants. In legumes, phenolic compounds have many important roles including flavonoid signal molecules required for nodulation, antibiotic phytoalexins for defense against pathogens, and precursors for the structural and defensive cell wall polymer lignin. In forages, there is a negative correlation between lignin content and digestibility, and this is particularly exacerbated in tropical forages, which generally have higher lignin contents (Minson, 1990). It has been demonstrated in transgenic tobacco that reduction of PAL activity achieved via co-suppression of PAL gene expression leads to a reduction in lignin content (Bate et al., 1994), but as yet this type of experiment has not been undertaken in a forage plant in which effects on digestibility can be tested. We are interested in manipulating lignin content and digestibility in tropical forages using antisense and ribozyme strategies in transgenic plants (McIntyre et al., 1993). In this work we have chosen to study Stylosanthes humilis Kunth as a model tropical pasture legume (McIntyre et al., 1995) because of its welldeveloped transformation system (Manners and Way, 1989) and its inbreeding and diploid genetics. As a starting point in this project we have cloned and sequenced a full-length cDNA encoding PAL from S. humilis. A cDNA library of S. humilis (McIntyre et al., 1995) was screened under low-stringency hybridization conditions with a partia1 cDNA of PAL isolated from French bean (Edwards et al., 1985). The longest hybridizing cDNA clone was subcloned into pBluescript SK+ and sequenced. The cDNA comprises a 2443-bp insert with a major reading frame of 2145 bp encoding 715 amino acids. The amino acid sequence of the deduced protein was confirmed as a PAL homolog by comparison with other complete PAL amino acid sequences. Homologies ranging between 79 and 87% were obtained with PAL amino acid sequences from sweet potato, alfalfa, pea, and tomato (Table I). Southern hybridization analysis with genomic DNA of S. humilis indicated that two or three