Changes In Phenylalanine Ammonia-lyase Activity Research Articles

Effects of light quality, photoperiod, CO2 concentration, and air temperature on chlorogenic acid and rutin accumulation in young lettuce plants

Environmental stimuli modulate plant metabolite accumulation, facilitating adaptation to stressful conditions. In this study, the effects of blue and red light, photoperiod, CO2 concentration, and air temperature on the chlorogenic acid (CGA) and rutin contents of lettuce (Lactuca sativa L.) were evaluated. Under continuous blue light and a high CO2 concentration (1000 ppm), the CGA level increased. The increased expression of phenylalanine ammonia-lyase (PAL) and activity of its product were correlated with high expression of cinnamate 4-hydroxylase (C4H) and coumarate 3-hydroxylase (C3H). Furthermore, changes in PAL activity altered the CGA content in lettuce exposed to the three environmental factors, blue light, continuous lighting and high CO2 concentration. In addition, the expression levels of genes related to flavonoid biosynthesis increased in accordance with the promotion of CGA accumulation by the environmental factors. Under continuous blue light, 400 ppm CO2 promoted rutin accumulation to a greater degree compared to 1000 ppm CO2, by downregulating DFR expression. Low air temperature induced CGA accumulation in lettuce grown under continuous blue light and 1000 ppm CO2. Therefore, light quality, photoperiod, CO2 concentration, and air temperature exert synergistic effects on the CGA and rutin contents of lettuce by modulating activity in the corresponding biosynthesis pathways.

Accumulation of Phenolic Compounds at the Initial Steps of Ontogenesis of Fagopyrum esculentum Plants That Differ in Their Ploidy Levels

Plants of buckwheat Fagopyrum esculentum possessing diploid and tetraploid genotypes were studied at the initial ontogenetic stages. They were compared in their morphophysiological characteristics, accumulation of phenolic compounds (including their main classes—phenylpropanoids and flavonoids), and activity of L-phenylalanine ammonia-lyase. An apparent resemblance in morphophysiological characteristics of seedlings was found between the two specimens, but diploid plants tended to faster linear growth than tetraploid ones. Differences in the accumulation of phenolic compounds in the hypocotyl and cotyledonous leaves were revealed. In most cases, in the course of seedling growth, the changes in phenylalanine ammonialyase activity did not correlate with the changes in the levels of these secondary metabolites. The effects of gene dosage were established towards accumulation of phenylpropanoids and anthocyanins in hypocotyls of seedlings and flavonoid accumulation in cotyledonous leaves. It is concluded that buckwheat seedlings with a tetraploid genotype have higher capacity than diploid seedlings for biosynthesis of phenolics.

Salinity Stress Is Beneficial to the Accumulation of Chlorogenic Acids in Honeysuckle (Lonicera japonica Thunb.).

Honeysuckle (Lonicera japonica Thunb.) is a traditional medicinal plant in China that is particularly rich in chlorogenic acids, which are phenolic compounds with various medicinal properties. This study aimed to examine the effects of salinity stress on accumulation of chlorogenic acids in honeysuckle, through hydroponic experiments and field trials, and to examine the mechanisms underlying the effects. NaCl stress stimulated the transcription of genes encoding key enzymes in the synthesis of chlorogenic acids in leaves; accordingly, the concentrations of chlorogenic acids in leaves were significantly increased under NaCl stress, as was antioxidant activity. Specifically, the total concentration of leaf chlorogenic acids was increased by 145.74 and 50.34% after 30 days of 150 and 300 mM NaCl stress, respectively. Similarly, the concentrations of chlorogenic acids were higher in the leaves of plants in saline, compared with non-saline, plots, with increases in total concentrations of chlorogenic acids of 56.05 and 105.29% in October 2014 and 2015, respectively. Despite leaf biomass reduction, absolute amounts of chlorogenic acids per plant and phenylalanine ammonia-lyase (PAL) activity were significantly increased by soil salinity, confirming that the accumulation of chlorogenic acids in leaves was a result of stimulation of their synthesis under salinity stress. Soil salinity also led to elevated chlorogenic acid concentrations in honeysuckle flower buds, with significant increases in total chlorogenic acids concentration of 22.42 and 25.14% in May 2014 and 2015, respectively. Consistent with biomass reduction, the absolute amounts of chlorogenic acid per plant declined in flower buds of plants exposed to elevated soil salinity, with no significant change in PAL activity. Thus, salinity-induced chlorogenic acid accumulation in flower buds depended on an amplification effect of growth reduction. In conclusion, salinity stress improved the medicinal quality of honeysuckle by promoting accumulation of chlorogenic acids, however, the mechanisms underlying this process were not consistent in flower buds and leaves. Honeysuckle appears to be a promising plant for cultivation in saline land. Our study deepens knowledge of medicinal plant ecology and may provide a guide for developing saline agriculture.

Phenylalanine ammonia lyase activity in fresh cut lettuce subjected to the combined action of heat mild shocks and chemical additives

Enzymatic browning reduces the visual quality of lettuce. In producing minimally processed lettuce, the stresses caused by wounding associated to cutting operations enhance enzymatic activity. Mild heat shocks with chlorinated water with calcium ions or ascorbic acid were assayed as a mean of reducing phenylalanine ammonia lyase (PAL) activity. Heat shocks reduced four times the rate of change in PAL activity during the first hours of storage over the rate for samples not subjected to thermal treatment. The addition of ascorbic acid resulted in an increase in PAL activity over samples where only chlorinated water was used. When calcium ions were introduced in the thermal baths, the PAL activity was reduced about four times, especially when the cutting operation was performed after the thermal shocks. Short heat shocks is a physical preservation technology that could be applied in countries with low technological development. This technology was efficient to control vegetable browning by reducing PAL activity.

Cloning and expression analysis of phenylalanine ammonia-lyase in relation to chilling tolerance in harvested banana fruit

Bananas are highly susceptible to chilling injury (CI) and phenylalanine ammonia-lyase (PAL), as a key enzyme involved in plant phenylpropanoid metabolism, has been associated with low temperature stress in plant tissues. However, little is known about the role of PAL (including PAL activity, gene and protein expression) in postharvest chilling tolerance of banana fruit. Two partial cDNAs sequences ( MaPAL1 and MaPAL2) with about 760 bp were cloned from banana pulp by RT-PCR. Western and northern hybridizations were used to investigate expression of PAL protein and PAL genes in fruit stored for 10 days at 7 °C (chilling temperature) and then transferred to 22 °C (room temperature). The effects of propylene (a functional ethylene analog) on their expression in relation to CI were also examined. Northern and western blot analyses revealed that mRNA transcripts of MaPAL1 and MaPAL2 and PAL protein levels in banana fruit during storage increased, reaching a peak at about day 8, and finally decreased at chilling temperature. Prior to low temperature storage, pretreatment with propylene could alleviate CI and enhance PAL activity, protein amount and mRNA transcripts of MaPAL1 and MaPAL2. Moreover, changes in PAL activity, protein amount and accumulation of MaPAL1 and MaPAL2 exhibited almost the same patterns. The results suggest that the induction of PAL in banana fruit during low temperature storage is regulated at transcriptional and translational levels, and is related to reduction in CI symptoms.

Changes in Phenylalanine Ammonia-lyase Activity and Gene Expression during Storage of Asparagus Spears

A cDNA clone coding phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library prepared from asparagus spears (Asparagus officinalis L. cv. Welcome) using the reverse transcription-polymerase chain reaction (RT-PCR). The partial cDNA clone encoded an mRNA of 527 bp and the derived amino acid sequence was found highly homologous to PAL from rice, maize and barley. Northern blot analysis showed an increase of pAS-PAL mRNA until 24 h at 20 degrees C, which coincided well with PAL activity and fiber development, suggesting that the increase is a response to the wounding associated with harvest.

Phenylalanine ammonia lyase activity in minimally processed Romaine lettuce

The effect of the intensity of damage on the phenylalanine ammonia lyase (PAL) activity, on browning of midrib tissue and on the overall visual quality (OVQ) of minimally processed Romaine lettuce was studied. In highly wounded leaves two peaks of PAL activity were observed. In whole and in less-damaged leaves only one peak was detected. The rate of change in PAL activity during the first day of storage was found to be related to the intensity of damage. Browning in midrib tissue developed in the most highly damaged leaves after 4 days of storage. Dehydration and wilting reduced the OVQ of lettuce before the onset of browning and the occurrence of peaks in PAL activity.

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.

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

A comparison of changes in phenylalanine ammonia-lyase activity, lignin and phenolic synthesis in the roots of Eucalyptus calophylla (field resistant) and E. marginata (susceptible) when infected with Phytophthora cinnamomi

Inoculation of primary roots of Eucalyptus marginata (jarrah, susceptible) and E. calophylla (marri, field resistant) with Phytophthora cinnamomi zoospores caused a lesion to develop within 12–16 h. Lesions in roots of E. calophylla were restricted within 3–4 days and ceased extending whereas those in roots of E. marginata continued to extend. The temporal and spatial changes in phenylalanine ammonia lyase activity, lignin concentration and the concentration of soluble phenolics in roots were followed for 96 h after inoculation. PAL activity increased in inoculated roots of E. calophylla within 24 h of inoculation, beginning at the root tip and then declining to below control levels over the succeeding 24–48 h. A similar pattern of increase in PAL activity, followed by a decline was recorded for four of the five successive 1 cm root segments distal to the root tip. There were no increases in PAL activity in inoculated roots of E. marginata. After inoculation, the amount of lignin m roots of E. calophylla was increased above control levels by as much as 53%. Lignin concentrations in inoculated roots of E. marginata were unchanged. The concentration of suberin remained at pre-inoculation levels throughout the experiment in roots of both species. There were rapid increases in total soluble phenolics in roots of E. calophylla after inoculation, but only minor increases in roots of E. marginata. In the third root segment from the root tip in E. calophylla levels of total phenolics were at their maximum of 97% above controls. Thin layer chromatography of an aqueous methanol extract of infected roots of E. calophylla revealed the presence of several new compounds which were not present in control roots of E. marginata. Treatment of roots of E. calophylla with the PAL inhibitor, aminooxyacetate, altered the resistance response through changes in the concentration of lignin and phenolics and produced what phenotypically resembled susceptibility. These results implicate lignin and phenolic synthesis in the resistance of cucalypts to P. cinnamomi.

Blackspot bruise dependent changes in enzyme activity and gene expression in Lemhi russet potato

Blackspot bruise-induced changes in enzyme activity and gene expression were examined in the bruise susceptible cultivar Lemhi. The activities of several enzymes involved with the synthesis and metabolism of phenolic intermediates were examined over the time course of blackspot bruise development in tubers. A large, transient increase (200-fold) in phenylalanine ammonia lyase (PAL) activity was observed, while other enzymes examined (chorismate mutase, tyrosine ammonia-lyase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, and polyphenol oxidase) showed either modest or no activation. Maximal PAL activity was observed 48 hours after bruise induction, well after the discoloration reactions were complete. The change in PAL activity was associated with a transient increase in messenger RNA coding for PAL. In addition, messenger RNAs encoding two stress-induced genes, ubiquitin and the 70 kD heat shock protein, were transiendy induced by bruising. Physical impact also induced a marked decrease, followed by recovery, in the messenger RNA level for patatin, a primary storage protein in the tuber. These results suggest that the stress resulting in blackspot bruising elicits a wound response similar to those observed in other injuries to plant tissues.

Phenolic biosynthesis during grain development in wheat: changes in phenylalanine Ammonia-lyase activity and soluble phenolic content

Changes in phenylalanine ammonia lyase (PAL) activity and the total phenolic content of several wheat cultivars have been monitored during grain development. PAL activity was maximal during the early milk stage of development and then declined. The level of soluble phenolics peaked 20-24 days after the PAL peak, just prior to the appearance of mature grain colour. Red cultivars showed higher PAL activities during early grain development and lower germination rates during maturation.

Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae

Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.The changes in phenylalanine ammonia-lyase activity in parsley cells were caused by corresponding changes in the mRNA activity for this enzyme. Phenylalanine ammonia-lyase mRNA activity increased much faster and transiently reached a much higher level in elicitor-treated than in irradiated cell cultures. In contrast to irradiation, treatment of the cells with the elicitor did not induce the enzymes of the flavonoid glycoside pathway, as demonstrated for acetyl-CoA carboxylase and chalcone synthase. Induction of these enzymes by light was abolished by simultaneous application of the elicitor.

Modifications of wound-induced changes in phenylalanine ammonia-lyase activity in potato tuber tissue

Potato tuber parencyma discs that are incubated in vitro in darkness at 20°C exhibit a ‘wounding time course’ where phenylalanine ammonia-lyase (PAL; E.C.4.3.1.5) activity peaks 24 h after excision and then declines to a stable plateau level. In contrast, if the discs are restored to the tuber environment immediately after excision, by a process of placing them inside a ‘host’ tuber, PAL activity remains at a constant low level for at least 48 h. When these discs are transferred from their host tuber to in vitro incubation, PAL activity immediately rises and follows a time course in which there are now two peaks within 48 h. Although the particular timing of the peaks is dependent on the length of the in situ preincubation, this pattern is established after only 4 h in situ. Both peaks are sensitive to actinomycin D and cycloheximide, which also abolish the low basal PAL activity.

Light-induced phenylalanine ammonia-lyase in cell-suspension cultures of Petroselinum hortense: Quantitative comparison of rates of synthesis and degradation

Immunological methods were used to determine in vivo the rates of synthesis and degradation of phenylalanine ammonia-lyase (EC 4.3.1.5) in irradiated cell-suspension cultures of parsley ( Petroselinum hortense Hoffm.). Irradiation of the cells with ultraviolet light induced an immediate, exponential increase in the rate of enzyme synthesis. In contrast to previous assumptions, this increase occurred with no detectable lag period. The increase continued at a high rate for several hours even in the dark period following short-term irradiation for 2.5 h. The maximally induced rate of enzyme synthesis in cells from a linear growth period was much larger than in cells from the stationary phase of the culture, both after short-term and continuous irradiation. The half-life of the enzyme, as determined by labeling studies in vivo, varied between about 10 h and 5 h for the two different growth stages. The curves for light-induced changes in enzyme activity coincided with corresponding curves calculated from the data obtained for the rates of enzyme synthesis and degradation. This coincidence supports the previous hypothesis that large and rapid changes in the rate of synthesis and an essentially constant rate of degradation determine the shapes of the curves for light-induced changes in phenylalanine ammonia-lyase activity at a given growth stage of a parsley cell culture.

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

Fluctuations in the activity of phenylalanine ammonia-lyase (PAL) in tissues of buckwheat seedlings were monitored by an intact cell assay and compared with results obtained with the conventional in vitro assay. Good correlation between measurements with the different assays was found for PAL activity changes during 1) growth of seedlings in the dark, 2) illumination of seedlings, and 3) incubation of hypocotyl segments. When leaf disks of buckwheat, strawberry, or sunflower were allowed to float on either water or a sugar solution, PAL activities determined with the intact cell assay increased irrespective of the presence of sugar, while PAL activities determined in cell homogenates increased only in disks floating on a sugar solution. Possible explanations for these discrepancies are discussed.

Specific effects on enzyme activities upon dilution of Petroselinum hortense cell cultures into water

Large increases in the specific activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and p-coumarate:CoA ligase (EC 6.2.1.-) occurred within a few hours after dilution of cultured Petroselinum hortense cells into water. No significant changes in the total amount of extractable protein and in the activities of chalcone isomerase (EC 5.5.1.6) and glutamate dehydrogenase (EC 1.4.1.2) were observed under the same conditions. The time course for the change in phenylalanine ammonia-lyase activity included a lag period of 2–3 h, a peak about 13 h after the onset of induction, and a subsequent period of rapid decline. The inducible amount of enzyme activity was greatly dependent upon the degree of dilution of the cells into water. Simultaneous induction by dilution and irradiation of the cells with white, fluorescent light resulted in an increase in the phenylalanine ammonia-lyase level of activity which exceeded that calculated from the sum of the separately induced levels. Consecutive inductions, first by dilution and then 5 h later by irradiation, each required a lag period of 2–3 h. Actinomycin D or cycloheximide were inhibitors of the induction. While the total protein-synthesizing capacity in vitro was not significantly influenced by the dilution of cells, analysis of the labeled products on polyacrylamide gels demonstrated small but significant changes in the radioactivity profiles. The results are consistent with the hypothesis that dilution of the cells into water reduces the concentration of one or more compounds of cellular origin, thereby stimulating the rate of de novo synthesis of a limited number of proteins.

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of trans-hydroxycinnamic acids to the cis-isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammonia-lyase activity in the hypocotyl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activity caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hydroxycinnamic acids.

Changes in phenylalanine ammonia-lyase activity in Satsuraa mandarin fruit during ontogeny

Changes in phenylalanine ammonia-lyase activity in Satsuraa mandarin fruit during ontogeny

Changes in phenylalanine ammonia-lyase activity during xylem differentiation in Coleus and soybean

Xylem differentiation was induced in cultured Coleus internode slices when grown in the light on a simple agar/sucrose/IAA medium and in darkgrown soybean callus tissue when cultured on a complex defined medium containing 5×10(-7) M kinetin. In the Coleus system, the activity of phenylalanine ammonialyase followed the same time course as the formation of lignified wound vessel members. The specific activity of PAL was higher in the soybean callus tissues grown on 5×10(-7) M kinetin, which produced tracheary elements, than in the soybean tissue grown on 10(-8) M kinetin, which did not produce tracheids. These observations suggest that PAL is a "marker enzyme" for xylogenesis and that PAL activity may be a rate limiting step in lignification.