Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase from Rhodosporidium toruloides

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.     

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-α-aminooxy-β-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L--aminooxy--phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283–287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.Abbreviations CAH trans-cinnamate 4-hydroxylase - L-AOPP L--aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase     

The ectopic expression of phenylalanine ammonia lyase with ectopic accumulation of polysaccharide-linked hydroxycinnamoyl esters in internode parenchyma of rice mutant Fukei 71

Both polysaccharide-linked hydroxycinnamoyl esters (PHEs) and lignin are biosynthesized via the phenylpropanoid pathway. In the abnormal internode parenchyma of the rice (Oryza sativa L.) mutant Fukei 71, which has a defective recessive gene (d50), the biosynthesis of lignin and PHEs differs. . The polysaccharide-linked ferulate and p-coumarate have been shown to accumulate to high levels in the irregularly shaped and collapsed internode parenchyma cells of Fukei 71 without an accompanying overaccumulation of lignin as a result of the defective d50 gene. In the present study we demonstrated that in this abnormal parenchyma tissue of Fukei 71 the expression of phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS) were ectopically induced with the ectopic accumulation of PHEs, suggesting that the d50 gene may play a role as a controlling element in the biosynthesis of PHEs during cell-wall formation in the grasses.     

Effects of cycloheximide, actinomycin D and ethylene on the increase and subsequent decrease in acid invertase activity in wounded sweet potato

In wounded sweet potato root tissue, the mechanisms of the increaseand subsequent decrease of acid invertase activity were examinedin compariosn with phenylalanine ammonia lyase (PAL) and peroxidaseactivities. The increase in acid invertase and PAL activitiesin response to wounding was inhibited by both cycloheximideand actinomycin D. However, the increase in peroxidase activitywas inhibited by cycloheximide but was not inhibited by actinomycinD, which repressed RNA synthesis 60 to 70%. The increase inacid invertase activity was supressed by ethylene, while PALand peroxidase activities were stimulated. The results of cycloheximideco-treatment suggest that the fluctuation in enzyme activitiesby ethylene treatment involves de nove protein synthesis. Further,the effect of ethylene on enzyme activities was under competitionby carbon dioxide. The decrease in acid invertase activity atthe late stage of incubation was stimulated by cycloheximidetreatment, but that of PAL activty was prevented. The resultssuggest the existence of different inactivating systems betweenacid invertase and PAL. ¹ This paper constitutes Part 117 in the Series "The PhytopathologicalChemistry of Sweet Potato with Black Rot and Injury". (Received September 2, 1974; )     

Sequential Induction of mRNAs for Phenylalanine Ammonia-lyase in Slices of Potato Tuber

Induction of the mRNA that encodes for phenylalanine ammonia-lyase(PAL, EC 4.3.1.5 [EC] ), which catalyzes the first reaction in thebiosynthesis of a wide variety of phenylpropanoid natural productsfrom phenylalanine, was investigated in. wounded tuber tissuesof the potato (Solanum tuberosum L. cv. Irish Cobbler). Northernblot analysis showed that hybridizable RNA was not present inunwounded tissue, but the amount of hybridizable PAL-specificmRNA increased rapidly in the polysomal RNA fraction with asharp, high peak at the early stage (0 h to 6 h) and two broadlower peaks at the later stage (6 h to 48 h) of the wound response.Addition of actinomycin D to the tissue prevented the appearanceof hybridizable mRNA in the total RNA fraction, confirming thatthe increase resulted from synthesis of PAL mRNA de novo. Levelsof translatable PAL mRNA activity in vitro increased in thepolysomal RNA fraction in parallel with the changes in levelsof hybridizable mRNA, with a subsequent increase in levels ofPAL subunit polypeptides and enzymatic activity in wounded tissues.PAL subunits synthesized both in vivo and in vitro had the samemolecular masses, of about 79 kDa, on SDS-polyacrylamide gelelectrophoresis, but isoelectric focusing revealed the presenceof isoforms of the native tetrameric enzyme with different pIvalues and changes in the relative levels of the isoforms afterwounding. Furthermore, two-dimensional gel electrophoresis ofPAL subunits synthesized in vitro showed that at least eightmRNAs that encoded subunit isoforms with different pI valueswere expressed sequentially after wounding. (Received May 24, 1990; Accepted October 24, 1990)     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

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     

Induction by light of hydroxycinnamoyl-CoA: Quinate hydroxycinnamoyl transferase in buckwheat (Fagopyrum esculentum Moench): Absence of feed-forward control by trans-cinnamate

The light-induced increase in the activity of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) in hypocotyls of etiolated buckwheat seedlings is not suppressed by aminooxy acetic acid, an inhibitor of cinnamic acid formation in this tissue. Incubation of hypocotyls in 2 mmol l^-1 trans-cinnamate in the dark does not increase CQT activity. Thus, trans-cinnamate does not appear to mediate the effect of light on CQT in buckwheat hypocotyls.Abbreviations AOA aminooxy acetic acid - CA4H cinnamic acid-4-hydroxylase - CQT hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase - PAL L-phenylalanine ammonialyase     

Expression of phenylalanine ammonia-lyase gene during tracheary-element differentiation from cultured mesophyll cells ofZinnia elegans L.

Expression of the phenylalanine ammonialyase (PAL) gene during tracheary-element differentiation was studied in mesophyll cell-suspension cultures ofZinnia elegans. Dose-response curves of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) were obtained for the cultures in order to achieve the highest percentage of differentiation. The optimal concentrations of BA and NAA were 0.1 mg·l^-1 and 0.06 mg·l^-1, respectively, which normally stimulated about 40% differentiation by 96 h of culture. The effects of the same ratio but different amounts of BA and NAA on tracheary-element formation have been tested and the results indicate that the absolute amounts of BA and NAA rather than the ratio of them were important for tracheary-element formation in theZinnia cultures. The cells when cultured in the presence of 0.001 mg·l^-1 of BA and 0.06 mg·l^-1 of NAA expanded and divided but did not differentiate. The level of PAL activity, synthesis of PAL protein and the level of PAL mRNA peaked during 72 to 96 h when lignin was actively deposited. This indicated that the PAL gene was temporally and preferentially expressed in association with the lignification during tracheary-element differentiation and thus it can be regarded as a molecular marker for the process. We thank Bejing Agricultural University, the Lundgren Research fund (University of Cambridge) and an ORS award for financial support.     

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     

Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

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).     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

Five new geminal aminocycloalkanephosphonic acids ( 4 – 8 ) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia‐lyase (PAL). Within the set of compounds which are related to 2‐aminoindane‐2‐phosphonic acid (AIP, 3 ), a known powerful inhibitor of PAL, racemic 1‐aminobenzocyclobutene‐1‐phosphonic acid ( 4 ), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine‐derived anthocyanin synthesis in buckwheat.     

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria

Summary. Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG₂ [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.