Extractable Phenylalanine Ammonia-lyase Research Articles

The mycoherbicide Atlernaria cassiae infects and alters phenolic metabolism of Cassia alata seedlings

The mycoherbicide Alternaria cassiae, a pathogen of the weed sicklepod (Cassia obtusifolia), was found to infect a previously untested Cassia species, Cassia alata (ringworm bush or seven golden candlesticks). When 2-wk-old, growth chamber-grown C. alata seedlings were inoculated with 105 spores mL-1 followed by a 12-h dew period, infection was apparent 16-20 h after treatment. Infection severity was proportional to spore concentration, and 106 spores mL-1 caused complete mortality 4-5 d after treatment. Plant age was also a factor in disease severity and mycoherbicidal efficacy. At 106 spores mL-1, 95% of 1- to 3-wk-old seedlings were killed (with severe damage to others); 3- to 4-wk-old plants had multiple lesions, growth reduction, and some leaf abscission; and 5- to 7-wk-old plants were infected with many lesions, but they outgrew these effects. Extractable phenylalanine ammonialyase in treated C. alata seedlings was increased three-fold above that of untreated seedlings 17-96 h after spore application. Soluble hydroxyphenolic levels were also increased in the A. cassiae-treated seedlings, but not until 48-96 h after treatment. Results indicate an expanded host range for this plant pathogen and suggest that phenolic metabolism is increased in défense of pathogen invasion.

Chemical Interactions with Bioherbicides to Improve Efficacy

Bioherbicides can be defined as plant pathogens, phytotoxins derived from pathogens or other microorganisms, augmentatively applied to control weeds. Although many pathogens with bioherbicidal potential have been discovered, most lack sufficient aggressiveness to overcome weed defenses to achieve adequate control. Plants use various physical and biochemical mechanisms to defend against pathogen infectivity, including callose deposition, hydroxyproline-rich glycoprotein accumulation, pathogenesis-related proteins (PR-proteins), phytoalexin production, lignin and phenolic formation, and free radical generation. Some herbicides, plant growth regulators, specific enzyme inhibitors, and other chemicals can alter these defenses. Various pathogens also produce chemical suppressors of plant defenses. Secondary plant metabolism is a major biochemical pathway related to several defense processes. Increased activity of a key enzyme of this pathway, phenylalanine ammonia-lyase (PAL), is often a response to pathogen attack, as demonstrated in two weeds and their associated bioherbicidal pathogens:Alternaria cassiaeon sicklepod andA. crassaon jimsonweed. Weakening of physical and biochemical defenses, and lowering of resistance to pathogen attack, may result from reduced production of phenolics, lignin, and phytoalexins caused by herbicides and other chemicals that affect cuticular component biosynthesis and/or key aspects of secondary plant metabolism. Potent PAL inhibitors [aminooxyacetic acid, α-aminooxy-β-phenylpropionic acid, and (l-amino-2-phenylethyl)phosphonic acid] have some regulatory action on secondary plant metabolism and pathogenicity. Various herbicides and other chemicals dramatically affect extractable PAL activity levels and/or substantially alter PAL product production. Some non-pathogenic organisms can alter herbicide efficacy, and some herbicides influence disease development in plants. Research has shown some synergistic interactions of microbes and chemicals with relevance to weed control. Further research on pathogen interactions with agrochemicals (or other chemicals/regulators) could result in increased efficacy of pathogen-herbicide combinations, reduction of herbicide and pathogen levels required for weed control, and expanded pathogen host range.

Pathogenic Interactions of Alternaria crassa and Phenolic Metabolism in Jimsonweed (Datura stramonium L.) Varieties

Alternaria crassa, a mycoherbicide for jimsonweed control, was tested for differential effects on growth, infectivity, and phenolic metabolism of two field-collected jimsonweed varieties. Seeds from field-grown red- and green-stemmed jimsonweed varieties were grown in the greenhouse and spores were applied in aqueous solutions. Infection studies showed no significant differences between the two weed varieties when tested at various growth stages from cotyledonary to 8–leaf stage at 2.5 × 104spores ml−1. Spore dose response tests with A. crassa spores (6.25 to 100 x103spores ml−1) showed no significant varietal differences in disease ratings. Extractable phenylalanine ammonia-lyase activity increased nearly equally (2- to 3-fold above control) in both varieties (8- to 9-leaf stage) 48 to 72 h after treatment with 5 × 104spores ml−1. The only significant differences between treated varieties occurred 72 h after treatment. Exposure to A. crassa lowered total ethanol-soluble phenolic levels in both varieties 72 h after treatment. Spectrophotometric scans (400 to 700 nm) of ethanol extracts (72 h after treatment) indicated that infection caused some absorbance increases, especially in red-stemmed jimsonweed. Anthocyanin content was about 11-fold higher in red-stemmed than green-stemmed jimsonweed, and A. crassa treatment slightly increased the anthocyanin absorption peak of the red-stemmed variety.

L-Phenylalanine Ammonia-Lyase from Sunflower Hypocotyls: Modulation by Cinnamic Acids

Summary Trans -cinnamic acid and other metabolites of the phenylpropanoid pathway, namely p -coumaric, caffeic, ferulic and sinapic acids, inhibited the increase in extractable phenylalanine ammonia-lyase (PAL) activity present in sunflower hypocotyls in response to light-sucrose and excision-light-sucrose treatments. Otherwise, in vitro inhibitors of PAL activity, such as D-phenylalanine, and 2-aminooxyacetic and phenylpropiolic acids, superinduced extractable PAL activity and prevented the enzyme activity decay typically observed in the light-sucrose induction system. In both inducing treatments, delayed transfer of hypocotyl tissue to t -cinnamic acid caused a rapid decline in PAL activity, while a superinduction effect was observed upon transfer of the tissue to phenylpropiolic acid. These results indicate that in sunflower hypocotyls cinnamic acid(s) could act as in vivo modulator(s) of PAL, affecting the mechanisms responsible for both the increase and decrease in enzyme activity.

Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.

Interaction of indoleacetic acid and glyphosate on phenolic metabolism in soybeans

This study examined indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) for effects and possible interactions with glyphosate on growth, extractable phenylalanine ammonialyase (PAL, EC 4.3.1.5) activity, soluble hydroxyphenolic compound levels, and anthocyanin accumulation in intact plants. Root feeding of soybean [ Glycine max (L.) Merr.] seedlings in liquid culture with IAA (10 μ M) and 2,4-D (2 μ M) in combination with glyphosate (0.5 m M) in the light (200 μE · m −2 · sec −1, PAR) did not reverse glyphosate's reduction of elongation or fresh weight. IAA plus glyphosate caused higher levels of hydroxyphenolics (per axis) than did glyphosate alone under light or dark growth. Glyphosate plus 2,4-D in the dark resulted in a phenolic content lower than that of glyphosate alone. Glyphosate's reduction of anthocyanin accumulation in hypocotyls was only marginally reversed by IAA (ca. 15%) and by 2,4-D (ca. 8%). IAA had little effect on extractable PAL in the light or dark, but 2,4-D was a potent inhibitor. Glyphosate increased extractable PAL activity in light- and dark-grown plants and IAA (in light) and 2,4-D (in light and dark) were antagonistic. Alterations of PAL activity, anthocyanin, and phenolic content caused by IAA and 2,4-D were not commensurate with growth reductions, suggesting that IAA (and the parameters altered by IAA) is not primarily involved in glyphosate's mode of action.

Metabolic fate of cinnamic acid in elicitor treated cell suspension cultures of Phaseolus vulgaris

Exogenously supplied [ 14C]cinnamic acid (1 mM) was rapidly taken up and metabolized by bean ( Phaseolus vulgaris) cell suspension cultures treated with fungal elicitor. Uptake of the cinnamic acid correlated with a reduction in extractable phenylalanine ammonia-lyase activity and was accompanied by the metabolism of the acid by glycosidic conjugation and ring-hydroxylation. Formation of soluble metabolises was followed by the accumulation of wall-bound radioactivity in the cellulosic and hemicellulosic fractions in the form of esterified cinnamates. A cytosolic uridine diphosphate glucose (UDPG): cinnamic acid glucosyl transferase with a K m toward cinnamic acid of 400, μM was identified in suspension cultures of both bean and alfalfa ( Medicago sativa). This enzyme was induced by fungal elicitor in both species and by cinnamic acid in bean. The possible function of the glucosyl transferase is discussed.

Acifluorfen Action on Growth and Phenolic Metabolism in Soybean (Glycine max) Seedlings

Three-day-old soybean seedlings were treated with acifluorfen via liquid culture [50 μM in dark, 1 μM in light (200 μE·m–2·s–1)]. Root elongation in the dark was inhibited only slightly (6%) after 96 h. In the light, acifluorfen inhibited root elongation after 48 h; after 96 h, inhibition was 32%. Hypocotyl length was not affected in either the light or dark. Soluble hydroxyphenolic content per axis was unaffected in the dark but was reduced by about 13 to 25% at 24 to 96 h in the light. Extractable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity per axis was unaltered by herbicide in the dark. A brief transient increase in PAL activity (4 to 24 h) in axes of light-grown seedlings in the presence of acifluorfen was followed by reduced enzyme levels compared to light-grown controls at 72 and 96 h. Although light is required for maximal herbicide action, there was no effect on anthocyanin or chlorophyll accumulation or on the chlorophyll a/b ratio in hypocotyls or light-grown seedlings.

A single glycoprotein from Puccinia graminis f. sp. tritici cell walls elicits the hypersensitive lignification response in wheat

Con A-binding glycoproteins were detected in germ tube walls of Puccinia graminis f. sp. tritici and appeared to be potent inducers of the hypersensitive lignification response. This response, which is typical of the large resistance reaction in wheat leaves, was preceded by a increase in extractable phenylalanine ammonia-lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway. Germ tube glycoproteins were isolated from homogenized fungal cell walls by differential centrifugation and affinity chromatography on a Con A-Sepharose column. The most active glycoprotein was purified on a Mono Q anion-exchange column. The relative molecular mass of the molecule was determined in SDS-polyacrylamide gels to be 67 kD. The carbohydrate portion consists mainly of mannose (50%) and galactose (47%) and contains the active part of the glycoprotein as demonstrated by the inability of pronase and trypsin digestion to influence activity. An elicitor-active glycoprotein with identical molecular mass and Con A-binding properties was isolated from intercellular fluids of rust-infected plants of the susceptible wheat cultivar Little Club indicating that the elicitor is released from fungal cell walls during infection. We discuss the role of Con A-binding proteins as inducers of non-host resistance in cereals.

Glutathione Causes a Massive and Selective Induction of Plant Defense Genes

The reduced form of glutathione (GSH), when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 to 1.0 millimolar, stimulates transcription of defense genes including those that encode cell wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) involved in lignin (PAL) and phytoalexin (PAL, CHS) production. Transcriptional activation of these genes leads to marked accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles that previously observed in response to fungal elicitor. GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, constituent amino acids, or other reducing agents are inactive. Possible roles of GSH in signaling biological stress are discussed.

Modification of the phosphite induced resistance response in leaves of cowpea infected with Phytophthora cryptogea by α-aminooxyacetate

Treatment of detached cowpea leaves with phosphite, the active breakdown product in plant tissues of fosetyl-Al, leads to the cessation of growth of Phytophthora cryptogea within 24 h of inoculation. Pretreatment of leaves with α-aminooxyacetate (AOA), an inhibitor of the phenylpropanoid pathway, increases the size of lesions in phosphite treated leaves and induces a complete susceptibility at 5 mM. By 24 h after inoculation, phenylalanine ammonia-lyase (PAL) activity is higher in phosphite treated leaves than in untreated leaves. The effects of AOA on PAL activity are paralleled with the effects of the increase of the spread of infection. AOA treatment does not affect phosphite uptake by fungal cells and leaf tissues, while it inhibits kievitone and phaseollidin accumulation in infected treated leaves and causes a reduction in extractable PAL activity. These results suggest that cessation of fungal growth in vivo is not as a result of a direct effect of phosphite on the fungus and strongly support a role of the host defence reactions in the mode of action of phosphite.

Effects of Calcium and Auxin on Russet Spotting and Phenylalanine Ammonialyase Activity in Iceberg Lettuce

Abstract Application of 0.3-0.5 M Ca or 0.1−1.0 mM 2,4-D strongly inhibited russet spotting (RS) in iceberg lettuce (Lactuca sativa L.) leaf tissues and significantly reduced extractable phenylalanine ammonia-lyase (PAL) activity. Since neither Ca nor 2,4-D reduced PAL activity in vitro, they may inhibit RS development by inhibiting in vivo PAL biosynthesis. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4-D); and 1H-indol-3-acetic acid (IAA).

Relationships Between Phenylalanine Ammonia-Lyase Activity and Physiological Responses of Soybean (Glycine max) Seedlings to Herbicides

The effects of 16 herbicides from 14 different chemical classes on levels of soluble protein, hydroxyphenolics, anthocyanin, and chlorophyll were determined in light- and dark-grown soybean [Glycine max(L.) Merr. ‘Hill’] seedlings. Growth-reducing concentrations of the herbicides were supplied to 3-day-old dark-grown soybean seedlings in liquid culture. Soluble protein (per axis) was reduced by fluridone {1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone}, paraquat (1,1′-dimethyl-4,4′-bipyridinium ion), perfluidone {1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl] methanesulfonamide}, and propanil (3′,4′-dichloropropionanilide) 24 or 48 h after treatment. In light-grown plants, soluble hydroxyphenolic compound levels were decreased on a per axis basis after 48 h by all chemical treatments except by atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], the methyl ester of diclofop {2-[4-(2,4-dichlorophenoxy)phenoxy] propanoate}, DSMA (disodium methanearsonate), fluridone, MH (1,2-dihydro-3,6-pyridazinedione), nitralin [4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline], TCA (trichloroacetic acid), and 2,4-D [(2,4-dichlorophenoxy)acetic acid]. Total chlorophyll content in hypocotyls of these seedlings was decreased by fluridone, metribuzin [4-amino-6-tert-butyl-3-(methylthio)-as-triazin-5 (4H)-one], norflurazon [4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3 (2H)-pyridazinone], paraquat, and 2,4-D, but others had no significant effect. Anthocyanin accumulation in hypocotyls of 48-h light-grown seedlings was decreased by atrazine, fenuron (1,1-dimethyl-3-phenylurea), metribuzin, norflurazon, paraquat, propanil, and propham (isopropyl carbanilate). Phenylalanine ammonia-lyase (PAL) activity (previously reported work) was positively correlated with anthocyanin levels in tissues after treatment with these 16 herbicides, but not with glyphosate [N-(phosphonomethyl)glycine]. There was, however, no correlation between extractable PAL activity and chlorophyll, soluble hydroxyphenolic compounds, or soluble protein content. These results indicated that extracted PAL activities usually reflect relative in vivo activities and that PAL activity is limiting to phenylpropanoid synthesis in vivo.

Induction of phenylalanine ammonia‐lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far‐red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA3, 0.1 mM), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis‐4‐cyclohexene‐l, 2‐dicarboximide (CHDC, 1.0 mM) both in light and dark. Both GA3 and benzyladenine (BA, 0.1 mM) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light‐sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.

Diphenyl ether herbicides: Effects of acifluorfen on phenylpropanoid biosynthesis and phenylalanine ammonia-lyase activity in spinach

Spinach leaves treated with acifluorfen synthesize >25-fold the normal level of the phenolic amide ( I) derived from ferulic acid and 3-methoxytyramine. The content of I is increased within 18 hr and at acifluorfen concentrations as low as 0.2 ppm. Biosynthesis experiments with precursor 14C-amino acids establish that the elevated content of I is due to de novo synthesis with the acyl moiety derived from phenylalanine, the amine moiety from tyrosine, and the O-methyl groups from methionine. Elevation of I content is followed by the appearance of necrotic lesions in spinach leaves but is preceded by an increase in extractable phenylalanine ammonia-lyase activity, detectable 9 hr after treatment, reaching 13-fold of normal values at 20 hr, and dropping to near control activity at 48 hr. Phenylalanine ammonia-lyase activity is also elevated by acifluorfen in soybeans and six other crops or weeds. Alterations in aromatic amino acid metabolism and phenylpropanoid biosynthesis involving secondary phenolic compounds may contribute to the phytotoxic effects of diphenyl ether herbicides.

Interference of L-?-aminoocy-?-phenylpropionic acid with cold-induced sphagnorubin synthesis in Sphagnum magellanicum BRID

The ability of the phenylalanine ammonia-lyase (PAL)-inhibitor L-α-aminooxy-β-phenyl-propionic acid (AOPP) to suppress the synthesis of the main reddish-violet wall pigment of Sphagnum magellanicum (sphagnorubin) was investigated. Fifty percent inhibition is achieved with 14 μM AOPP in mosses stimulated to intensive coloring by sugar feeding. AOPP does not affect the content of free amino acids, except for phenylalanine, during cold-induced sphagnorubin synthesis. AOPP dramatically amplifies the increase in extractable PAL activity in response to cold treatment. Phenylalanine applied in vivo causes an eminent increase in PAL activity, above the level of the cold-treated mosses. The results from the feeding experiments are discussed in connection with a possible end-product repression in PAL activity with sphagnorubin-synthesizing mosses. These results are correspond best to the theory that the enzyme level is regulated independently from a mechanism of feedback repression.

Effects of DPX-4189 (2-chloro-N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl)benzenesulfonamide) on anthocyanin synthesis, phenylalanine ammonia lyase activity, and ethylene production in soybean hypocotyls

The effects of the herbicide DPX-4189 (2-chloro-N-((4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl)benzenesulfonamide) on anthocyanin accumulation, phenylalanine ammonia lyase (PAL) activity, and ethylene production in seedlings of soybean (Glycine max L.) were investigated. Application of 1 μg DPX-4189 per plant led to an increase in anthocyanin content in soybean hypocotyls. The increase in anthocyanin content became evident 4 days after application of the herbicide. Accompanying the increase in anthocyanin content was an eightfold increase in extractable PAL activity. An increase in endogenous ethylene evolution also accompanied the increase in anthocyanin content. Application of silver nitrate (an inhibitor of ethylene action) to herbicide-treated seedlings did not prevent the increase in anthocyanin content. Application of 2-chloroethylphosphonic acid (an ethylene-releasing compound) to soybean seedlings stimulated PAL activity but had no effect on anthocyanin content. These results indicated that ethylene did not play a role in DPX-4189 mediated anthocyanin accumulation.

Effects of Herbicides on Growth and Extractable Phenylalanine Ammonia-Lyase Activity in Light- and Dark-Grown Soybean (Glycine max) Seedlings

Effects of 16 herbicides representing 14 herbicide classes on growth and extractable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) were examined in light- and dark-grown soybean [Glycine max(L.) Merr. ‘Hill’] seedlings. High purity (96 to 100%) herbicides were supplied via aqueous culture at various concentrations: 0.5 mM amitrole (3-amino-s-triazole), 0.1 mM atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], 0.07 mM diclofop-methyl {methyl ester of 2-[4-(2,4-dichlorophenoxy)phenoxy] propanoicacid}, 0.5 mM DSMA (disodium methanearsonate), 0.2 mM fenuron (1,1-dimethyl-3-phenylurea), 0.05 mM fluridone {1-methyl-3-phenyl-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone}, 0.5 mM MH (1,2-dihydro-3,6-pyridazinedione), 0.5 mM metribuzin [4-amino-6-tert-butyl-3-(methylthio)-as-triazin-5(4H)-one], 1.8 μM nitralin [4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline], 0.5 mM norflurazon [4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H)-pyridazinone], 0.05 mM paraquat (1,1′-dimethyl-4,4′-bipyridinium ion), 0.15 mM perfluidone {1,1,1-trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl] methanesulfonamide}, 0.2 mM propanil (3′,4′-dichloropropionanilide), 0.1 mM propham (isopropyl carbanilate), 0.5 mM TCA (trichloroacetic acid), and 0.05 mM 2,4-D [(2,4-dichlorophenoxy)acetic acid]. Dark-grown soybean seedlings (3-day-old) were transferred to control solutions (2 mM CaSO4) or to herbicide solutions (in 2 mM CaSO4) and grown at 25 C in continuous white light (200 μE•m-2•s-1) or continuous darkness until harvested 24 or 48 h after transfer. After 48 h, growth (fresh weight, dry weight, elongation) was inhibited by most of the chemicals. Other signs of toxicity (necrosis, secondary root stunting, and root tip swelling) were noted for some treatments. Roots were most affected, although hypocotyls were generally not changed. Hypocotyl elongation was stimulated by atrazine, fluridone, and norflurazon after 48 h light. Extractable PAL activity from soybean axes was decreased by atrazine, fenuron, metribuzin, norflurazon, propanil, propham, and 2,4-D. Amitrole and paraquat were the only herbicides that increased extractable PAL activity. Other compounds tested had no effect on the enzyme. None of the herbicides significantly affected in vitro PAL activity.

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10(-5)M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30-40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4-5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with α-aminooxy-β-phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.

Effects of Glyphosate on Metabolism of Phenolic Compounds: VI. Effects of Glyphosine and Glyphosate Metabolites on Phenylalanine Ammonia-Lyase Activity, Growth, and Protein, Chlorophyll, and Anthocyanin Levels in Soybean (Glycine max) Seedlings

The growth regulator, glyphosine [N,N-bis(phosphonomethyl)glycine], and other possible metabolites of glyphosine and glyphosate [N-(phosphonomethyl)glycine] [glycine, sarcosine, and aminomethylphosphonic acid (AMPA)] were tested individually (0.5 mM) or as a mixture (each at 0.5 mM) for their effects on growth, extractable phenylalanine ammonia-lyase (PAL) activity, hydroxyphenolic-compound production, chlorophyll and anthocyanin contents, and on soluble-protein levels in soybean [Glycine max(L.) Merr. ‘Hill’] seedlings. Most chemical treatments caused some inhibition of growth either on fresh weight accumulation or on root elongation in the light and dark over 72 h. Glyphosine was generally the most inhibitory and caused the greatest inhibition on axis dry-weight accumulation. Glyphosine significantly increased extractable PAL activity in axes of light- and dark-grown soybeans to a lesser extent than did glyphosate. AMPA had some inhibitory effects on extractable PAL activity whereas other compounds had little influence on the enzyme. These compounds had little effect on total soluble protein in axes or on soluble protein in PAL preparations from 12 to 72 h in light-or dark-grown seedlings. No in vitro effect of the chemicals on PAL activity was found at concentrations up to 0.5 mM. Hydroxyphenolic compound levels increased within 24 to 72 h (per gram fresh weight basis) in light- or dark-grown soybean axes treated with glyphosine, AMPA, or a metabolite mixture (AMPA, sarcosine, and glycine). Anthocyanin content was decreased by glyphosate and to a lesser extent by glyphosine, but was increased by AMPA and the mixture. Glyphosate significantly increased the chlorophylla/bratio and decreased total chlorophyll, but glyphosine decreased the chlorophyll content to a lesser degree.