Changes in Polyamine Titer in Etiolated Pea Seedlings Following Red Light Treatment

The polyamines agmatine, cadaverine, putrescine, spermidineand spermine were measured by means of thin layer chromatographyand high performance liquid chromatography in buds and in 5mm long subapical sections of the 3rd internode of 6-day-oldetiolated pea seedlings. The polyamine pattern of each organwas specific, relative quantities varying with age and growth.While agmatine, putrescine, spermidine and spermine were presentin buds and in tissues of the 3rd internode, cadaverine wasfound in the 3rd internode only. Concentrations of spermidineand spermine were higher in the bud than in the 3rd internode,and the highest putrescine titer was found in the internode.Short exposure of etiolated seedlings to red light (5 min) increasedbud development while inhibiting growth of the 3rd internode.In general, exposure to red light increased the titer of putrescine,agmatine and spermidine in the bud, whereas in the internodea reverse pattern was found, i.e., internodes of seedlings growingin the dark yielded higher titer of polyamines in general, andagmatine in particular. These results are particularly pronounced18 hr after exposure to red light. A link between phytochrome-controlledgrowth and polyamine titer is suggested. ² On sabbatical leave from the Hebrew University of Jerusalem,Department of Horticulture, Rehovot, Israel. ³ Supported by a grant from the Turkish Government; Permanentaddress: Department of General Botany, University of Istanbul,Suleymaniye, Istanbul, Turkey. ¹ Supported by a grant from NSF to A.W.G. (Received August 24, 1981; Accepted October 22, 1981)     

Polyamine Biosynthesis during Vegetative and Floral Bud Differentiation in Thin Layer Tobacco Tissue Cultures

Levels of putrescine, spermidine and spermine increase whenvegetative or floral buds form in cultures derived from surfaceexplants of inflorescences of Nicotiana tabacum L. cv. Wisconsin-38.Concomitantly, the activity of arginine decarboxylase (ADC)rises and that of ornithine decarboxylase (ODC) declines. DL--Difluoromethylarginine(DFMA), a specific suicide inhibitor of ADC, inhibits bud initiation,while DL--difluoromethylornithine (DFMO), the analogous suicideinhibitor of ODC, does not. On the other hand, DFMO inhibitsthe subsequent development of newly regenerated floral buds,while DFMA does not. It thus appears that polyamines derivedthrough ADC may be involved in bud initiation, while polyaminesderived through ODC are required for subsequent growth and developmentof such buds. Especially large increases of spermidine are associatedwith floral bud differentiation, indicating a possible specialmorphogenetic role for that polyamine. ¹Present address: Laboratori de Fisiologia Vegetal, Facultadde Farmacia, Universitat de Barcelona, 08028 Barcelona, Spain (Received April 25, 1988; Accepted August 12, 1988)     

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.     

Opposite effects of fusicoccin and IAA on putrescine synthesis of rice coleop tiles

Changes in polyamine biosynthesis and elongation of etiolated rice coleoptiles ( Oryza sativa L. cv. Taichung Native 1) in response to fusicoccin (FC) and indoleacetic acid (IAA) were investigated. FC stimulated coleoptile elongation at concentrations higher than 1 μ M but caused a decrease in the levels of free putrescine, spermidine and sper-mine, as well as in the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and S -adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50). The extent to which FC caused these effects was dependent on its concentration. Treatment with 100 μ M IAA also induced coleoptile elongation and resulted in a decrease in free spermidine/sper-mine and SAMDC activity. However, treatment with IAA resulted in an increase in free putrescine levels and ADC activity. The extent of coleoptile elongation and putrescine accumulation also depended on IAA concentration. α-Difluoromethylarginine (DFMA), an irreversible inhibitor of ADC. but not α-difluoromethylornithine (DFMO). an irreversible inhibitor of ODC (EC 4.1.1.17), inhibited the LAA-stimulated coleoptile elongation and putrescine accumulation. Addition of putrescine could not reverse the effect of DFMA.     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

Promotion by gibberellic Acid of polyamine biosynthesis in internodes of light-grown dwarf peas

When gibberellic acid (GA₃; 5-35 micrograms per milliliter) is sprayed on 9-day-old light-grown dwarf Progress pea (Pisum sativum) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA₃ on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA₃ also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA₃ or by AMO-1618.     

Purification and Synthesis under Anaerobic Conditions of Rice Arginine Decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19 [EC] ) which catalyzes thesynthesis of putrescine, is involved in the responses of plantsto stress. The enzyme was purified 1,561-fold from rice coleoptilesby steps that included ammonium sulfate fractionation, gel filtration,ion-exchange chromatography and chromatofocusing. The purifiedenzyme had a pI of 5.3, a molecular mass of 176 kDa and appearedto be composed of three subunits of 63 kDa. A polyclonal antibodywas raised in a rabbit and the IgG fraction was purified fromserum. On Western blots the antibody recognized the ADC fromboth rice and E. coli. Immunoprecipitation with the ADC-specificantibodies allowed detection of radiolabelled ADC in extractsfrom aerobically and anaerobically grown rice seedlings thathad been supplied with a mixture of ¹⁴C-amino acids. This resultis discussed in relation to the role of ADC under anaerobicconditions. (Received April 28, 1994; Accepted September 27, 1994)     

Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)     

Environmental and Chemical Control of Vegetative Dormant Bud Production in Hydrilla verticillata

Bud production was investigated using algal-free cultures ofthe aquatic weed, Hydrilla verticillata. Vegetative dormantbud production in hydrilla is stimulated by a shortened photoperiodand is not temperature-dependent between 15 and 30 C. The phytochromesystem appears to be involved in this process and ABA stimulatesbud production. Artificial illumination throughout the nightprevented dormant bud production. Ethylene, applied as ethephon,reduced bud production in greenhouse cultures by 80 per centwhen applied at a level of 0.1 mgl^–1 ethephon at 2-d intervals. Hydrilla verticillata, aquatic plants, dormant buds, ethephon, ethylene, abscisic acid     

Polyamine Biosynthetic Enzymes in Chlorella: Characterization of Ornithine and Arginine Decarboxylase

In a Chlorella culture grown asynchronously under autotrophicconditions, two biosynthetic enzymes of putrescine—ornithinedecarboxylase (ODC) and arginine decarboxylase (ADC)—weredetected. Both enzymes require pyridoxal phosphate and dithiothreitolfor their activity but differ in their optimal pH, the ionicstrength of their buffer, temperature of inactivation, and Km.In addition, L-canaline was found to inhibit the activity ofODC but not that of ADC. During the logarithmic phase of growth,ODC activity increased sharply, then decreased before the onsetof the stationary phase. ADC activity changed only slightlyduring growth. ³The work was performed in partial fulfillment of the requirementsfor the Ph.D. Thesis of E.C. (Received September 25, 1982; Accepted June 1, 1983)     

Pith Autolysis in Plants: IV. The Activity of Polygalacturonase and Cellulase during Drought Stress Induced Pith Autolysis

The water potential, amount of pith autolysis and activitiesof apoplastic cellulase and polygalacturonase of tomato stemswere measured during 24 h of drought stress (DS) and for 24h following reirrigation. During DS the water potential droppedfrom —5.5 to —10.4 bars and rose to —8.3 barssoon after reirrigation. Drought stress induced considerablepith autolysis, more of which occurred after reirrigation. Pretreatmentwith mechanical perturbation (MP) of the stems or applicationof exogenous ethephon on the buds hardened the tomato plantsagainst DS-induced pith autolysis. Drought stress caused anincrease in apoplastic polygalacturonase and an even greaterincrease in apoplastic cellulase. Reirrigation caused a largetransient increase in the former and a decrease in the latter.The apoplastic reducing sugar content (as galacturonic acid)of the stem rose in parallel with the activity of the enzymes.Both DS and MP caused an increase in ethylene evolution, althoughthe former was significantly greater than the latter. However,when MP preceded DS, the amount of ethylene produced was significantlyless than DS alone induced. Pretreatment with either MP or exogenousethephon inhibited the increase in apoplastic cellulytic enzymes. It is concluded that DS induces ethylene evolution from thetomato stem, causing an increase in the stem apoplastic cellulyticenzymes, which in turn start the autolysis of the pith cellwalls. Pretreatment with MP or ethephon, each of which inducesethylene evolution, hardens the stem so that it does not producemore ethylene during DS, and thus becomes resistant to DS-inducedpith autolysis. ¹Supported by Bi-national Agricultural Research and Developmentgrant I-127, NASA grant NAGW 96 and NSF grant 8003689to MJJ. ²Permanent address: Horticulture Department, Faculty of Agriculture,The Hebrew University Rehovot, Israel ³Permanent address: Vegetable Crops Department, AgriculturalResearch Organization, the Volcani Center Bet Dagan, Israel     

Arginine and ornithine decarboxylases, the polyamine biosynthetic enzymes of mung bean seedlings

General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. alpha-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity.High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio.It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process.     

Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation

We studied the effects of dl-α-difluoromethylarginine (DFMA) and dl-α-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.     

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Summary Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL -difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL -difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - DFMA DL -difluoro-methylarginine - DFMO DL -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

Polyamine metabolism,floral initiation and floral development in Chrysanthemum (Chrysanthemum morifolium Ramat.)

In the short-day plant Chrysanthemum (Chrysanthemum morifolium Ramat. variety Pavo) putrescine and spermidine conjugates appeared in the apical bud before the first observable transformation of the meristem into floral structures. These compounds accumulated on floral initiation and well before floral evocation. Spermidine conjugates were predominant during floral initiation whereas free amines did not accumulate to any significant extent. Different associations of amides were observed during floral initiation as compared with the reproductive phase. 3,4-Dimethoxyphenethylamine conjugates (water-insoluble compounds) were the predominant amine conjugates observed during flower development. These compounds decreased drastically after fertilization. In vegetative buds from plants grown in long days polyamine conjugates were very low and appeared as plants aged. We present evidence that ornithine decarboxylase (ODC) regulates putrescine biosynthesis during floral initiation and floral development. When ODC action was blocked by DFMO (-DL-difluoromethylornithine, a specific, irreversible inhibitor of ODC), flowering was inhibited, and free and conjugated polyamines were not detected. This treatment led to a slight enhancement of ADC activity. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with DFMA (-DL difluoromethylarginine, a specific, irreversible inhibitor of ADC) did not affect flowering and the polyamine titers. The results suggest that ODC and polyamine conjugates are involved in regulating floral initiation in Chrysanthemum.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)     

Tiller Bud Suppression in Reproductive Plants of Lolium multiflorum Lam. Cv. Westerwoldicum

The control of tiller bud growth during reproductive developmentwas investigated in experimental plants ofLolium multiflorumLam. cv. Westerwoldicum that were reduced to a main axis havinga developing but unemerged ear, elongating stem internodes,a series of expanded leaves, slow-growing tiller buds and aroot system. Isolation of the ear by excision of its base, ordecapitation so as to remove the ear together with the upperleaves, promoted the movement of ¹⁴C-assimilates to tiller buds,decapitation being the more effective treatment. Applicationof 0.1 per cent indol–3yl-acetic acid (IAA) to cut tissuesof decapitated plants diverted ¹⁴C-assimilates to upper internodesbut did not reduce import by buds, whereas application of 1.0per cent IAA both diverted labelled assimilates to upper internodesand reduced bud import. Radioactivity from [¹⁴C] IAA appliedto the upper leaves or to the ear base was recovered from budsin very small amounts; larger amounts were recovered from budsfollowing the application of labelled IAA to an elongating internode,especially from the bud at the base of the treated internode.It is suggested that tiller bud suppression may be influencedby the movement of inhibitory levels of auxin into buds fromnearby elongating stem internodes, whose activity in turn maybe controlled by the developing inflorescence and upper leaves.     

Epicormic Bud Development in Quercus robur L. Studies of Endogenous IAA, ABA, IAA Polar Transport and Water Potential in Cambial Tissues10

Seasonal measurements of IAA,³ made using GC-MS, ⁴ indicatedthat in Q. robur the spring initiation of cambial activity andonset of visible bud outgrowth in the canopy is preceded byan increase in cambial region IAA. The effects of notch-girdlescut into the bole indicated that IAA in the cambial region laterwas present in separate physiological pools, with only the polar-transportedfraction affecting epicormic bud outgrowth. The stage in thespring when the epicormic buds grew out coincided with an increaseboth in cambial region IAA and in the capacity of cambial explantsfor IAA polar transport. Thus the stimulus needed by the epicormicbuds to overcome inhibition by polar-transported IAA appearedto be self-generated. The observed effects of exogenous hormoneson epicormic bud outgrowth from stem explants indicated thatthis stimulus might be cytokinin. The seasonal changes detectedin cambial region ABA³ were consistent with a role for stress-inducedABA in the induction of epicormic bud dormancy after canopydevelopment during the summer. No consistent effects of standthinning on cambial region IAA, ABA, water potentials or watercontents were detected, although polar transport of exogenousIAA by cambial region explants removed in the spring was reducedby thinning. Key words: Epicormic buds, cambium, hormones