Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6–1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

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

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33–40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments ³H is stereospecifically released from the pro-3S-position of L-[2,3-³H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, ³H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.Abbreviations AOA -aminooxyacetic acid - L-AOD L-aminoacid oxidase (EC 1.4.3.2) - D-AOD D-amino-acid oxidase (EC 1.4.3.3) - L-AOPP L--aminooxy--phenylpropionic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TAL tyrosine ammonia-lyase     

Phenylalanine and tyrosine ammonia-lyase activity in Sporobolomyces pararoseus

Phenylalanine ammonia-lyase from Sporobolomyces pararoseus was purified more than 450-fold. Polyacrylamide disc gel electrophoresis of this purified enzyme gave a single major protein band. Tyrosine ammonia-lyase activity was monitored during the purification of phenylalanine ammonia-lyase. Deaminating activities for phenylalanine and tyrosine were not separated during the purification process. The existence of one ammonia-lyase with bisubstrate activity is postulated.     

Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and tyrosine ammonia-lyase (TAL, 4.3.1.), the key enzymes of the phenylpropanoid pathway, are inducible in response to biotic (such as chitin from fungal cell walls) and abiotic cues. Application of chitin and chitosan to soybean leaf tissues caused increased activity of PAL and TAL enzymes. The elevation of enzyme activity was dependent on the chain length of the oligomers and time after treatment. The hexamer of chitin and pentamer of chitosan produced the maximum activities at 36 h after treatment as compared to controls. Total phenolic content of soybean leaves increased following chitosan and chitin oligomer treatments, showing a positive correlation between enzyme activity and total phenolic content.     

棉花抗蚜性与苯丙氨酸解氨酶活性的关系

为探讨苯丙氨酸解氨酶活性与棉花抗蚜性的关系,在室内人工接蚜危害和田间自然蚜群2种条件下,测定不同抗蚜性棉花品种受棉蚜Aphis gossypii Glover危害后叶片的PAL活性。结果表明:未受棉蚜危害时,抗、感品种PAL活性无明显差异;棉蚜危害胁迫棉花PAL活性升高,抗蚜品种受到棉蚜危害诱导所产生的PAL活性远比感蚜品种高。田间有蚜株率与棉花叶片苯丙氨酸解氨酶活性的呈显著的负相关关系。研究说明PAL酶活性对棉花的抗蚜性有一定的影响。     

Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots

The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract     

Phenylalanine ammonia-lyase: Purification and characterization from soybean cell suspension cultures

Soybean cell suspension cultures (Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 M_r. The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis, and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-C_n-NH₂, n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis. The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.     

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.     

Changes of antioxidant enzyme and phenylalanine ammonia-lyase activities during Chimonanthus praecox seed maturation

Changes in peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were studied during Chimonanthus praecox seed maturation. According to our findings the protein content increased steadily from 8 to 12 weeks after flowering, and thereafter decreased significantly. Similarly, SOD and POD activities increased gradually up to 12 weeks after flowering and then declined. PAL activity declined gradually during seed maturation. CAT activity, however, showed no changes during seed maturation. By means of polyacrylamide gel electrophoresis (PAGE), SOD and POD isoenzymes were observed during seed maturation. The staining intensities of SOD and POD isoenzymes correlated well with SOD and POD activities as obtained by an assay in solution. These findings suggest that POD, SOD and PAL may be involved in the growth and development during Chimonanthus praecox seed maturation.     

Changes in the levels of peroxidase and phenylalanine ammonia-lyase inBrassica napus cultivars showing variable resistance toLeptosphaeria maculans

Peroxidase and phenylalanine ammonia-lyase activities (PAL) were determined in leaves of healthy and inoculatedBrassica napus cultivars, showing differential disease reaction towards a virulent and a weakly virulent strain ofLeptosphaeria maculans, the black leg pathogen. Both enzymes showed increased activities as the result of inoculation, PAL activity increasing as early as 12 h after inoculation. The most significant increase in both peroxidase and PAL activity was observed when the moderately resistant cultivar, Cresor, was challenged with the weakly virulent strain. Highest activity of the two enzymes was detected 2 d after inoculation. Very low peroxidase activity was detected in both strains ofL. maculans, while no PAL activity was detectable in either of the strains. Cytochemical tests revealed increased peroxidase activity following inoculation, mainly in the epidermal and guard cells.     

Synthesis of phenylalanine ammonia-lyase in anthocyanin-containing and anthocyanin-free callus cells of Daucus carota L.

When callus cells of Daucus carota are grown on a medium containing gibberellic acid (GA₃) in a physiological concentration of 3x10^-6 M the cells cease to accumulate anthocyanins. This anthocyanin-free cell line has a very low activity of phenylalanine ammonia-lyase. After density labelling with D₂O an intensive de novo synthesis of the phenylalanine ammonia-lyase (E.C. 4.3.1.5; PAL) in the anthocyanin-containing cells does occur. 58% of the C-bound H-atoms are replaced by deuterium. The anthocyanin-free cells show only a very low enzyme synthesis which is difficult to detect with density labelling experiments. To ascertain that de novo synthesis occurs in the anthocyanin-free cells, the incorporation of ¹⁴C-labelled amino acids into the partially purified enzyme protein was measured after separation of the protein a) in CsCl gradients and b) on polyacrylamide gels. In both cases the enzyme bears ¹⁴C-label. These results suggest that in the anthocyanin-free cells de novo synthesis of PAL is still occuring but the synthesis is reduced in comparison to the anthocyanin-containing cells.Abbreviations GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase (E.C.4.3.1.5) - DCb anthocyanin-containing cells - DCw anthocyanin-free cells     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.     

Regulation of induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

Total RNA was extracted from fast growing suspension cells of bean, the mRNA was translated and the products of protein synthesis analysed by gel electrophoresis. Actinomycin D (20 g ml^–1) added to the cultures 12 h before the induction of phenylalanine ammonia-lyase (PAL) activity by naphthylacetic acid (NAA) (1 mg/l) and kinetin (0.2 mg/l) failed to prevent the increased activity of the enzyme usually produced by this ratio of the plant growth hormones. PAL was isolated and purified from suspension cultured bean cells. The purified enzyme ran as a single band on polyacrylamide gel electrophoresis. The protein translated from RNA prepared from induced and non-induced cells was separated by gel electrophoresis and the bands of protein on the gels were compared. There was no evidence for an increase in the amount of PAL synthesised in vitro from the mRNA of induced cells even though these had 5 times the amount of activity of the enzyme compared with that of the non-induced cells. The results indicate that the induction of PAL activity is not immediately preceeded by an increase in the synthesis of PAL-mRNA by the cells. The control of the activity of the enzyme is discussed with respect to this finding.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3naphthylacetic acid - DEAE Diethylamino ethyl - EDTA Ethylenediamine tetraacetate - SDS Sodium dodecyl sulphate     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).     

Phenylalanine ammonia-lyase,chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree

Summary During the first growth phase of walnut (Juglans sp.) stump shoots, the concentrations of the two major phenolic compounds are not correlated with an increasing rate of shoot growth. The concentration of hydrojuglone glucoside (naphtoquinone) decreases as shoot growth rate increases, whereas the concentration of myricitrin (flavonol) remains constant. In contrast, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity is proportional to the growth rate of shoots. Rejuvenation, which induces a higher growth rate and vegetative propagation ability, results in an increase of both PAL and chalcone synthase (CHS, EC 2.3.1.74) activities and hydrojuglone glucoside/myricitrin ratio. Moreover, physiological ageing is characterized by an accelerated functioning of polyphenolic metabolism. Fluctuations in PAL activity are associated with changes in shoot growth rate and with rejuvenation, but PAL does not directly control the accumulation of flavonoid compounds during rejuvenation. On the contrary, mathematical correlation of CHS activity and flavonoid accumulation during annual shoot growth of both adult and rejuvenated trees, indicates that CHS is the rate-limiting enzyme of the pathway.