Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco.

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N(6)-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation.

Binding of spermidine to a unique protein in thin-layer tobacco tissue culture.

The mechanism by which spermidine induces the appearances of floral buds in thin-layer tobacco (Nicotiana tabacum) tissue culture was studied by following the fate of the radioactive compound. [3H]Spermidine was taken up rapidly by the tissue, and after a brief lag, a portion was bound to trichloroacetic acid precipitable macromolecules. Such binding increased to a maximum on day 4 of culture, coinciding with the onset of bud differentiation, and declined thereafter until shortly before flowering. About 82% of the label in the trichloroacetic acid precipitate remained as spermidine, 14% was metabolized to putrescine, 3% to spermine, and 1% to gamma-aminobutyric acid. Spermidine was covalently bound to a protein with a molecular size of about 18 kilodaltons. Hydrolysis of this protein and analysis of the labeled entities revealed 81% spermidine, 16% putrescine, and 3% spermine. This post-translational modification of a unique protein by attachment of spermidine may be causally connected to the appearance of flower buds in thin-layer tobacco cultures.

Spermidine and flower-bud differentiation in thin-layer explants of tobacco.

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.

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-alpha-diffluromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL-alpha-difluromethylorinithine (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.

Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas.

When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.

Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

Cadmium-induced accumulation of putrescine in oat and bean leaves.

The effects of Cd2+ on putrescine (Put), spermidine (Spd), and spermine (Spm) titers were studied in oat and bean leaves. Treatment with Cd2+ for up to 16 hours in the light or dark resulted in a large increase in Put titer, but had little or no effect on Spd or Spm. The activity of arginine decarboxylase (ADC) followed the pattern of Put accumulation, and experiments with alpha-difluoromethylarginine established that ADC was the enzyme responsible for Put increase. Concentrations of Cd2+ as low as 10 micromolar increased Put titer in oat segments. In bean leaves, there was a Cd(2+)-induced accumulation of Put in the free and soluble conjugated fractions, but not in the insoluble fraction. This suggests a rapid exchange between Put that exists in the free form and Put found in acid soluble conjugate forms. It is concluded that Cd2+ can act like certain other stresses (K+ and Mg2+ deficiency, excess NH4+, low pH, salinity, osmotic stress, wilting) to induce substantial increases in Put in plant cells.

Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa.

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.

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) alpha-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) alpha-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.

Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content.

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg(2+) to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca(2+) to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca(2+) before exerting its antisenescence action.

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves : II. Inhibition of Ethylene Biosynthesis and Possible Mode of Action.

The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methionine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chl loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity; thus, treatments which promote deterioration of membranes inhibit both processes. Ca(2+) in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence.

Relation of polyamine synthesis and titer to aging and senescence in oat leaves.

Polyamine biosynthesis in senescing leaves of Avena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during light exposure of excised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the light, irrespective of senescence. S-Adenosyl-l-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and decline with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of light exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spermidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development.

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers.

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.

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

When gibberellic acid (GA(3); 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 GA(3) 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. GA(3) 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 GA(3) or by AMO-1618.The results support the hypothesis that ADC and polyamine content are important regulators of plant growth.

Polyamine oxidase in oat leaves: a cell wall-localized enzyme.

The localization and activity of polyamine oxidase (PAO; EC 1.5.3.3), was investigated in leaves and protoplasts of oat seedlings. Activity of the enzyme is highest with spermine as substrate; spermidine is also oxidized, but putrescine and cadaverine are unaffected by the enzyme. Protoplasts isolated following digestion of leaves with cellulase in hypertonic osmoticum showed no PAO activity, and about 80% of the total leaf PAO activity could be accounted for in the cell wall debris. Histochemical localization experiments showed intense PAO activity in guard cells and in vascular elements whose walls are not digested by cellulase. When protoplasts were cultured in a medium suitable for regeneration of cell wall, PAO activity could be detected as the cellulose wall developed. Thus, PAO appears to be localized in cell walls.Since applied spermine and spermidine prevent senescence of detached leaves, PAO activity was investigated during leaf senescence. The specific activity of PAO declines with increasing age of attached leaves and with increasing senescence of excised leaves incubated in darkness. This decline in enzyme activity, which parallels the decreases in chlorophyll and protein content used as measures of leaf senescence, suggests that the enzyme is not involved in the control of senescence of oat leaves.

Polyamine-induced DNA Synthesis and Mitosis in Oat Leaf Protoplasts.

Freshly isolated protoplasts from leaves of oat seedlings (var. Victory) which do not divide when cultured on a wide range of media are capable of incorporating tritiated leucine, uridine, and thymidine into trichloroacetic acid-insoluble macromolecules. Over 70% of the leucine and uridine incorporated over an 18-hour period are found in protein and RNA, respectively, as shown by hydrolysis of the macromolecular products with a specific protease or RNase. In contrast, little or none of the tritiated thymidine is incorporated into macromolecules hydrolyzable by DNase over an 18- to 96-hour period. Incorporation of thymidine into trichloroacetic acid-insoluble material declines sharply with increasing time of culture after 18 hours. However, addition of diamines or polyamines to the medium not only prevents the decline, but actually increases net thymidine incorporation, including a fraction going into DNA. A significant increase in mitoses and binucleate protoplasts is also observed in 72- to 168-hour cultures.The inability of oat leaf protoplasts to synthesize significant quantities of DNA suggests that they are arrested at the G(1) phase of the cell cycle. Treatment with polyamines appears to promote both DNA synthesis and the inception of mitotic activity in oat protoplasts, as in numerous animal and microbial cells.

Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts.

Dibasic amino acids and polyamines added to oat (Avena sativa L.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, which is manifested even when the added polyamine is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually follows protoplast isolation. Polyamines, but not dibasic amino acids, also decrease RNase activity in vitro. This in vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyamine, the greater is its inhibitory effect in vitro. The activity of dibasic amino acids when added during protoplast isolation probably results from their conversion to polyamines.

Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence.

Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml-arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis.