Maximum Phenylalanine Ammonia Lyase Research Articles

Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt disease

Plant immunization for resistance against a wide variety of phytopathogens is an effective strategy for plant disease management. Seventy-nine plant growth-promoting fungi (PGPFs) were isolated from rhizosphere soil of India. Among them, nine revealed saprophytic ability, root colonization, phosphate solubilization, IAA production, and plant growth promotion. Seed priming with four PGPFs exhibited early seedling emergence and enhanced vigour of a tomato cultivar susceptible to the bacterial wilt pathogen compared to untreated controls. Under greenhouse conditions, TriH_JSB27 and PenC_JSB41 treatments remarkably enhanced the vegetative and reproductive growth parameters. Maximum NPK uptake was noticed in TriH_JSB27-treated plants. A significant disease reduction of 57.3% against Ralstonia solanacearum was observed in tomato plants pretreated with TriH_JSB27. Furthermore, induction of defence-related enzymes and genes was observed in plants pretreated with PGPFs or inoculated with pathogen. The maximum phenylalanine ammonia lyase (PAL) activity (111U) was observed at 24h in seedlings treated with TriH_JSB27 and this activity was slightly reduced (99U) after pathogen inoculation. Activities of peroxidase (POX, 54U) and β-1,3-glucanase (GLU, 15U) were significantly higher in control plants inoculated with pathogen after 24h and remained constant at all time points. A similar trend in gene induction for PAL was evident in PGPFs-treated tomato seedlings with or without pathogen inoculation, whereas POX and GLU were upregulated in control plus pathogen-inoculated tomato seedlings. These results determine that the susceptible tomato cultivar is triggered after perception of potent PGPFs to synthesize PAL, POX, and GLU, which activate defence resistance against bacterial wilt disease, thereby contributing to plant health improvement.

Foliar application of l-phenylalanine and ferulic acids to pea plants: induced phenylalanine ammonia lyase activity and resistance against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of l-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100 and 150 ppm) of l-phenylalanine caused increased activity of PAL in comparison to the control. In pea leaves treated with 50 ppm l-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves as compared to the control. Maximum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt. after 24 h at 50 ppm and then increased with time. Treatment with both the compounds significantly reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. The conidial germination on l-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid-treated leaves was 34% as compared to the control (46%). Foliar application of different concentrations of l-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum accumulation of ferulic acid (79.3 and 83.5 μg/g fresh wt.) was observed following the application of l-phenylalanine after 24 h and 48 h, respectively. At 50 ppm, ferulic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt. and 74.3 and 86.5 μg/g fresh wt. at 100 ppm.

Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.

Modeling and Optimization of Phenylalanine Ammonia Lyase Stabilization in Recombinant Escherichia coli for the Continuous Synthesis of l-Phenylalanine on the Statistical-Based Experimental Designs

Some approaches for improving recombinant phenylalanine ammonia lyase (PAL) stability in Escherichia coli during the enzymatic methods of l-phenylalanine (l-Phe) production were developed following preliminary studies by means of statistical-based experiment designs (response surface method). The traditional non-statistical technology was used to screen four critical factors for PAL stability during the bioconversion process, viz., glycerin, sucrose, 1,4-dithiothreitol (DTT), and MgSO(4). The central composite design (CCD) was applied to optimize the combined effect of critical factors for recombinant PAL stability and understand the relationship between the factors and PAL stability. The optimum values for testing variables were 13.04 mM glycerin, 1.87 mM sucrose, 4.09 mM DTT, and 69 mM Mg(2+). A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory because the coefficient of determination was 0.88. The maximum PAL activity was retained as 67.73 units/g after three successive cycles of bioconversion. In comparison to initial PAL activity, the loss of PAL activity was only 22%. PAL activity was enhanced about 23% in comparison to the control (without any stabilizer additives). PAL stability was significantly improved during successive bioconversion. The results obtained here verified the effectiveness of the applied methodology and may be helpful for l-Phe production on an industrial scale.

Growth Promotion and Induction of Systemic Resistance in Rice Cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp.

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), persistent colonizers of plant leaf surfaces, belong to the genus Methrlobacterium and are mostly transmitted through seeds. Plant growth-promoting activity of methylotrophic bacteria and their effects on disease suppression were evaluated on rice under greenhouse conditions. Rice seeds were inoculated with Methylobacterium sp. strain PPFM-Os-07 and seed germination was evaluated in terms of morphometric measurements, seedling growth, rate of germination (R(subscript G)), and seedling vigour index (SVI). Another experiment was carried out to study the induction of pathogenesis-related proteins (PR-proteins) in rice plants that were inoculated with methylotrophic bacteria by seed imbibition or foliar spray. In the third experiment, sixty-day-old rice plants grown in pots were challenge inoculated with Rhizoctonia solani strain TNAU-01. Methylobacterium inoculation promoted seed germination and plant growth. Increased plant height, number of tillers, plant biomass, and grain yield were observed. The average yield increases for seed imbibition and phyllosphere spray were, respectively, 22.1% and 24.3% greater than control. The bacteria also significantly reduced the sheath blight incidence when applied as either bacterial culture through seed imbibition and or phyllosphere spray. The percent disease reduction recorded for seed imbibition alone and for combined applications of seed imbibition and phyllosphere spray were 17.8% and 23.5%. Rice plants sprayed with PPFM-Os-07 strain showed increased presence of PR-proteins and phenolic contents on day 1 after application. Maximum phenylalanine ammonia lyase (PAL) and peroxidase activity on day 4 and β-1,3-glucanase and chitinase activity on day 5 were recorded. The results suggest that Methylobacterium inoculation may alter rice susceptibility to R. solani. This work emphasizes the importance of evaluating induced systemic resistance while studying plant-associated growth promoting bacteria.

Influence of maturity and bagging on the relationship between anthocyanin accumulation and phenylalanine ammonia-lyase (PAL) activity in ‘Jonathan’ apples

Abstract The influence of maturing and ripening on phenylalanine ammonia-lyase (PAL) activity and anthocyanin accumulation was examined using bagged, non-bagged and shaded ‘Jonathan’ ( Malus domestica ) apples. Bagged fruit were covered with paper bags from mid-June at an early stage of fruit growth. Shaded apples were insulated from sunlight by newspaper at the beginning of colouring (middle September). The apples were harvested at different maturity stages and were irradiated with UV+white light for 96 h at 15°C after which anthocyanin content and PAL activity were measured. In non-bagged and shaded apples, anthocyanin accumulation and maximal PAL activity increased as the apples developed from the immature to the initial ripe stage, then decreased at full ripe stage. A positive linear relationship between the maximum PAL activity and the amount of anthocyanin accumulated suggested that the change in PAL activity is involved in the change of anthocyanin accumulation. In the bagged apples in the immature stage, anthocyanin accumulation and peak PAL activity after irradiation was higher than that in non-bagged apples. However, as the apples ripened, anthocyanin accumulation was less than for less mature fruit, even though PAL activity was relatively high. Therefore, PAL is not the only regulating factor for anthocyanin accumulation in bagged mature and ripe fruit.

97/00061 Tribomechanical aspects of movement of coal-charge materials over a heated surface

The periderm formation of wound tissue of potato tuber was inhibited temporarily after exposure to S-carvone. In wound tissue of control tubers a suberized cell layer was visible after 4 days whereas in S-carvone treated tissue this was observed only after 14 days in most of the tubers. At day 21 all the S-carvone treated tubers had formed suberin but only 10 % of the tubers had developed a (begin of a) cambium layer. The appearance of suberin was related to the activity of phenylalanine ammonia lyase (PAL); in control and S-carvone treated tissue, maximum PAL activity preceded the appareance of the first suberized cell layers by 2 and 5 days, respectively. The specific activity reached the same level in both control and S-carvone treated wound tissue. A short term treatment (2, 4 and 7 days) of tuber wound tissue with S-carvone delayed suberin formation. All the treated tubers showed suberization about 4 days after removal of S-carvone and this was closely related to the increase in PAL-activity. After the S-carvone treatment, the glutathione metabolism increased: after an initial decrease, as observed in the control, the total glutathione content increased fourfold after 9 days and remained high during the treatment. However, the length of the wound healing period before the start of a S-carvone treatment was of great influence on the increase of the glutathione metabolism and on the induction of glutathione reductase.

S-Carvone Inhibits Phenylalanine Ammonia Lyase (PAL) Activity and Suberization During Wound Healing of Potato Tubers

Summary The periderm formation of wound tissue of potato tuber was inhibited temporarily after exposure to S-carvone. In wound tissue of control tubers a suberized cell layer was visible after 4 days whereas in S-carvone treated tissue this was observed only after 14 days in most of the tubers. At day 21 all the S-carvone treated tubers had formed suberin but only 10 % of the tubers had developed a (begin of a) cambium layer. The appearance of suberin was related to the activity of phenylalanine ammonia lyase (PAL); in control and S-carvone treated tissue, maximum PAL activity preceded the appareance of the first suberized cell layers by 2 and 5 days, respectively. The specific activity reached the same level in both control and S-carvone treated wound tissue. A short term treatment (2, 4 and 7 days) of tuber wound tissue with S-carvone delayed suberin formation. All the treated tubers showed suberization about 4 days after removal of S-carvone and this was closely related to the increase in PAL-activity. After the S-carvone treatment, the glutathione metabolism increased: after an initial decrease, as observed in the control, the total glutathione content increased fourfold after 9 days and remained high during the treatment. However, the length of the wound healing period before the start of a S-carvone treatment was of great influence on the increase of the glutathione metabolism and on the induction of glutathione reductase.

Phenylalanine ammonia lyase as a precursory enzyme of legume stem lignification

In some instances, lignin content may not be significantly correlated with phenylalanine ammonia lyase (PAL) activity because: (1) PAL is not committed exclusively to lignin, and (2) the time of maximum PAL activity may not coincide with maximum lignin deposition. This study evaluates correlations and timing of PAL activity and lignin deposition during legume stem maturation. Three forage legumes, alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), and red clover (Trifolium pratense L.), were established, and basal stem regrowth was sampled, biweekly, for 10 wk, for dry weight (DW), cell wall (CW), lignin, and PAL analyses. Nonlinear regression of lignin content by the Gompertz function indicated that lignin increased sigmoidally, and PAL activity by the third-order quadratic demonstrated rapid initial increases in activity, followed by decreases, as a function of regrowth days. First derivative of the Gompertz function demonstrated that changes in lignin deposition closely resembled changes in PAL activity. Among species, peak deposition of DW and CW content occurred 3–11 d prior to maximum lignin deposition. Time of maximum PAL activity occurred 8 d prior to maximum lignin deposition in birdsfoot trefoil and red clover and 3 days after maximum lignin deposition in alfalfa. Across species, lignin content was not positively correlated with PAL activity on a protein basis. However, lignin deposition was positively correlated with PAL per unit protein (r = 0.76, P < 0.05) and lignin content was positively correlated with PAL on a per plant basis (r = 0.60, P < 0.05). These results indicate that the activity of PAL is related to lignin deposition in a cause-and-effect relationship. Key words: Alfalfa, birdsfoot trefoil, cell wall, lignin, phenylalanine, ammonia lyase, red clover

932 PB 530 PAL INDUCTION AND FRESH-CUT LETTUCE QUALITY

Phenylalanine ammonia-lyase (PAL) activity in iceberg lettuce (Lactuca sativa L.) is increased in response to several kinds of stress, including wounding, exposure to ethylene, and fungal infection. Ethylene-induced PAL activity is correlated with the discoloration and shelf-life of fresh cut lettuce (Couture et al. 1993. HortScience 28:723). The objective of this research was to further characterize the kinetics of wound-induced PAL in fresh cut lettuce. Leaves of different cvs were cut into salad pieces (1.5 × 3 cm), rinsed in chlorinated water, centrifuged manually, and placed into containers at 5 or 15C through which humidified air flowed. Samples were evaluated for overall visual quality and specific types of discoloration. Midrib tissue was also finely cut (1 × 0.5 cm) for enhanced wound-induction of PAL, which was assayed spectrophotometrically. The kinetics of PAL in midrib tissue of fine cut and salad cut lettuce were similar, with maximum activities obtained within 12-16 h at 15C and within 40-60 h at 5C. Maximum PAL levels in the fine cut lettuce were 1.5-2.0 times those observed in the salad cut pieces, and similar to those induced by ethylene. The usefulness of PAL as a predictor of the storage life of fresh cut lettuce depends on simplifying and expediting the PAL assay.

Phenylalanine ammonia-lyase, phenolic acids and ethylene in alfalfa (Medicago sativa L.) cell cultures in relation to their embryogenic ability

Phenylalanine ammonia-lyase (PAL) activity, contents of phenolic acids and ethylene production during the lag-phase, and contents of phenolic acids at the late exponential phase, showed significant differences in embryogenic (EC) and non-embryogenic (NEC) suspension cultures of Medicago sativa L. Maximum PAL activity at 6 h after inoculation was followed by an increase in the level of phenolic acids from 9.6 μg g(-1) fresh mass to 21 μg g(-1) fresh mass in NEC at 12 h. Thereafter the level of phenolic acids decreased to 5.2 μg g(-1) fresh mass at 72 h. The decline was caused predominantly by the decrease of ester-bound cinnamic acid derivatives, the decrease ranging from 83 to 20% of total phenolics. Two maxima of ethylene production were observed in NEC: the first one immediately after inoculation and the second at 6 h, coinciding with the peak of PAL activity. In NEC, most of the phenolic acids occurred in esterified form. Ability to form somatic embryos (EC) was associated with the absence of the second peak of ethylene production as well as of the peak of PAL activity at 6 h. The level of phenolic acids during the lag-phase remained low (7.2 μg g(-1) FM) and did not change. The proportion of cinnamic acid derivatives was very low (18% of total phenolics), mostly due to the extremely low level of ferulic acid. In EC, phenolic acids bound to methanol insoluble material formed the major fraction. Loss of embryogenic potential of the embryogenic culture (ECL) was associated with qualitative and quantitative changes in the contents of phenolic acids insignificantly increased PAL activity after inoculation was followed by a moderate increase in the contents of phenolic acids from 9.35 μg g(-1) fresh mass to 12.42 μg g fresh mass. A high rate of ethylene production was observed only immediately after the transfer of the culture to fresh medium. The loss of embryogenicity correlated also with changes in the relative amounts of the investigated fractions of phenolic acids. A distinct increase in the level of methoxy-substituted phenolic acids is a characteristic feature of the ECL culture.

The effect of ethylene on phenylalanine ammonia lyase (PAL) induction by a fungal elicitor in Phaseolus vulgaris

The effects of ethylene on the induction of phenylalanine ammonia-lyase (PAL) by cell wall elicitors of Colletotrichum lindemuthianum was investigated in leaves of susceptible and resistant cultivars of Phaseolus vulgaris (cultivars Pinto and Kievit respectively). Induction of PAL by exogenously supplied ethylene and associated test substances alone, without elicitor, was negligible. With elicitor alone, cv. Kievit responded to lower concentrations than cv Pinto. Exogenously supplied ethylene augmented the response of cv. Kievit only in the presence of limiting concentrations of elicitor. When concentrations of elicitor giving maximum PAL induction were used, ethylene was without effect in cv. Kievit also. Aminoethoxyvinylglycine (AVG) partially inhibited the induction of PAL in cv. Kievit and this inhibition could be reversed by aminocyclopropane-1-carboxylic acid (ACC) and ethylene. However, since cv. Pinto did not respond to ethylene, the application of AVG in the presence of elicitor was, as expected, without effect. Unless introduced within 2 h of elicitor application, ethylene was without effect on PAL production in cv. Kievit also; likewise, the inhibitory effect of AVG was minimal if not injected within the same time period following elicitor. Elicitor rapidly induced the synthesis of ACC during this 2 h period leading to the production of stress ethylene and this was completely inhibited by AVG and completely reversed by ACC. The effect of the elicitor, after injection into the leaf of cv. Kievit, was to change both the distribution and relative concentrations of the soluble proteins. Ethylene enhanced this effect and the 40 % increase in PAL activity observed in the presence of the hormone can largely be accounted for by the increase in the concentration of the enzyme. Changes brought about by the elicitor were eliminated by inhibitors of protein and nucleic acid synthesis, the effects of which could not be reversed by ethylene. These results are discussed in relation to other studies. It has been proposed that elicitors induce the biosynthesis of ethylene and PAL independently. Ethylene is involved in the activation of the induced response leading to the synthesis of PAL, and thereby phytoalexins, particularly when the response to elicitor is sub-optimal.

Effects of Exogenous Factors on Phenylalanine Ammonia-Lyase Activity and Accumulation of Anthocyanins and Total Phenolics in Grape Berries

The effects of ethephon (E), sucrose (S), and light (L) on phenylalanine ammonia-lyase (PAL) activity and on the concentration of anthocyanins and total phenolics in the berry skins of <i>Vitis vinifera</i> L. Cardinal berries were studied at temperatures of 14°, 22°, and 35°C over a period of 72 hours. Maximum PAL activity was obtained in the light/ethephon/sucrose-treated (LES) and light/sucrose-treated (LS) berries at 36 and 48 hours. In berries kept in the dark, PAL activity decreased over the experimental period. Anthocyanin concentration increased rapidly after a phase of slow accumulation of 24 to 60 hours, depending on the temperature, in the presence of light but not in the dark. Sucrose plus ethephon had a synergistic effect on synthesis of anthocyanins at 22°C. The period of slow anthocyanin accumulation was shortened as temperature increased. Cycloheximide (CHI) applied to LES-treated berries at 22°C caused a significant reduction in the concentration of anthocyanins in berry skins. Rapid phenolic accumulation in grape berry skins occurred after 48 hours at all temperatures. The pattern of changes in total phenolics in berry skins was similar for all temperature treatments with the maximum concentration reached after 60 hours. There were no consistent effects of sucrose and ethephon on total phenol accumulation in berry skins.

The biosynthesis of benzoic acid in Bramley's Seedling apples infected by Nectria galligena Bres

Radioactive benzoic acid was synthesized from glucose- 14C and l-phenylalanine- 14C in apples infected with Nectria galligena and in apples infused with protease extracted from tissue rotted by N. galligena. Phenylalanine ammonia-lyase (PAL) activity was rapidly induced in apples infused with the fungal protease but a lag phase occurred following inoculation with the pathogen. Production of benzoic acid was maximal approximately 3 days after maximum PAL production. Enzymes extracted from necrotic apple tissue converted phenylalanine to cinnamic and benzoic acids. P. expansum, a non-protease producing apple pathogen, did not induce either PAL activity or elicit benzoic acid accumulation in apples.