Carole L. Cramer

Phenylalanine ammonia-lyase gene organization and structure

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) genomic sequences were isolated from bean (Phaseolus vulgaris L.) genomic libraries using elicitor-induced bean PAL cDNA sequences as a probe. Southern blot hybridization of genomic DNA fragments revealed three divergent classes of PAL genes in the bean genome. Polymorphic forms were observed within each class. The nucleotide sequences of two PAL genes, gPAL2 (class II) and gPAL3 (class III), were determined. gPAL2 contains an open reading frame encoding a polypeptide of 712 amino acids, interrupted by a 1720 bp intron in the codon for amino acid 130. gPAL3 encodes a polypeptide of 710 amino acids showing 72% similarity with that encoded by gPAL2, and contains a 447 bp intron at the same location. At the nucleotide level, gPAL2 and gPAL3 show 59% sequence similarity in exon I, 74% similarity in exon II, and extensive sequence divergence in the intron, 5′ and 3′ flanking regions. S1 nuclease protection identified transcription start sites of gPAL2 and gPAL3 respectively 99 bp and 35 bp upstream from the initiation codon ATG, and showed that gPAL2 but not gPAL3 was activated by elicitor, whereas both were activated by wounding of hypocotyls. The 5′ flanking region of both genes contain TATA and CAAT boxes, and sequences resembling the SV40 enhancer core. gPAL2 contains a 40 bp palindromic sequence and a 22 bp motif that are also found at similar positions relative to the TATA box in 5′ flanking regions of other elicitor-induced bean genes.

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid.

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [35S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzymes active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of35S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzymes active site.

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: partial degradation of enzyme subunits in vitro and in vivo

Abstract L -Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from suspension cultured cells of French bean ( Phaseolus vulgaris L.) which had been exposed to polysaccharide elicitor preparations from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum . After preliminary purification by ammonium sulphate fractionation and gel filtration, the enzyme was further purified by (a) ion-exchange chromatography followed by chromatofocussing, (b) chromatography on rabbit anti-(phenylalanine ammonia-lyase) IgG, or (c) affinity chromatography on L -aminooxy( p -hydroxyphenyl)propionic acid (or L -tyrosine) linked to epoxy-activated Sepharose 6B via the phenolic hydroxyl group. The purified enzyme preparations exhibited subunit M r values of 77 000, 70 000 and 53 000, the relative proportions of these depending upon the enzyme source, length of time taken for purification, and inclusion of freeze-thaw steps. Four forms of the enzyme, differing in p I value, were resolved by chromatofocussing, although all forms from the same preparation consisted of similar proportions of the different subunit M r forms. Peptide mapping and freeze-thaw studies indicate that the M r 77 000 native phenylalanine ammonia-lyase subunit is inherently unstable in vitro and breaks down to yield the lower M r partial degradation products. Such products could also be observed following in vitro translation of phenylalanine ammonia-lyase mRNA. Pulse-chase experiments indicated that the 77 000 → 70 000 → 53 000 subunit interconversion also occurs in vivo.

Organization, Structure and Activation of Plant Defence Genes

Plants exhibit natural resistance to disease which has been exploited by breeders to reduce crop losses and hence increase yield. Disease resistance involves not only static protection, but also inducible defence mechanisms including: (i) accumulation of host-synthesized phytoalexins; (ii) deposition of lignin-like material; (iii) accumulation of hydroxyproline-rich glycoproteins and (iv) increases in the activity of certain hydrolytic enzymes such as chitinase and glucanase [1]. Although the genetics, physiology and cytology of plant:pathogen interactions have been extensively studied, until recently relatively little was known at the biochemical level about how plants respond to infection to activate these defence responses.

Accumulation of Hydroxyproline-Rich Glycoprotein mRNAs in Biologically Stressed Cell Cultures and Hypocotyls

Hydroxyproline-rich glycoproteins (HRGPs) are structural components of plant cell walls and may also function in the processes of plant development, growth, and disease resistance [reviewed in 1,2]. In higher plants, these cell wall HRGPs consist of 35–45% hydroxyproline (Hyp) and are also relatively rich in serine, valine, tyrosine and lysine. A repeating pentapeptide sequence, Ser-(Hyp)4, further characterizes these unusual glycoproteins which are often referred to as “extensins”. Most of the hydroxyproline residues are O-glycosidically attached to short oligoarabinosides, while some of the serine residues are O-glycosidically linked to galactose.