Occurrence of uncommon polyamines in cultured tissues of maize

Summary The uncommon polyamines, norspermidine and norspermine, were detected in maizein vitro cultures of three different genotypes. The common polyamines, spermidine and spermine, along with the diamine, putrescine, were also observed. The total amounts of the uncommon polyamines, norspermidine and norspermine, were comparable to the total amounts of the common polyamines, spermidine and spermine, in the maize tissues. The titer for norspermidine was 6- to 15-fold greater than that of its common counterpart (spermidine) in the three genotypes. Norspermidine was the predominant polyamine among all triamines and tetramines detected in cell cultures of two of the three genotypes of maize examined and was predominant along with spermine in the third genotype. Enzyme assays performed with extracts from callus of one of the genotypes suggested a likely mechanism to account for the biosynthesis of the uncommon polyamines in cultured maize cells, through the actions of putrescine aminopropyltransferase, polyamine oxidase, and Schiff-base reductase/decarboxylase enzyme activities. This is the first report of the detection of uncommon polyamines in maize tissues, as well as the first report of these uncommon polyamines in a monocotyledonous plant.     

Changes in chlorophyll,polyamines and chloroplast ultrastructure of Puccinia striiformis induced ‘green islands’ on detached leaves of Triticum aestivum

Biochemical and ultrastructure features of ‘green islands’ were investigated using detached wheat leaves infected with the yellow rust Puccinia striiformis. Chlorophylls appear to culminate 10 d after inoculation at which point ‘green islands’ were fully developed. These changes were paralleled by an increase in spermidine and spermine content which play an important role in formation of ‘green islands’. Retention of chlorophyll was demonstrated in leaf tissues of wheat plants supplied with exogenous putrescine, spermidine and spermine. Putrescine was least and spermidine and spermine most effective in retarding loss of chlorophylls. Ultrastructural observation revealed that chloroplasts were regenerated in ‘green islands’ where many proplastids were detected. The regeneration of chloroplasts coincided with the high concentration of chlorophylls and polyamines particularly spermidine and spermine. The ultrastructural changes of chloroplasts in leaf cell containing infection structures were parallel to physiological changes.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F_V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg²⁺. It is also demonstrated that the increase in F_V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Improvement of in vitro androgenesis in niger using amino acids and polyamines

The effects of amino acids (arginine, aspargine, cysteine, glutamine, glycine and proline) and polyamines (putrescine and spermidine) on embryogenesis and plant regeneration from cultured anthers of Guizotia abyssinica (L. f.) Cass. cv. Ootacamund was studied. Supplementation of amino acids (0.5–5.0 mM) to the induction medium individually and in combination, improved embryo yield. B5 medium supplemented with 2 mM proline, 10 µM 2,4-dichlorophenoxyacetic acid, 2 µM kinetin and 0.2 M sucrose induced highest number of embryos (63 per 60 anthers cultured). Addition of polyamines (5–200 µM) to the same medium also enhanced the rate of embryogenesis.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

Screening South African wheat germplasm for androgenic competence

Microspore and anther cultures provide an opportunity to create haploid and doubled haploid plants within a single season, thereby reducing the time and cost of cultivar development. Microspore and anther culture has been widely used and incorporated into wheat breeding programs in many countries, but little is known about the effectiveness of these techniques on South African germplasm. By using two responsive genotypes, isolated microspore culture was shown as more effective at revealing androgenic competence, and was used to evaluate the response of four South African inbred lines and two hybrids. Inbred lines A and B were highly responsive (336 and 207 embryo-like structures [ELS] per 100 anthers, respectively), line D was slightly responsive (5.1 ELS per 100 anthers) while line C was recalcitrant. The hybrid A × C was highly responsive (274 ELS per 100 anthers), and B × D did not respond at all. Green plant regeneration in a local genotype was very low (1% for line B) compared to that of foreign genotype (17% for Pavon 76). Similarly to other wheat genotypes grown around the world, the responsiveness of the South African varieties is also very variable. Thus, more efforts are needed so that isolated microspore culture can become a general tool in breeding programs.     

An efficient procedure for embryogenic callus induction and double haploid plant regeneration through anther culture of Thai aromatic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. subsp. <Emphasis Type="Italic">indica</Emphasis>)

Anther culture is a biotechnology technique that can be used for the production of pure lines. The aims of this investigation were to induce embryogenic callus from major and minor culms of Thai aromatic rice cultivars and to subsequently regenerate double-haploid green plantlets by the application of exogenous polyamines. Embryogenic callus derived from anther culture was successfully induced in varieties KDML105, Homjan (HJ), and Pathumthani 1 (PT1). Production of embryogenic callus from anthers collected from the major culms was greater than those collected from the minor culms, especially in cultivar HJ. Plantlet regeneration in the three rice cultivars was observed from embryogenic callus and was highest, at 12.1%, from variety HJ treated with 0.5 mM spermidine. Plantlet regeneration from anther-derived embryogenic callus was dependent on the plant genotype, the types of exogenous polyamines, and the interactions of these factors. The percentage of haploid plantlets regenerated in PT1, KDML105, and HJ were 68.1%, 70.7%, and 78.5%, respectively. Only haploid plantlets were treated with colchicine for double-haploid production. This investigation has increased the knowledge of both embryogenic callus induction and plantlet regeneration in aromatic rice and has lead to the development of a pure, double-haploid line for the use in rice breeding programs in Thailand.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Assessment of the polyamines modulation on cytokinins and ethylene and its effect in lemon (Citrus limon) de novo regeneration

A highly productive and robust adventitious organogenesis protocol is key as an initial step when lemon in vitro mutagenesis or genetic transformation are addressed. Many molecules play a major role on plant regeneration, and in this study, the addition of the main naturally-occurring polyamines (spermine, spermidine, and putrescine) and their effect on endogenous cytokinins and on ethylene production have been analyzed in explants of the lemon (Citrus limon) cultivar ‘Verna 51’.The three polyamines were added to the organogenesis media, and the regeneration rate and percentage were recorded. Changes produced in the endogenous cytokinins along with ethylene production were studied. Polyamines were shown to play an important role in the adventitious shoot regeneration of ‘Verna 51’. However, this effect would not be related to ethylene production since changes were not detected. Comparing the three studied polyamines, it is possible to conclude that, within the studied doses, spermine (0.2 mM) and spermidine (0.3 and 0.6 mM) are more useful to induce organogenesis than putrescine in ‘Verna 51’. In the most regenerative treatments, lower concentrations of cytokinins were found, which could indicate a correlation between the addition of polyamines to the culture media, the adventitious regeneration, and the metabolism of cytokinins.

     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Interconversion of putrescine,spermidine and spermine in goldfish and rat retina

Labelled putrescine is converted to spermidine and spermine in the retina of both the goldfish and of the rat, but the bulk remains as putrescine and spermidine in the goldfish retina whereas the bulk is present as spermine in the rat retina. Labelled spermidine is converted to spermine and to putrescine in the retina of both species, most remaining as spermidine in the goldfish retina whereas most is converted to spermine in the rat retina. Labelled spermine is converted to both spermidine and putrescine in the retina of both species with a greater conversion in the goldfish retina than in the rat retina. These results provide direct evidence of the interconversion of putrescine, spermidine and spermine in neural tissue from both fish and mammals and suggest that spermine should not be regarded solely as an end-product of putrescine metabolism but also as a source of spermidine and putrescine.The pattern of distribution of putrescine and the polyamines, spermidine and spermine, in goldfish retina is the reverse of that in rat retina: Putrescine is the most abundant in goldfish retina whereas spermine is most abundant in rat retina suggesting that the individual polyamines are of different importance in the two species.     

24-Epibrassinolide ameliorates the saline stress and improves the productivity of wheat (Triticum aestivum L.)

Two wheat (Triticum aestivum L.) cultivars, Sids 1 and Giza 168, were grown under non-saline or saline conditions (4.7 and 9.4 dS m⁻¹) and were sprayed with 0.00, 0.05 and 0.10 mg l⁻¹ 24-epibrassinolide (EBL). Salt stress considerably decreased plant productivity, membrane stability index, photochemical reactions of photosynthesis, the content of relative water, chlorophyll and nitrate, the activity of nitrate reductase and carbonic anhydrase and the level of carbohydrate and protein. The reduction was more pronounced in Giza 168. The follow-up treatment with 0.1 mg l⁻¹ EBL detoxified the stress generated by salinity and significantly improved the above parameters, especially in Sids 1. Glycinebetaine concentration was sharply elevated by salt stress and/or EBL treatments, particularly in Sids 1. Salinity increased putrescine level in Sids 1 and Giza 168, however, spermidine and spermine increased in Sids 1 and decreased in Giza 168. Exogenously applied EBL had a varying effect on polyamines pool under saline condition, an increase in putrescine level associated with low contents of spermidine and spermine in Giza 168 was observed, while Sids 1 showed a decrease in putrescine and high increase in spermidine and spermine. EBL prevented diamine oxidase and polyamine oxidase inhibition, indicating a positive correlation between salt tolerance and polyamines accumulation. Obviously, EBL can be a practical strategy toward generating high-yielding plants under saline condition by enhancing carbon and nitrogen metabolisms. This is the first report dealing with EBL effect on polyamines pool under salt stress.     

Specific interaction of spermine with cibacron blue F3GA

The polyamines, spermine, spermidine, and putrescine, have been shown to bind to Cibacron blue F3GA generating a difference spectrum with a maximum at 685 nm and a minimum at 585 nm, which is characteristic of ionic interactions between the dye and the polyamines. The difference spectral signal vanishes when the charges on the amino groups of the polyamines are neutralized. The magnitude of perturbation of the dye spectrum by the polyamines and, by inference, the capacity to bind to the dye, decrease in the order spermine > spermidine > putrescine. For spermine, the spectral signal of the dye-spermine complex is dependent on the charge state of an aminium group with a pK_a = 8.2.