Polyamines,floral induction and floral development of strawberry (Fragaria ananassa Duch.)

In the short-day plant, strawberry (Fragaria ananassa Duch.), polyamines (putrescine, spermidine and spermine), conjugated spermidine (water-insoluble compounds) and bound amines (putrescine, spermidine, phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine) accumulated in the shoot tips during floral induction and before floral emergence. Different associations of free amines and conjugated amines were observed during floral induction, as compared with the reproductive phase. During the whole period of floral development, phenylethylamine (an aromatic amine) was the predominant amine, representing 80 to 90% of the total free amine pool. Phenylethylamine conjugates (water-insoluble compounds) were the predominant amides observed prior to fertilization. These substances decreased drastically after fertilization. In vegetative shoot tips from plants grown continously under long days, free polyamines (putrescine, spermidine) and bound polyamines (putrescine, spermidine) were low and no change was observed. Free amines (spermine and phenylethylamine), bound aromatic amines (phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine), conjugated spermidine and phenylethylamine did not appear. Male-sterile flowers were distinguished by their lack of conjugated spermidine and phenylethyalamine and by a decrease in free phenylethylamine. In normal and sterile strawberry plants -DL-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), caused inhibition of flowering and free and polyamine conjugates. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with -DL-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), did not affect flowering and polyamine titers. These results suggest that ornithine decarboxylase (ODC) and polyamines are involved in regulating floral initiation in strawberry. The relationship between polyamines, aromatic amines, conjugates, floral initiation and male sterility is discussed.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - Put putrescine - Spd spermidine - Spm spermine - Phen phenylethylamine - 3H4M Phen 3-hydroxy, 4-methoxyphenylethylamine     

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 during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Polyamines and related enzymes in rice seeds differing in germination potential

In ungerminated rice seeds, (Japonica rice variety, CV Tapei 309), the content of free amines (putrescine, spermidine, spermine, tyramine) was higher in seed lots having a low germination frequency compared to those with high germination potential. Conversely, amine conjugates (di-feruloylputrescine, di-feruloylspermidine, diferuloyldiaminopropane and feruloyltyramine) decreased with loss of viability. Thus, these compounds appeared to constitute biochemical markers of seed viability. In seeds with high germination potential, conjugates decreased drastically during germination, with an early and rapid increase in free amines (putrescine, spermidine, tyramine). Arginine decarboxylase (ADC) activity was highest during the germination of high germination potential seeds, its activity gradually declining with loss of viability and being closely correlated with agmatine content. The polyamine biosynthetic inhibitors (-DL-difluoromethylarginine, DFMA, a specific and irreversible inhibitor of ADC; -DL-difluoromethylornithine, DFMO, a specific irreversible inhibitor of ornithine decarboxylase (ODC); cyclohexylammonium sulfate, CHA, inhibitor of spermidine synthase) neither depleted putrescine and spermidine levels nor inhibited germination in high germination potential seeds. In low germination potential seeds, the germination process was inhibited by DFMA or CHA. Application of agmatine resulted in a reversal of inhibition. DFMA inhibited ADC activity in both categories of seeds. In low germination potential seeds treated with CHA no ADC activity was found. These results suggest that amines are involved in the germination process of rice seeds. It appears that amine conjugates may serve as a storage form of amines which, upon enzymatic hydrolysis, could supply the cell with an additional amine reserve and influence cell division and/or cell elongation.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

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)     

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.Abbreviations ADC arginine decarboxylase - Dap diaminopropane - DFMA -difluoromethylarginine - MGBG methylglyoxal-bis-guanylhydrazone - MTA 5-deoxy-5-methylthioadenosine - ODC ornithine decarboxylase - PA polyamines - PAO polyamine oxidase - PCA perchloric acid - PLP pyridoxal phosphate - Put putrescine - SAM S-adenosylmethionine - dSAM decarboxylated S-adenosylmethionine - SAMDC S-adenosylmethionine decarboxylase - Spd spermidine - Spm spermine     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL--difluoromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL--difluoromethylornithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.     

Modulator effect of DL-α-difluoromethylarginine treatments on differentiation processes of young maize calluses

The effect of pretreatments with 0.5, 1, 3, 5 and 10 mM of DL--difluoromethylarginine (DFMA), an irreversible suicide inhibitor of the arginine decarboxylase (ADC) activity, on the differentiation process of young maize calluses was studied. Callus protein, total polyamine content and ADC activity decreased versus control in all the assayed treatments. Furthermore, ornithine decarboxylase (ODC) activity was significantly lower in the treated calluses, which was probably due to the arginase activity detected in them. Short tratments at high doses of DFMA significantly increased the number of regenerated buds versus the control (four times more in 10 mM and almost two times more in 5 mM). By contrast, long treatments at low doses (0.5, 1 and 3 mM) reduced the number of plantlets. Conjugated putrescine seems to be implicated in the regeneration response of control and high DFMA-treated calluses.     

Recent advances in the understanding of polyamine functions during plant development

Suggested roles for polyamine function, and the evidence for these functions, is reviewed. These include membrane stabilization, free radical scavenging, effects on DNA, RNA and protein synthesis, effects on the activities of RNase, protease and other enzymes, the interaction with ethylene biosynthesis, and effects on second messengers. It is concluded that in addition to interacting with plant hormones, polyamines are able to modulate plant development through a fundamental mechanism(s) common to all living organisms.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ADC arginine decarboxylase - Chl chlorophyll - DAP diaminopropane - DFMA DL--difluoromethylarginine - DFMO DL--difluoromethylornithine - PAs polyamines - Put putrescine - SAM S-adenosylmethionine - Spd spermidine - Spm spermine     

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)     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Cytological events induced by the inhibition of polyamine biosynthesis in thin cell layers of tobacco

Summary The proliferative growth of thin cell layers ofNicotiana tabacum cultured on a rhizogenic medium was markedly disturbed when polyamine biosynthesis was inhibited. Treatments with polyamine inhibitors led to cell expansion, accompanied by thinning of the cell wall and inhibition of cell division, and frequent cases of nucleolar extrusion, mainly in the parenchymal layer in contact with the medium. Nucleolar extrusion was not correlated with cell expansion. The highest incidence of nucleolar extrusion occurred when the pathways of putrescine biosynthesis were inhibited and when spermidine synthesis, via S-adenosylmethionine decarboxylase, was blocked. The duration of the growth phase with nuclear amitotic divisions was prolonged in the presence of the inhibitors and root meristem formation delayed. When polyamines were added with the inhibitors, all reactions proceeded as in the controls.Abbreviations CHA cyclohexylamine - DFMA DL--difluoromethyl-arginine - DFMO DL--difluoromethylornithine - LS longitudinal section - MGBG methylglyoxal-bis(guanylhydrazone) - PA polyamine - Pu putrescine - RLS radial longitudinal section - S.E. standard error - Spd spermidine     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

The effect of submergence,ethylene and gibberellin on polyamines and their biosynthetic enzymes in deepwater-rice internodes

Submergence and treatment with ethylene or gibberellic acid (GA₃) stimulates rapid growth in internodes of deepwater rice (Oryza sativa L. cv. Habiganj Aman II). This growth is based on greatly enhanced rate of cell-division activity in the intercalary meristem (IM) and on increased cell elongation. We chose polyamine biosynthesis as a biochemical marker for cell-division activity in the IM of rice stems. Upon submergence of the plant, the activity of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in the IM increased six- to tenfold within 8 h; thereafter, SAMDC activity declined. Arginine decarboxylase (ADC; EC 4.1.1.19) showed a similar but less pronounced increase in activity. The activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in the IM was not affected by submergence. The levels of putrescine and spermidine also rose in the IM of submerged, whole plants while the concentration of spermine remained low. The increase in SAMDC activity was localized in the IM while the activity of ADC rose both in the node and the IM above it. The node also contained low levels of ODC activity which increased slightly following submergence. Increased activities of polyamine-synthesizing enzymes in the nodal region of submerged plants probably resulted from the promotion of adventitious root formation in the node. Treatment of excised rice-stem sections with ethylene or GA₃ enhanced the activities of SAMDC and ADC in the IM and inhibited the decline in the levels of putrescine and spermidine. We conclude that SAMDC and perhaps also ADC may serve as biochemical markers for the enhancement of cell-division activity in the IM of deepwater rice.Abbreviations ADC arginine decarboxylase - GA gibberellin - IM intercalary meristem - ODC ornithine decarboxylase - SAM S-adenosylmethionine - SAMDC SAM decarboxylase