Phenylalanine Ammonia-lyase Enzyme Activity Research Articles

Phenylalanine ammonia lyase mRNA accumulation, enzyme activity and cytoplasmic responses in barley isolines, differing at Ml-a and Ml-o loci, attacked by Erysiphe graminis f.sp. hordei

Seedling leaves of two pairs of near-isogenic barley lines were inoculated with conidia of the powdery mildew fungus, Erysiphe graminis D.C. f.sp. hordei Marchal, race 3. One set of isolines (RISO 5678-R and RISO 5678-S) differed at the Ml-o locus where the recessive allele ( ml-o ) confers a high degree of race non-specific, penetration-based and papilla-associated resistance to E. graminis , while the dominant allele ( Ml-o ) allows a proportion of attacking fungal germlings to succeed in infecting. The second isoline set (Algerian-R and Algerian-S) differed at the Ml-a locus where the dominant allele confers race-specific, epidermal cell death resistance visible only by light microscopy. The recessive allele ( ml-a ) allows a proportion of attacking fungal germlings to succeed in infecting. Leaf samples were taken at 0, 2, 4, 6, 8, 10, 12, 15, 18, 21 and 24 h after inoculation to examine the timing of host epidermal cell cytoplasmic aggregate responses (visible by light microscopy) relative to phenylalanine ammonia-lyase (PAL) mRNA transcript accumulation (determined by quantitative northern blots), and PAL enzyme activity (using radiolabellcd phenylalanine). Erysiphe graminis produced primary germ-tubes (PGTs) within 2 h and appressorial germ-tubes within 6–10 h of inoculation. In all isolines, host epidermal cell cytoplasmic aggregates formed and subsequently dispersed beneath PGTs, between 2 and 10 h, and beneath appressoria between 6 and 15 h. Concurrently, biphasic patterns of PAL transcript accumulation, typified by peaks at 4 and 12 h occurred in all isolines. Temporal patterns of PAL enzyme activity were roughly similar to those of PAL transcript accumulation. Fungal germ-tube contact initiated host epidermal cell cytological responses common to all isolines, induced PAL transcript accumulation, and increased PAL activity regardless of the Mendelian inheritance of “major gene resistance factors” in the barley isoline sets. Thus, there was PAL induction associated with a general defence to infection, but no unusually strong correlation between PAL induction and major gene resistance was found.

A transient increase of phenylalanine ammonia-lyase transcript in kinetin-treated tobacco callus.

In tobacco cell culture (Nicotiana tabacum L. "Bright Yellow" T-13), phenylalanine ammonia-lyase (PAL) activity was induced in response to an exogenously added kinetin. RNA blot hybridization analysis showed that a single species of PAL transcript 2.9-kb in size was detected using the PAL cDNA cloned from kinetin-treated cells. The cellular content of this transcript increased transiently (2.5-fold) 4 h after the addition of kinetin followed by an increase in PAL enzyme activity at 16 h.

Stress Responses in Alfalfa (Medicago sativa L.) (XIV. Changes in the Levels of Phenylpropanoid Pathway Intermediates in Relation to Regulation of L-Phenylalanine Ammonia-Lyase in Elicitor-Treated Cell-Suspension Cultures).

We have used high-resolution gas chromatography to determine the levels of trans-cinnamic acid (CA) and trans-4-coumaric acid (4CA) in alfalfa (Medicago sativa L.) cell-suspension cultures to address the role of these phenylpropanoid pathway intermediates as potential negative regulators of phenylalanine ammonia-lyase (PAL) in vivo. Exogenous addition of CA to elicitor-treated cultures resulted in rapid increases in endogenous CA, 4CA, and CA-conjugate levels associated with inhibition of the appearance of PAL transcripts. Treatment of elicited cultures with [alpha]-aminooxy-[beta]-phenylpropionic acid (AOPP), a potent and specific inhibitor of PAL activity in vivo, resulted in reductions of CA and 4CA, with concomitant increases in PAL transcripts and extractable enzyme activity. In contrast, treatment with tetcyclacis, an inhibitor of CA 4-hydroxylase, resulted in increased CA and CA-conjugate levels, decreased 4CA levels, and decreased PAL transcript levels and enzyme activity. In tetcyclasis-treated cells, the inhibition of PAL transcript appearance preceded the increase in the levels of free CA and its conjugates. In elicited cells in which the phenylpropanoid pathway was not perturbed by metabolic inhibitors, PAL transcripts accumulated rapidly and transiently, beginning to decline by 2 h postelicitation. Changes in levels of total free or conjugated CA or 4CA did not consistently correlate with these changes in transcript levels. We propose that regulation of PAL transcript levels by endogenous phenylpropanoid pathway intermediates could involve compartmentalized pools that may exist because of the microsomal localization of cinnamic acid 4-hydroxylase.

Activity of Enzymes Involved in Capsaicin Biosynthesis in Callus Tissue and Fruits of Chili Pepper ( Capsicum annuum L.)

In order to examine the functionality of the capsaicinoid biosynthetic pathway in callus cultures of chili pepper (Capsicum annuum L.), we investigated the enzyme activity of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (CA4H), p-coumaric acid-3-hydroxylase (CA3H), caffeic acid-O-methyltransferase (CAOMT) and capsaicinoid synthetase (CS). These enzymes were also determined in developing and mature fruits. The maximum activities of PAL, CA4H and CA3H in callus cultures were similar to those of chili pepper fruits, but the values of CAOMT and CS were 6 times lower than those of fruits. The significance of these data in relation to capsaicin accumulation is discussed.

Inhibition of Phytoalexin Biosynthesis in Elicitor-Treated Tobacco Cell-Suspension Cultures by Calcium/Calmodulin Antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [(35)S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.

Vanadate mimics effects of fungal cell wall in eliciting gene activation in plant cell cultures

Cell-suspension cultures of peanut (Arachis hypogaea L.) can be used as a very sensitive and rapidly responding physiological system for monitoring extracellular signals. Elicitors effect the activation of the genes that code for a set of enzymes synthesizing stilbenes. Within 2-6 h after administering micromolar, concentrations of orthovanadate to the suspended cells, the enzyme activities of phenylalanine ammonia-lyase, stilbene synthase, and cinnamate 4-hydroxylase increased 10-to 100-fold. The transient time course of induction, and the quality and quantity of gene expression found with vanadate as artificial elicitor were very similar to those observed after biotic stress generated by fungal cell walls. The dose-response of vanadate as an elicitor of gene expression in intact cells matched precisely its inhibitory effect on the ATPase activity of isolated plasma membrane. By concentrating, on the profiles of cinnamate 4-hydroxylase activity, we observed differences between the effects elicited by fungal cell wall or vanadate when different stages of cell development were analyzed. Unlike the fungal elicitor, vanadate did not induce the hydroxylase activity when cells at the stationary phase of the cell cycle were used. This lack of response was not the result of a decrease in membrane biosynthesis. The finding, that the effects of vanadate and fungal elicitor are additive indicates that vanadate does not interfere negatively with the perception of the biotic signal but rather addresses the same intracellular intermediate of the signalling process. We hypothesize that membrane potentials created or modulated by ATPases may be intermediates in the signal chain, starting with the recognition process at the plasma membrane and eventually leading to the production of stilbenes as low-molecular-weight plant-defence products.

Lignin biosynthetic enzymes in stem rust infected, resistant and susceptible near-isogenic wheat lines

Near-isogenic lines of the wheat cultivar Prelude, carrying either the Sr5 gene for resistance or the sr5 allele for susceptibility were inoculated with the stem rust fungus Puccinia graminis f. sp. tritici race 32, which possesses the P5 gene for avirulence. The time-course of the enzyme activities of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), as well as the enzyme activities of the specific pathway leading to the biosynthesis of lignin, cinnamyl-alcohol dehydrogenase (CAD) and peroxidase (PO), were determined. The resistant and the susceptible isoline both exhibit a first maximum in the coordinately regulated enzyme activities of PAL and 4CL at a time when the fungus is still growing on the surface of the leaves. This maximum is followed by a decrease to the levels of uninoculated controls. In the resistant isoline, a second increase is observed at the time of the hypersensitive resistant reaction. In contrast, enzyme activities in susceptible plants continue to decline, even falling below control levels until the onset of sporulation when a second, late increase can be detected. The observed changes in CAD and PO activities are consistent with a feed-forward regulation by products of the general phenylpropanoid pathway. However, no late increase in PO activity can be observed in the compatible interaction. An interpretation of these results is proposed in terms of non-host resistance, basic compatibility and race-cultivar specific resistance.