Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

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).     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.     

Ethylene biosynthesis in Regnellidium diphyllum and Marsilea quadrifolia

The pathway of ethylene biosynthesis was examined in two lower plants, the semi-aquatic ferns Regnellidium diphyllum Lindm. and Marsilea quadrifolia L. As a positive control for the ethylene-biosynthetic pathway of higher plants, leaves of Arabidopsis thaliana (L.) Heynh. were included in each experiment. Ethylene production by Regnellidium and Marsilea was not increased by treatment of leaflets with 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene in higher plants. Similarly, ethylene production was not inhibited by application of aminoethoxyvinylglycine and -aminoisobutyric acid, inhibitors of the ethylene biosynthetic enzymes ACC synthase and ACC oxidase, respectively. However, ACC was present in both ferns, as was ACC synthase. Compared to leaves of Arabidopsis, leaflets of Regnellidium and Marsilea incorporated little [¹⁴C]ACC and [¹⁴C]methionine into [¹⁴C]ethylene. From these data, it appears that the formation of ethylene in both ferns occurs mainly, if not only, via an ACC-independent route, even though the capacity to synthesize ACC is present in these lower plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - AIB -aminoisobutyric acid - AVG aminoethoxyvinylglycine This research was supported by the U.S. Department of Energy through grant No. DE-FG02-91ER20021 and, in part, by a fellowship of the National Engineering and Research Council of Canada to Jacqueline Chernys.     

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip Apeldoorn have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS sodium dodecyl sulfate Some of the results were presented at the meeting of German Botanical Society in Freiburg, FRG, September 1982, and at the meeting of the Groupe Polyphenols in Toulouse, France, September/October 1982     

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Detection of the putative<Emphasis Type="Italic"> cis-</Emphasis>region involved in the induction by a<Emphasis Type="Italic"> Pyricularia oryzae</Emphasis> elicitor of the promoter of a gene encoding phenylalanine ammonia-lyase in rice

A rice PAL (phenylalanine ammonia-lyase) gene sequence (rPAL-P5), which is highly similar to and likely the same as a previously described rice ZB8PAL gene, including the 5-upstream and exon I coding regions of PAL, was isolated using PCR amplification. The expression of several PALs, including rPAL-P5, was strongly induced following inoculation with Pyricularia oryzae or treatment with a P. oryzae elicitor. To identify the promoter region induced by the P. oryzae elicitor, we constructed and subsequently transformed rPAL-P5 promoter deletion series into rice calli using particle bombardment. Results from both elicitor-inducible reporter gene and gel mobility shift assays demonstrated that the sequence –349 to –256 of the rPAL-P5 promoter includes a cis-element involved in the induction of P. oryzae.Abbreviations CTAB Cetyltrimethylammonium bromide - 2,4-D 2,4-Dichlorophenoxyacetic acid - GUS -Glucuronidase - 4-MU 4-Methylumbelliferone - 4-MUG 4-Methylumbelliferyl glucuronide - NOS Nopaline synthase - PAL Phenylalanine ammonia-lyase Communicated by J.C. Register III     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

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     

Rapidly induced ethylene formation after wounding is controlled by the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis

Bean leaves from Phaseolus vulgaris L. var. Pinto 111 react to mechanical wounding with the formation of ethylene. The substrate for wound ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC). It is not set free by decompartmentation but is newly synthesized. ACC synthesis starts 8 to 10 min after wounding at 28°C, and 15 to 20 min after wounding at 20°C. Aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene formation from methionine via ACC, inhibits wound ethylene synthesis by about 95% when applied directly after wounding (incubations at 20°C). AVG also inhibits the accumulation of ACC in wounded tissue. AVG does not inhibit conversion of ACC to ethylene. Wound ethylene production is also inhibited by cycloheximide, n-propyl gallate, and ethylenediaminetetraacetic acid.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine - EDTA ethylenediaminetetraacetic acid     

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese,a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn²⁺ substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn²⁺ enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl₂. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The R_F of cofactor on cellulose plates developed in butanol: acetic acid: H₂O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl₂. Chelators, anti-oxidants, and catalase were inhibitors of Mn²⁺-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95–98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH₄)₂SO₄-fractionation.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid - DDTC diethyldithiocarbamate     

Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.     

Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs

The increase in ethylene formation and in 1-aminocyclopropane-1-carboxylic acid (ACC) content in flavedo tissue of grapefruit (Citrus paradisi Macfad. cv. Ruby Red) in response to excision was markedly inhibited by exogenous ethylene. Ethylene treatment inhibited the synthesis of ACC, but increased the tissue's capability to malonylate ACC to N-malonyl-ACC, resulting in further reduction in the endogenous ACC content. The development of extractable ACC-malonyl-transferase activity in the tissue was markedly promoted by treatment with exogenous ethylene. These results indicate that the autoinhibition of ethylene production in this tissue results not only from suppression of ACC synthesis, but also from promotion of ACC malonylation; both processes reduce the availability of ACC for ethylene synthesis.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine (2-amino-4-(2-aminoexthoxy)-trans-3-butenoic acid) - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid     

The effect of light pretreatments on phytochrome-mediated induction of anthocyanin and of phenylalanine ammonia-lyase

Induction by light of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and of anthocyanin in cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by a light pretreatment which operates through phytochrome. If PAL or anthocyanin is induced by a light pulse, the effectiveness of phytochrome (P_fr) is strongly increased by a light pretreatment; however, if the increase of the PAL level or synthesis of anthocyanin is elicited by continuous far-red light (operating via phytochrome in the High Irradiance Response), effectiveness of light is strongly reduced by the same light pretreatment. This reduction of effectiveness is correlated with a decrease of total phytochrome (P_tot) caused by the light pretreatment. It is argued that the observations are compatible only with the open phytochrome-receptor model as suggested by Schäfer (J. Mathem. Biol. 2, 41–56, 1975). The peaks of the time courses of the PAL levels under continous far-red light are located at 48 h after sowing and do not depend on the original level of phytochrome. The decrease of the PAL levels beyond 48 h after sowing takes place independently of phytochrome and of the actual level of PAL.Abbreviations P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - P_tot total phytochrome (P_r+P_fr) - {ie369-1} [P_fr] /[P_tot], photoequilibrium of phytochrome at wavelength - HIR High Irradiance Response - PAL phenylalanine ammonialyase (EC 4.3.1.5)     

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl₂; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid