Free and conjugated polyamines during de novo floral and vegetative bud formation in thin cell layers of tobacco

The concentrations of three classes of polyamines, trichloroacetic acid-soluble (free), TCA-soluble conjugated (to small molecules) and TCA-insoluble conjugated (to macromolecules), was examined during de novo floral and vegetative bud formation in thin cell layers of Nicotiana tabacum L. cv. Samsun. Explants (consisting of 5–6 layers of epidermal, subepidermal and parenchyma cells) were excised either from floral pedicels or from stem internodes at the unripe fruit stage and cultured on the same medium. In the former, the first de novo formed flower buds appeared on day 8 of culture, while in the latter the first vegetative domes appeared on day 10. In both cases the number of floral and vegetative buds increased up to day 12 and 15, respectively. Changes in dry weight were determined throughout the culture period. Free and conjugated putrescine titer increased 5–60 times in both types of culture and in the three classes of polyamines examined; spermidine content also increased, while spermine, when present, did not show significant changes. TCA-soluble conjugated polyamines were most abundant, being about 2-fold the TCA-insoluble conjugated ones and 10-fold the free ones. The major increment in putrescine and spermidine content occurred in stem internode explants developing vegetative buds. In pedicel explants the maximum putrescine level was reached before or on day 8 in culture (emergence of the first flower buds with calyx initials), while in stem internode explants the maximum level was reached on day 12, at the emergence of the first vegetative buds with leaf primordia. While spermidine prevailed on day 0, putrescine was the most abundant polyamine during both differentiation processes. The putrescine content rapidly increased immediately after the onset of culture. Thus conjugated polyamines, especially putrescine, and not only the free ones, seem to be involved in both the reproductive and vegetative phases of tobacco growth and development.     

Effects of ethylene and auxin on polyamine levels in suspension-cultured tobacco cells

The effects of ethylene and auxin on polyamine levels were studied in suspension-cultured cells of tobacco ( Nicotiana tabacum . L). Treatment of 4-day-cultured cells with ethylene increased the levels of spermidine and spermine. The activities of arginine decarboxylase (ADC; EC 4.1.1.19), ornithine decarboxylase (ODC: EC 4.1.1.17), and S -adenosylmethionine decarboxylase (SAMDC: EC 4.1.1.50) rapidly increased between 3 and 12 h. An auxin, indole-3-acetic acid (IAA), increased polyamine levels and activities of ADC, ODC and SAMDC. The spermine level continued to increase significantly during a 24-h incubation with IAA. The increases in polyamine accumulation induced by ethylene were partially offset by an inhibitor of ethylene action, 2,5-norbornadiene. It is suggested that the auxin-induced polyamine accumulation occurred directly, without metabolic competition between ethylene and polyamine biosynthesis, and indirectly, through auxin-induced ethylene formation.     

Polyamines and carnation flower senescence: Endogenous levels and the effect of applied polyamines on senescence

The polyamines putrescine and spermine were effective in delaying the senescence of carnation buds, but were ineffective when applied to flowers of which the petals had already opened, and were 90° with respect to the stem. Higher levels of endogenous putrescine were detected in the open flowers than in the buds, and this may explain the negative effect obtained when polyamines were applied to open flowers that were picked for commercial distribution.Abbreviations AOA amino-oxyacetic acid - AVG amino-ethoxyvinyl-glycine - SAM S-adenosyl methionine - STS silver thiosulphate     

Protease activity, organisation of vegetative primordia and effect of putrescine in tobacco thin layers

Since in tobacco thin layers exogenous putrescine alters the physiological and mor-phogenic responses induced by IAA (indole-3-acetic acid) and/or BA (benzylade-nine), the effect of this polyamine on protease activity and on the formation of meristemoids and vegetative primordia was studied during morphogenesis. Superficial thin layer explants, excised from the stem of tobacco (Nicotiana tabacum L. cv. Samsun) plants in the vegetative stage, were cultured under various hormonal conditions (IAA, IAA+BA, BA) and in a hormone-free medium, in the presence or absence of 100 μM putrescine. Histological analysis showed that no meristemoids were formed on the control medium or with putrescine alone and only a few were formed on IAA-treated explants with or without putrescine. An increasing number of meristemoids was observed in IAA+BA and BA treatments during culture; in both cases this number was enhanced by the presence of exogenous putrescine. Protease activity was evaluated spectrophotometrically using two synthetic substrates, azocasein and N-benzoyl-DL-arginine-p-nitroanilide (BAPNA). In the former, maximum protease activity was observed in IAA+BA- and BA-treated explants on days 10 and 15, respectively, while with IAA activity was lowest, the maximum occurring on days 5–10. In this case exogenous putrescine enhanced protease activity in the presence of IAA alone or with BA, while it decreased it in the presence of BA. BAPNA-mediated proteolytic activity (serine-proteases) was highest in IAA+BA-treated explants, intermediate in BA- and not different from controls in IAA-treated explants. Putrescine only affected proteolytic activity in IAA+BA treatments. The use of specific inhibitors of protease activities indicated that these enzymes belong to two main classes of proteases, that is serine- and thiol-proteases. The pattern of proteolytic activities during culture appeared to be related to the differentiation of meristemoids into vegetative primordia. The effect of exogenous putrescine on protease activity was different depending on different synthetic substrates, developmental patterns, pH and ionic strength.     

小菜蛾对多胺的嗅觉反应

在实验室条件下利用Y型嗅觉仪测试了小菜蛾Plutella xylostella成虫对多胺的嗅觉反应。结果表明：与蒸馏水对照比较,小菜蛾在交配前和交配后对精胺、亚精胺和腐胺这3种被测的多胺均有明显的趋性反应,但对3种多胺两两之间的选择则无显著差异;在对3种多胺和甘蓝Brassica oleracea叶汁之间的选择中,除未交配雄蛾对腐胺有明显的趋性（与甘蓝叶汁比较）和交配后雌蛾对甘蓝叶汁有明显的趋性（与精胺比较）外,其他均没有显著差异。表明多胺对小菜蛾具有引诱作用。     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.     

Biochemistry of the polyamines

The naturally-occurring polyamines exist in the free form, as N-acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.     

Replacement of Mg2+ by polyamines in the aminoacylation of tRNA from Phaseolus

The ability of putrescine, spermidine and spermine to replace Mg²⁺ ions in the charging reaction of tRNA was estimated for seventeen amino acids. The polyamines promoted only the transfer reaction in the case of Leu, Ile, Val, Tyr and Arg. A synergistic effect was observed when spermine was added to a suboptimal concentration of Mg²⁺ (charging at only 5% of the optimal level). This synergistic effect was not observed for Ala, Asp-NH₂, His, Lys and Ser. Kinetic studies showed a slower aminoacylation rate in those experiments when spermine and Mg⁺² (at 5% of the Mg²⁺ optimal concn) were used together than with Mg²⁺ (at the optimal concn) alone.     

Ethylene involvement in vegetative bud formation in tobacco thin layers

Summary The role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 M AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (twin) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 M) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - STS silver thiosulphate - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - BA benzyladenine - HF hormone-free     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Free and conjugated polyamines in embryogenic and non-embryogenic leaf regions of camellia leaves before and during direct somatic embryogenesis

Contents of endogenous free and conjugated polyamines were determined in embryogenic and non-embryogenic leaf regions of Camellia japonica leaf explants, before (day 0), and 20 and 45 days after the induction of direct somatic embryogenesis, to clarify whether or not polyamines are linked to the specific morphogenic responses previously reported in the leaf regions. The analysis was carried out by high-performance liquid chromatography. The results showed that there were no significant differences in the endogenous free and conjugated putrescine (Put), spermidine (Spd) and spermine (Spm) contents between the embryogenic and non-embryogenic leaf regions of the same leaf. Thus, leaf region-specific embryogenic response in C. japonica is not being determined/conditioned by the endogenous levels of Put, Spd and/or Spm. However, soluble and insoluble conjugated Put and soluble conjugated Spd seem to be related to the formation and development of globular embryos.     

Growth kinetics, polyamine pattern and biosynthesis in hairy root lines of Nicotiana tabacum

The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T' roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T' roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T' roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.     

Endogenous polyamines in apple and their relationship to fruit set and fruit growth

The distribution of free and bound polyamines was investigated from blooming until harvest on flower, fruitlets and fruits of Malus domestica Borkh cv. Golden Delicious, Relationships between polyamines and fruit set and growth were also investigated. The level of free polyamines was high only during the first weeks after full bloom and then decreased gradually. The amount of bound trichloroacetic acid-insoluble polyamines was much higher than free polyamines. Bound spermine in particular showed a high value for almost 40 days after full bloom, while spermidine and putrescine were no longer detectable even a few days after full bloom.
In relation to fruit set, it was possible to observe that abscission peaks took place when free polyamine levels were low or decreasing. Insofar as fruit growth is concerned, the most substantial variations in polyamine levels occurred very early in the season when fruit dry weight and protein amount were also changing rapidly and fruit diameter was almost impossible to measure.     

Changes in polyamine contents during root and nodule growth of Phaseolus mungo

In Phaseolus mungo seeds, polyamine content increased during early germination, being maximum after 24 hr; and the arginine decarboxylase showed a peak after 18 hr. During nodule initiation and growth two peaks of polyamine contents were noted-the first being 2 weeks after nodule initiation and a second one after 5 weeks. Arginine decarboxylase activity also followed the same pattern. In the roots the polyamine concentration as well as arginine decarboxylase increased up to week 2 after sowing followed by a gradual decrease. Estimation of RNA, DNA and protein contents showed a pattern similar to that of the polyamines.     

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.     

Polyamine concentrations in four Poa species, differing in their maximum relative growth rate, grown with free access to nitrate and at limiting nitrate supply

Polyamines are thought to play a role in the control of inherent or environmentally-induced growth rates of plants. To test this contention, we grew plants of four grass species, the inherently fast-growing Poa annua L. and Poa trivialis L. and the inherently slow-growing Poa compressa L. and Poa pratensis (L.) Schreb., at three levels of nitrate supply. Firstly, plants were compared when grown with free access to nitrate, allowing the plants to grow at their maximum relative growth rate (RGR_max). Secondly, we compared the plants when grown with relative nitrate addition rates of 100 and 50 mmol N (mol N)^–1 day^–1 (RAR₁₀₀ and RAR₅₀, respectively).The freely-occurring polyamines, spermine, spermidine and putrescine, were separated from their conjugates; the latter were further subdivided into a TCA-soluble and a TCA-insoluble fraction. Each of the three fractions responded differently to the nitrate supply. Under nitrogen limitation, the total concentration of polyamines (free and bound ones together) decreased in both leaves and roots of all Poa species, whereas that in the stem remained more or less the same. These effects were to a large extent determined by the free polyamines. For the conjugates there was more differentiation, both between plant organ and among polyamine structures. A positive correlation between the RGR, LAR (leaf area per plant mass), SLA (leaf area per leaf mass), LMR (leaf mass per plant mass) and SMR (stem mass per plant mass) with the polyamine concentration was found. The RMR (root mass per plant mass) showed a negative one. No significant differences were found between the inherently fast- and slow-growing grass species.The (putrescine)/(spermine + spermidine) ratio in the leaves increased with decreasing nitrate supply, which is associated with a decrease in leaf expansion, accounting for a decrease in LAR and SLA. For the roots, this ratio tended to decrease with decreasing nitrate supply, whereas for the stems the results were somewhat more variable.We found no evidence for a crucial role of polyamines in the determination of inherent variation of growth in spite of a positive correlation of especially the free polyamines with growth parameters.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.