Stress Responses in Alfalfa (Medicago sativa L.) 11. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures

L-Phenylalanine ammonia-lyase has been purified from elicitortreated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of Mr 311,000; the intact subunit Mr was about 79,000, although polypeptides of Mr 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different Km values for L-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction. L-Phenylalanine ammonia-lyase (PAL2, EC 4.3.1.5) catalyzes the first committed step in the biosynthesis of a range of phenylpropanoid secondary compounds from L-phenylalanine (reviewed in refs. 8 and 13). In legumes such as bean, soybean, and pea, rapid increases in PAL activity precede the accumulation of antimicrobial isoflavonoid phytoalexins in intact tissues or cell suspension cultures in response to fungal infection or treatment with microbial elicitors (7). In bean, increased PAL activity in response to elicitor has been shown to result from rapid transcriptional activation of PAL genes (19). Bean PAL is encoded by a small family of three genes (4), which are differentially activated in response to fungal infec'Permanent address: Department of Biochemistry and Molecular Biology, E.T.S. de Ingenieros Agronomos, University of Cordoba, Cordoba, Spain. J.J. was recipient of a NATO Research Fellowship. 2 Abbreviations: PAL, L-phenylalanine ammonia-lyase (EC 4.3.1.5); IEF, isoelectric focusing; pI, isoelectric point. tion, environmental conditions, and developmental cues (20). At the polypeptide level this is reflected by extensive subunit polymorphism and variation in the corresponding isoforms of the active PAL tetramer (1). Elicitation results in the specific accumulation of PAL isoforms with a low Km for the substrate L-phenylalanine (1). In other systems, however, PAL may exist in a single form (3, 11, 14), and the regulation of phenylpropanoid biosynthesis in these cases may be correspondingly less complex and flexible. We have recently shown that suspension cultured alfalfa cells accumulate the isoflavonoid phytoalexin medicarpin and related compounds on exposure to elicitor released from autoclaved cell walls of the bean pathogen Colletotrichum lindemuthianum (5). Phytoalexin accumulation results from rapid and extensive increases in the extractable activities of PAL, cinnamic acid 4-hydroxylase, 4-coumarate: CoA ligase, chalcone synthase, chalcone isomerase, and isoflavone 0methyl transferase. The system is an attractive one for studying defense gene activation because, unlike bean, alfalfa is amenable to genetic transformation (27). The rate of increase in PAL activity has also recently been correlated with resistance of alfalfa callus lines to infection with the vascular wilt fungus Verticillium albo-atrum (17). In an attempt to define better the biochemical basis of induced defense in alfalfa, we here report on the complexity of PAL at the enzyme level and confirm the de novo synthesis of the enzyme in response to elicitor. MATERIALS AND METHODS