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.     

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.     

Increased formation of cinnamoyl putrescines by fedbatch fermentation of cell suspension cultures of Nicotiana tabacum

The tobacco cell line TX1 (Nicotiana tabacum) accumulates up to 10 mg cinnamoyl putrescines/g dry weight. This level was further increased under various growth limiting conditions. The negative effect of accumulated phosphate on the optimal expression of cinnamoyl putrescine biosynthesis was prevented by the fedbatch fermentation technique. A batch fermenter yielded 160 mg cinnamoyl putrescines/1 while a phosphate fedbatch fermentation produced 400 mg/1. This was mainly due to the fact that P in the cells was kept low during the whole growth cycle.Abbreviation 2,4-D 2,4-Dichlorophenoxyacetic acid     

A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Metabolism of l[U-14C]-arginine and l[U-14C]-ornithine in maturing and vernalised embryos and megagametophytes of Picea abies

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these 14 C-labelled amino acids into liberated CO₂, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.     

Characterization of a carrot callus line resistant to high concentrations of putrescine

A mutant carrot callus line resistant to a high concentration (1 mM) of putrescine has been isolated. A high level of endogenous putrescine, about 13-fold higher than in the controls grown in the absence of putrescine, characterized this resistant mutant. Ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were similar in the resistant line and in the controls by the fifth subculture. The uptake of putrescine when supplied to the medium at high concentration (1 mM), was similar in both the putrescine-treated calli and in the untreated controls. At low concentrations (0.64 M) however the putrescine absorbed by the resistant calli was less than that absorbed by the controls. Putrescine uptake took place almost always against a concentration gradient and might be due to an active mechanism.Abbreviations ADC arginine decarboxylase - 2,4D 2,4-dichlorophenoxyacetic acid - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - TCA trichloroacetic acid - TLC thin layer chromatography     

Mg2+ restores membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate movements

Cellular ornithine biosynthesis could be expected to play a significant role in putrescine formation and hence in growth. Two enzymes are involved in ornithine biosynthesis: arginase and transamidinase. These enzyme activities were studied in two human melanoma cell lines differing in their K_m of diamine oxidase for putrescine and in their tumorigenicity in nude mice. Arginase activity accounts for the majority of ornithine formed in the highly tumorigenic cell line, while the majority of ornithine is derived from transamidinase action in the poorly tumorigenic cell line, with concomitant formation of methyl guanidine, a potent inhibitor of diamine oxidase.     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : I. Metabolism of l-Serine

Three levels of free amines and the activities of their biosynthetic enzymes were measured in subcellular fractions of two cell lines of Nicotiana tabacum L. cv Xanthi. The TX4 cell line, a p-fluorophenylalanine resistant culture which accumulates high levels of cinnamoylamides, was compared to the wild-type culture TX1. In cells harvested on day 6 of the growth cycle, nearly all free putrescine, spermidine, and tyramine was found in the supernatant fraction of both cell lines. Although a consistent portion of ornithine decarboxylase activity was detected in the nuclear-enriched fractions of TX1 and TX4, the largest levels of activity were in the supernatants of both lines. In TX1, arginine decarboxylase activity was low relative to that of ornithine decarboxylase, but, in the TX4 line arginine decarboxylase levels in the cytosol were substantially elevated. Tyrosine decarboxylase was not detected in 6-day-old TX1 cells, but significant amounts of activity were measured in the 1000g and supernatant fractions of TX4. S-Adenosylmethionine decarboxylase activity was low in both cell lines and was located predominantly in the supernatant.     

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.

     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.     

Putrescine Accumulation in Wine: Role of Oenococcus oeni

Putrescine, the most abundant biogenic amine in wine, was proved to be produced by Oenococcus oeni strains in wine not only from ornithine but also from arginine. In this case, putrescine may originate from strains possessing the complete enzyme system to convert arginine to putrescine or by a metabiotic association, with an exchange of ornithine, between strains capable of metabolizing arginine to ornithine but unable to produce putrescine and strains capable of producing putrescine from ornithine but unable to degrade arginine. Putrescine production by this metabiotic association occurred once the malolactic fermentation was completed, whereas conversion of ornithine to putrescine by a single culture of the ornithine decarboxylating strain concurred with the degradation of malic acid. Moreover, in the former case, putrescine formation proceeded more slowly than in the latter. Metabiosis may play an important role in the accumulation of putrescine in wine, arginine being one of the major amino acids found in wine.     

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.     

Studies on the biosynthesis of sym-homospermidine in sandal (Santalum album L.)

The biosynthesis of the newly isolated polyamine, sym-homospermidine (NH(2)-[CH(2)](4)-NH-[CH(2)](4) -NH(2)), was studied by using radioactive amino acids. Arginine was the most effective precursor, being about 10 times as active as ornithine. Unlabelled agmatine and putrescine markedly inhibited the incorporation of [(14)C]arginine into homospermidine. Similarly the incorporation of ornithine was inhibited by unlabelled arginine and putrescine. gamma-Aminobutyraldehyde, the oxidation product of putrescine, was considered to be one of the intermediates in the biosynthesis of homospermidine. The biosynthesis may involve a Schiff-base formation of putrescine with gamma-aminobutyraldehyde and subsequent reduction. A limited synthesis of spermidine also takes place under these conditions.     

The role of polyamines in potato tuber formation

Summary The involvement of free and conjugated polyamines in tuber formation was studied in in vitro cultured node explants ofSolanum tuberosum cv. Superior. Tubers developed from the axillary buds in 100% of the explants cultured in MS medium containing high sucrose levels and supplemented with kinetin (Kin) and chlorocholine chloride (CCC). The addition of growth regulators was not essential for tuber formation, although smaller tubers were formed in the medium devoid of Kin and CCC. Tuber formation was inhibited in about 75% of node explants treated with 0.5 mM difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase. The inhibitory effect of DFMO was almost completely reversed by putrescine addition. Addition of difluoromethylarginine (DFMA), the analogous inhibitor of arginine decarboxylase, had no effect on tuber formation. DFMO, but not DFMA, also inhibited the development of axillary buds into shoots in light-grown node explants. Aminooxyphenylpropionic acid (0.1 to 0.25 mM), an inhibitor of phenylalanine ammonia lyase, caused a sharp reduction in cinnamoyl putrescines, but had no effect on tuber formation. Our results suggest that hydroxycinnamic acids are not causal in tuber formation but may serve as polyamine storage pools. Our findings support the hypothesis that polyamines derived via the ornithine decarboxylase-mediated pathway are necessary for tuber formation in vitro, probably at the early phase of morphogenesis involving active cell division.     

Comparative role of polyamines division and plastid differentiation of Euglena gracilis

Regulation of polyamine biosynthesis during growth and differentation of Euglena gracilis was investigated. Increased activity of l-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotropically or photoautotrophically grown cells. In photoatotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition from quiescence of active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decaboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena, i.e., a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. α-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed only by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Accumulation of putrescine and related amino acids in potassium deficient Sesamum

Sesamum indicum was grown in complete or potassium deficient nutrient solution and amino acids, amines, nitrogen and potassium were determined weekly in the leaves. The incorporation of l-arginine-[U-¹⁴C] into protein was also followed. The interconversions of the amino acids of the ordithine-urea cycle, and their contribution to the formation of amines, were studied in cell-free extracts and intact leaves using labelled amino acids. As the level of potassium in the leaves decreased, the levels of the amino acids ornithine, citrulline and arginine, and of the amines putrescine, N-carbamylputrescine and agmatine increased. Potassium deficiency also reduced the rate of protein synthesis. Putrescine appears to be formed preferentially from citrulline with N-carbamylputrescine as intermediate.     

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.