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     

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.Abbreviations PAs polyamines - Put putrescine - Spd spermidine - Spm spermine - S free polyamine fraction - SH conjugated polyamine fraction - PH bound polyamine fraction     

Host-Parasite Relations in Loose Smut of Wheat: III. The Utilization of 14C-Labelled Assimilates

Infection of wheat by Ustilago nuda caused an increased rateof export of radioactivity from the sixth leaf of infected plantsas compared with healthy plants. The ear acted as the most important‘sink’, in both infected and healthy plants. Erythntol,mannitol, and trehalose were present in the infected ears andnodes, but were not present in the healthy plants. Radioactivitywas incorporated into these compounds in the infected ear, andthis was reflected in a lower percentage in the insoluble fractionthan in the healthy ear.     

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.     

Host-Parasite Relations in Loose Smut of Wheat: II. The Distribution of 14C-Labelled Assimilates

The effect of infection by Ustilago nuda on the distributionof ¹⁴C-assimilates in spring wheat plants at several stagesof growth was examined Only small differences were detectedbetween healthy and infected plants at the early stages, butthere were marked differences between the older plants The amountof assimilate exported by the fed leaf appeared to be dependenton the demand for assimilates in all plants, and was greatestduring a period of rapid growth of host or parasite, such asthe sporulation of the fungus in the infected ear The distributionof exported assimilates from the fed leaf of healthy plantsvaried with the position of the fed leaf, its stage of development,and the age of the plant, the pattern was very similar to thatfound in other plant diseases This pattern was altered by thepresence of the pathogen, which acted as an alternative siteof accumulation Totally different sites received ¹⁴C-labelledassimilates in infected plants, in some cases assimilates weretranslocated over unusually long distances, for example fromthe main shoot flag leaf to infected tiller ears. The differencein distribution correlated well with the differences in growthcaused by the pathogen. In addition, the ratio between the radioactivityin alcohol-insoluble and that in alcohol-soluble compounds wasaffected by the infection, in young infected plants more radioactivitywas present in the insoluble fraction and in older infectedplants in the soluble fraction     

Suborganellar localisation and effect of light on <Emphasis Type="Italic">Helianthus tuberosus</Emphasis> chloroplast transglutaminases and their substrates

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.     

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-¹⁴C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.     

Polyamines and Morphogenesis - Effects of Methylglyoxal-bis(guanylhydrazone)

The effects of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis were studied on tuberization and cellular polyamine content using in vitro Solanum tuberosum (cv Binjte) plants. When MGBG was added to the culture medium, it produced a partial inhibition of the growth of stems and leaves; it totally blocked rhizogenesis and strongly stimulated tuber formation. Morphogenetic effects of MGBG were correlated to a 40 % decrease in free putrescine, spermidine, spermine content of the leaves and to a 28 % decrease in spermidine titer of the stems. In the tubers, this inhibitor did not change the free polyamine titer but increased by up to 85 % the titer of conjugated putrescine, spermidine, spermine. When the plants were grown in the dark, MGBG produced, like benzyladenine, a stimulation of the rate of tuberization and enhanced the content of conjugated polyamines in the tuber. These results support the hypothesis that polyamines play an important role in the morphogenesis of potato plants. The role of polyamine conjugation in tuber development is discussed.     

Urinary metabolites of polyamines in rats

Urinary metabolites of polyamines in rats were studied systematically by the intraperitoneal injection of radioactive polyamines. Urinary metabolites were fractionated into 4 fractions containing non-polar and acidic compounds, acidic and neutral ampholytes, basic ampholytes and polyamines. A large amount of radioactivity was detected in the fractions containing non-polar and acidic compounds and polyamines of urine of rats injected with radioactive putrescine, while in the case of the injection of radioactive spermidine or spermine a relatively large amount of radioactivity was found in the basic ampholyte fraction as well as in the polyamine fraction. Analysis of these fractions indicated that gamma-aminobutyric acid, N-monoacetylputrescine, 2(3)-hydroxyputrescine, putreanine, N-(3-aminopropyl)-4-aminobutyric acid (isoputreanine), spermic acid, N-(3-aminopropyl), N'-(2-carboxyethyl)-1,4-diaminobutane, and N-monoacetylspermidine A and B were excreted as urinary metabolites of the polyamines in addition to putrescine, spermidine and spermine.     

Taphrina maculans reduces the therapeutic value of turmeric (Curcuma longa)

Rhizomes of turmeric are used in several culinary preparations. They have been used as household remedies since time immemorial. Phenolics are found in plants only and they play a great role in human health. The high performance liquid chromatography of different parts of healthy and infected plants reveals that the phenolic acid content is reduced in diseased tissues including rhizomes which are commonly consumed by human beings in several countries of the world. Results also showed that the infected leaves had maximum phenolic acids as compared to healthy leaves which indicate that due to infection the amount of phenolic acids increased. We report for the first time that leaf spot (Taphrina maculans) of turmeric (Curcuma longa), which is an important disease, reduces the quantity and number of phenolics thereby damaging the therapeutic properties of turmeric.     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.     

Increased accumulation of phenolic metabolites in groundnut (Arachis hypogaea L.) genotypes contribute to defense against Sclerotium rolfsii infection

Abstract

Fungal infections cause several metabolic changes to the plants, which can affect its physiology and survival in various ways. In the present study, we have analysed various phenolic compounds and activity of oxidative enzymes in healthy and Sclerotium rolfsii-infected groundnut genotypes. Increased phenolics content and higher activity of oxidative enzymes was observed in the tolerant genotype (CS 19, GG 16) followed by susceptible genotype (GG 20, TG 37A). Among the phenolic compounds tested, chlorogenic acid content has increased greatly in leaf, stem and root of infected tolerant genotypes compared to the respective controls. In vitro growth of S. rolfsii showed significant inhibition at concentrations 500 and 1000 µg/mL of phenolic compounds in the radial growth inhibition assay. These results have strongly suggested that, higher accumulation of chlorogenic acid could be an important factor in imparting resistance and protecting groundnut against S. rolfsii infection in tolerant genotypes.     

Differential conjugation of polyamines to calf nuclear and nucleolar proteins

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.     

Effects of the Fungal Endophyte Acremonium coenophialum on Nitrogen Accumulation and Metabolism in Tall Fescue

Infection by the fungal endophyte Acremonium coenophialum affected the accumulation of inorganic and organic N in leaf blades and leaf sheaths of KY 31 tall fescue (Festuca arundinacea Schreb.) grown under greenhouse conditions. Total soluble amino acid concentrations were increased in either the blade or sheath of the leaf from infected plants. A number of amino acids were significantly increased in the sheath, but only asparagine increased in the blade. Infection resulted in higher sheath NH₄⁺ concentrations, whereas NO₃⁻ concentrations decreased in both leaf parts. The effects on amino acid, NO₃⁻, and NH₄⁺ concentrations were dependent upon the level of N fertilization and were usually apparent only at the high rate (10 millimolar) of application. Administration of ¹⁴CO₂ to the leaf blades increased the accumulation of ¹⁴C in their amino acid fraction but not in the sheaths of infected plants. This may indicate that infection increased amino acid synthesis in the blade but that translocation to the sheath, which is the site of fungal colonization, was not affected. Glutamine synthetase activity was greater in leaf blades of infected plants at high and low N rates of fertilization, but nitrate reductase activity was not affected in either part of the leaf. Increased activities of glutamine synthetase together with the other observed changes in N accumulation and metabolism in endophyte-infected tall fescue suggest that NH₄⁺ reassimilation could also be affected in the leaf blade.     

Expression of a humanS-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.     

Impact of the flower smut Ustilago cynodontis (Ustilaginaceae) on the performance of the clonal grass Cynodon dactylon (Gramineae)

Effects of the flower-infecting smut fungus Ustilago cynodontis on the growth, survival, and competitiveness of the clonal rhizomatous perennial grass Cynodon dactylon were explored through a series of glasshouse experiments. Ustilago cynodontis sterilized C. dactylon by replacing floral structures with a teliospore-producing fungal stroma. Infection caused a reduction in overall dry matter production and the growth rate of stolons, changed the allocation of resources between roots and shoots, and affected the survival of C. dactylon plants growing in pots under crowded conditions in a naturally lit glasshouse. In mixtures of healthy and infected plants, the growth of infected ones was disproportionally affected. Infection had no effect on the germination or emergence of seedlings. Transmission of the pathogen along stolons was incomplete, especially as the length of the stolon increased. The nutrient status of the soil had no significant effect on stolon growth. Results are discussed in terms of the differential effect of systemic floral smut diseases on tiller-forming and clonal rhizomatous perennial grasses.     

Role of polyamines in the ontogeny of plants and their biotechnological applications

Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to be involved in a wide range of plant processes, however their exact role is not completely understood. In this review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been focussed.     

Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, alpha-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.     

Floral morphogenesis in Rudbeckia hirta in relation to polyamine concentration

Changes in polyamine concentration in the long day (LD) plant Rudbeckia hirta were examined over the course of floral initiation and development. Plants of R. hirta were grown to maturity under 9h, non-floral-inductive photoperiods. At maturity, half the plants were placed in ambient day length plus a 4- h night interruption. Plants were sampled at 0, 4, 8, 12 or 16 days for polyamine content and floral initiation. Polyamines were extracted from fully expanded leaves and the meristems were examined histologically. In another experiment, polyamines were extracted from the meristems under paired LD and short day (SD) conditions every 2 days from 0 to 22 days. A rise in free polyamines was linked to important cytological events during floral initiation. Free putrescine and spermidine levels increased after 4 LD and continued until 14 to 16 LD when the levels in the meristem began to decline. Events of floral initiation began between 4 and 8 LD with cell proliferation and the start of stem elongation. Initiation was irreversible after 14 to 16 LD, the period when putrescine and spermidine began to decline. After 4 LD, the polyamine level was consistently higher in the photoinduced plants. Our results from this study, using direct histological comparisons of meristematic development and polyamine concentration, clearly demonstrate a correlation of polyamines and flowering.