Changes in polyamine concentration during seed germination

Phaseolus mungo seeds 0 to 10 days after germination contained putrescine, spermidine, spermine, cadaverine, agmatine and tyramine. The rate of biosynthesis of total polyamines, proteins and RNA in the developing seeds follows similar profiles, reaching maxima 3 hr from germination. Putrescine, cadaverine, spermidine, spermine and agmatine were the major amines found in Pisum sativum 0–7 days after germination. RNA and proteins seem to follow the same pattern as polyamines during the first 12 hr in the developing pea seeds. RNA reaches a peak at 15 hr and polyamines and proteins peak 24 hr after germination. A rise to total polyamine concentration was also observed in seeds of Tragopogon porrifolius, Zea mays and Triticum aestivum 2–12 hr after germination.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Polyamine oxidase from barley and oats

The pH optimum for the stability of the barley leaf polyamine oxidase is 4.8, which is also the pH optimum for its activity with spermine as substrate. Zonal centrifugation indicates that the enzyme is associated with a particle which is slightly more dense than chloroplasts, and the peak of activity corresponds with the peak of nucleic acid. Neither DNase nor RNase released the enzyme from the particles, despite the hydrolysis of more than 50% of the nucleic acid. The enzyme from the leaves of oat seedlings grown in the dark was purified 900-fold. Mg²⁺ and Ca²⁺ inhibited both barley and oat enzymes by ca 50% at 50 mM. The optimum pH for both spermine and spermidine oxidation by the oat enzyme was 6.5. The MW of the enzyme from both sources determined by gel chromatography was ca 85 000.     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Polyamine oxidase from Zea mays shoots

Polyamine oxidase of maize shoots purified 10-fold had a pH optimum of 6·3 with spermidine as substrate, and K_m of 6 × 10^?4 M. The enzyme was inhibited by the acridine compounds quinacrine, 6,9-diamino-2-ethoxyacridine and acriflavin, but carbonyl reagents, typical thiol inhibitors and copper-binding agents were without effect. Inhibition by quinacrine was reversed by FMN and FAD. Furthermore, about 50 % of the activity of the apoenzyme was restored by the addition of FAD, but not by FMN or riboflavin, indicating that the maize polyamine oxidase is an FAD-dependent flavoprotein.     

Further properties of the polyamine oxidase of barley leaves

Polyamine analogues have been studied as potential inhibitors or substrates of barley leaf polyamine oxidase. NH₂(CH₂)₃NH(CH₂)₁₀NH₂ was particularly effective as an inhibitor of spermine oxidation at pH 4·5 (K_i = 5 × 10^?6 M). Methylglyoxal-bis(guanylhydrazone) inhibited spermine oxidation only slightly (K_i = 10^?4 M). Activity with the polyamine analogues as substrates was generally 10% or less of the activity with spermine. The K_m for oxygen was 3 × 10^?4 M. The K_m for spermine oxidation was independent of oxygen concentration. Using the N-methyl-2-benzothiazolone hydrazine reagent, 1-(3-aminopropyl)pyrroline was shown to be formed stoichiometrically by the enzyme on oxidation of spermine. The enzyme will not function as a dehydrogenase in the presence of oxygen with either potassium ferricyanide or dichlorophenolindophenol as electron acceptors. Activity in the leaves increased with age, up to 4 weeks. In the leaves of 11-week-old plants activity was lower than in leaves of 1-week-old plants. The enzyme was mainly associated with an easily-sedimented particulate fraction, and relatively small proportions were found in the cell wall or soluble fractions.     

Polyamine Uptake and Translocation in Plants

Recently, evidence has increased for both long- and short-distance transport of polyamines (PAs) in living organisms, but the mechanisms involved and physiological significance of PAs translocation are still not well understood. This review deals with various aspects of polyamine uptake and transport in higher plant tissues.     

Changes in polyamine contents during development and germination of rice seeds

In rice seed, polyamine concentration on a fresh weight basis increased for 16 days after fertilization, followed by a gradual decline. Arginine decarboxylase activity also followed the same pattern. On germination, the polyamine concentration was greatest after 24 hr and the arginine decarboxylase showed a peak after 48 hr.     

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.     

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.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.     

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.     

Purification and properties of a polyamine oxidase from Zea mays

Polyamine oxidase, purified 260-fold from maize shoots, was light yellow in colour. Maximum light-absorption was at 450 nm and was decreased by the addition of either sodium dithionite or spermidine, but not by putrescine. Under aerobic conditions, the enzyme could use p-benzoquinone as an electron acceptor. Cu²⁺ inhibited the enzyme activity, while SO₃⁻ was stimulatory. Several metal-binding agents and thiol reagents were without effect.     

A specific role for spermidine in the initiation phase of somatic embryogenesis in Panax ginseng CA Meyer

Somatic embryogenesis of Panax ginseng CA Meyer was initiated from suspension aggregates of an embryogenic callus, in a liquid medium consisting of half strength Murashige and Skoog (1962) supplemented with the synthetic auxin benzoselenienyl-3 acetic acid. The addition of spermidine to this initiation medium significantly increased the production of somatic embryos. In this case, the total polyamine content of the embryogenic mass was higher than that of cultures without spermidine. At day 6 of the culture, a transient accumulation of free polyamines, mainly spermidine, was observed. After this peak, free and conjugated polyamines levels did not show significant variation nor did the polyamine oxidase activity. The results clearly demonstrated that spermidine supplied to the medium was oxidised by polyamine oxidase and partially metabolised into putrescine. The role of spermidine and its interaction with auxin in the initiation of the embryogenic process in Panax ginseng are discussed in relation to embryogenic potential.     

多胺代谢与癌肿的研究

研究多胺与癌肿的关系。这些癌肿包括Raji癌肿细胞 ,急性淋巴细胞白血病 ,妇产科癌肿 (卵巢癌等 ) ,卵巢癌HO— 891 0细胞 ,肺癌以及胃癌等。研究结果 :(1 )Raji癌细胞株及卵巢癌细胞株 (HO— 891 0 )在培养过程中 ,第 2 4～4 8h多胺水平出现高峰 ,它与这两种癌细胞的核酸合成 ,细胞增殖呈现正相关 ;(2 )急性淋巴细胞白血病患者淋巴细胞及红细胞中多胺水平均升高 ,这有助于对这些病的早期诊断及判断预后 ;(3 )妇科癌症 (尤其卵巢癌 ) ,肺癌 ,胃癌等患者尿液中多胺水平明显高于正常 ,所以尿液中多胺对这些癌肿也是一种有效的诊断标记物     

Polyamine biosynthesis and titer during various developmental stages of Phaseolus vulgaris

The activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC; EC 4.1.1.17) as well as polyamine content were examined in Phaseolus vulgaris L. cv. Taylor's Horticultural before and during anthesis, during fruit development and throughout vegetative growth. The specific activities of polyamine biosynthetic enzymes were highest in all rapidly growing tissues, e.g., root apices, hypocotyls, young internodes, young leaves, flower buds, young pods and pericarps. They were lowest in mature, non-growing tissues. Similarly, the content of the major polyamines (putrescine, spermidine, spermine) is highest in rapidly growing tissues, and lowest in mature tissue. These correlations reinforce the growing connection between polyamines and rates of cell division and metabolic activity during both vegetative and reproductive development.     

Effects of Ca2+ and polyamines on Na+ and Rb+ influx in excised maize roots

When 1 m M spermidine or spermine was included in an absorption solution which contained 20 m M Na⁺ and 1 m M Rb⁺, Na⁺ influx into excised maize roots ( Zea mays L. cv. Golden Cross Bantam) was reduced. Rb⁺ influx was reduced in the presence of spermidine and uneffected in the presence of spermine when compared with control solutions. When 1 m M Ca²⁺ replaced the polyamines, Na⁺ influx was strongly reduced and Rb⁺ influx was promoted. Rb⁺ influx from 1 m M Rb⁺ solutions which did not contain Na⁺ was also promoted by 1 m M Ca²⁺, but was inhibited by 1 m M spermidine. This Ca²⁺ promotion of Rb⁺ influx could be reversed by 10 times greater concentration of spermidine in the absorption solution. H⁺ efflux from excised roots was inhibited by spermidine when compared with Ca²⁺ or control solutions, however, the plasma membrane ATPase was not inhibited by spermidine. It is concluded that external Ca²⁺ plays two separate roles in membrane function, only one of which can be substituted for by polyamines. The first role, maintenance of membrane integrity, can be substituted for by spermidine or spermine. The second function, maintenance of the Rb⁺ transport mechanism, is Ca²⁺ specific and cannot be substituted for by spermidine or spermine. The results of this study are discussed in terms of electrostatic interactions between the plasma membrane and the Ca²⁺ or polyamines.