Formation of putrescine and cinnamoyl putrescines in tobacco cell cultures

A p-fluorophenylalanine- (PFP) resistant cell line of Nicotiana tabacum and wild type cells accumulating high and low levels of cinnamoyl putrescines, respectively, were used to study the formation of putrescine in the biosynthesis of cinnamoyl putrescines. Labelled arginine and ornithine were equally well incorporated into the main conjugates caffeoyl and feruloyl putrescine. Trapping experiments indicated that both amino acids were decarboxylated for putrescine biosynthesis. Nearly all alcohol-extractable radioactivity from the labelled amino acids was found as cinnamoyl putrescines in the PFP-resistant cell line, whereas wild type cells retained significant radioactivity in the amino acids. The enzyme activities of arginine and ornithine decarboxylases in the resistant cell line were increased 3- to 6-fold.     

Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.     

Transport kinetics and metabolism of exogenously applied putrescine in roots of intact maize seedlings

Putrescine metabolism, uptake, and compartmentation were studied in roots of hydroponically grown intact maize (Zea mays L.) seedlings. In vivo analysis of exogenously applied putrescine indicated that the diamine is primarily metabolized by a cell wall-localized diamine oxidase. Time-dependent kinetics for putrescine uptake could be resolved into a rapid phase of uptake and binding within the root apoplasm, followed by transport across the plasma membrane that was linear for 30 to 40 minutes. Concentration-dependent kinetics for putrescine uptake (between 0.05 and 1.0 millimolar putrescine) appeared to be nonsaturating but could be resolved into a saturable (V_max 0.397 micromoles per gram fresh weight per hour; K_m 120 micromolar) and a linear component. The linear component was determined to be cell wall-bound putrescine that was not removed during the desorption period following uptake of [³H]putrescine. These results suggest that a portion of the exogenously applied putrescine can be metabolized in maize root cell walls by diamine oxidase activity, but the bulk of the putrescine is transported across the plasmalemma by a carrier-mediated process, similar to that proposed for animal systems.     

Correlation between Polyamines and Pyrrolidine Alkaloids in Developing Tobacco Callus

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) α-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) α-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.     

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines

The incorporation of [¹⁴C]arginine and [¹⁴C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.

Both [¹⁴C]arginine and [¹⁴C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.

It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

     

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Diamine Oxidase from Cultured Roots of Hyoscyamus niger: Its Function in Tropane Alkaloid Biosynthesis

Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective K_m values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants V_max/K_m for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.     

Metabolism of polyamines in mouse neuroblastoma cells in culture: formation of GABA and putreanine.

—Polyamine metabolism of mouse neuroblastoma cells grown in culture was studied with special reference to the synthesis of GABA from putrescine and putreanine from spermidine. This study shows that neuroblastoma cells in the presence of a complete culture medium containing calf serum readily metabolized [¹⁴C]putrescine to GABA; the rate of synthesis is similar to the rate of synthesis of spermidine from putrescine. In the absence of serum the conversion of putrescine to GABA is minimal. In the presence of serum GABA formation is completely inhibited by the diamine oxidase inhibitor aminoguanidine. GABA synthesis does not occur in the absence of cells. The GABA synthesized is not readily metabolized to succinate or homocarnosine. Mouse neuroblastoma cells metabolized [¹⁴C]ornithine to putrescine, GABA, and spermidine. Spermidine was metabolized to putrescine, putreanine and spermine.     

Diamine Oxidase Activity in Different Physiological Stages of Helianthus tuberosus Tuber

Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Δ¹-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the K_m of the enzyme for putrescine was of the order of 10⁻⁴ molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Immunoaffinity purification and characterization of diamine oxidase from Cicer

Antiserum specific for diamine oxidase (DAO;EC 1.4.3.6) from Lens culinaris cross-reacted with DAO from several other members of the Leguminosae when tested by agar double diffusion. Antibodies purified by affinity chromatography were used to make an immunoadsorbent for the one-step purification of DAO from various species of the Leguminosae. This technique has made it possible to purify in one step the already characterized DAO from pea and lentil, and the unknown diamine oxidase from Cicer arietinum. This enzyme was partially characterized; it showed a pH optimum of 7.5 with putrescine as substrate and followed typical Michaelis-Menten kinetics with a K_m of 2.4 × 10^?4 M. Copper ligands and carbonyl group-directed reagents inhibited the enzyme.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [¹⁴C]Δ¹-pyrroline formation from [¹⁴C] putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased Δ¹-pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with yield of Δ¹-pyrroline and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rats prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. These results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzyme rather than to a slowing of its degradation.     

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH₄Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations > 1 mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis–Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (K_M) = 190 μM and maximum rate (k_cat) = 21.8 s^?1 for the oxidative deamination of putrescine with a lower K_M (=60 μM) and comparable k_cat (=18.2 s^?1) for the copper oxidase. MALDI–TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.