Intervening in sibling competition for assimilates by controlled pollination prevents seed abortion under postpollination drought in maize

During maize production, drought throughout the flowering stage usually induces seed abortion and yield losses. The influence of postpollination drought stress on seed abortion and its underlying mechanisms are not well characterized. By intervening in the competition for assimilates between kernel siblings under different degrees of postpollination drought stresses accompanied by synchronous pollination (SP) and incomplete pollination (ICP) approaches, the mechanisms of postpollination abortion were investigated at physiological and molecular levels. Upon SP treatment, up to 15% of the fertilized apical kernels were aborted in the drought-exacerbated competition for assimilates. The aborted kernels exhibited weak sucrose hydrolysis and starch synthesis but promoted the synthesis of trehalose-6-phosphate and ethylene. In ICP where basal pollination was prevented, apical kernel growth was restored with reinstated sucrose metabolism and starch synthesis and promoted sucrose and hexose levels under drought stress. In addition, the equilibrium between ethylene and polyamine in response to the drought and pollination treatments was associated with the abortion process. We conclude that competition for assimilates drives postpollination kernel abortion, whereas differences in sugar metabolism and the equilibrium between ethylene and polyamines may be relevant to the “live or die” choice of kernel siblings during this competition.     

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.

     

Effect of polyamines on ethylene production

The polyamines putrescine, cadaverine, spermidine and spermine reduced the amount of ethylene produced by senescing petals of Tradescantia but they did not prevent anthocyanin leakage from these same petals. These polyamines also inhibited auxin-mediated ethylene production by etiolated soybean hypocotyls to less than 7 % of the control. The basic amino acids lysine and histidine reduced the amount of auxin-induced ethylene produced by soybean hypocotyls by ca 50 %. In the hypocotyls, methionine was unable to overcome the inhibition caused by the polyamines. The polyamines spermidine and spermine inhibited ethylene production induced by the application of 1-aminocyclopropane-1-carboxylic acid and they also reduced the endogenous content of this amino acid in the treated tissues.     

Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts

Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca²⁺ in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.     

Metabolism of l[U-14C]-arginine and l[U-14C]-ornithine in maturing and vernalised embryos and megagametophytes of Picea abies

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these 14 C-labelled amino acids into liberated CO₂, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post-translationally as polyamines. Higher radioactivity was seen in the small ≤14-kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3-diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

Relationship Between Photosynthate Supply and Endosperm Development in Maize

Maize (Zea mays L., cultivar Pioneer 3925) plants were givenshaded, thinned and control light treatments during 10 d or20 d periods surrounding pollination. Glucose, sucrose, starch,and dry matter (DM) contents were measured at intervals in compositesamples of pericarp/nucellus (PN), and in endosperms taken fromdeveloping kernels. Total kernel DM per ear at maturity washigher in the thinned treatment than control and shaded treatmentsdue to higher kernel set in apical regions of ears. In PNs at11 d after pollination (DAP), DM and sucrose contents were slightlygreater in thinned than control and shaded plants. Glucose contentswere substantially greater than controls in PNs of thinned plantsand were less than controls in shaded plants. In endospermsfrom apical kernels at 8 to 12 DAP (during cell division), DM,glucose and sucrose contents were substantially less in shadedthan control and thinned plants. Sucrose contents were greaterin endosperms of thinned than control plants. Sugar contentsin endosperms from basal kernels were nearly the same in thethree light treatments. At 12 DAP, apical and basal endospermsin shaded plants had fewer nuclei than those of the other lighttreatments. The light treatments appeared to effect apical kernelgrowth by influencing the extent of cell division. Zea mays L, maize, light treatment, endosperm, cell division, glucose, sucrose, starch     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.)

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.     

Polyamine levels and tomato fruit development: possible interaction with ethylene

Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.     

The Effects of Inhibitors of Polyamine and Ethylene Biosynthesis on Senescence, Ethylene Production and Polyamine Levels in Cut Carnation Flowers

The role of polyamine metabolism in the regulation of senescenceand ethylene evolution was examined in cut carnations. Endogenousconcentrations of spermine and spermidine did not change asflowers aged, but putrescine, their immediate biosynthetic precursor,increased dramatically and paralleled a sharp rise in ethylene.When D-arginine, difluoromethylarginine and methylglyoxal bis(guanylhydrazone)were used to inhibit specific steps in polyamine synthesis,ethylene production and the onset of senescence were promoted.In contrast, inhibition of ethylene by aminooxyacetic acid increasedthe level of spermine, presumably by increasing the availabilityof aminopropyl groups derived from S-adenosylmethionine (SAM)and required for the synthesis of polyamines from putrescine.These results suggest that the ethylene and polyamine biosyntheticpathways compete for SAM during senescence of carnation flowers. (Received September 1, 1983; Accepted January 7, 1984)     

Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

Engineered polyamine accumulation in tomato enhances phytonutrient content,juice quality,and vine life

Polyamines, ubiquitous organic aliphatic cations, have been implicated in a myriad of physiological and developmental processes in many organisms, but their in vivo functions remain to be determined. We expressed a yeast S-adenosylmethionine decarboxylase gene (ySAMdc; Spe2) fused with a ripening-inducible E8 promoter to specifically increase levels of the polyamines spermidine and spermine in tomato fruit during ripening. Independent transgenic plants and their segregating lines were evaluated after cultivation in the greenhouse and in the field for five successive generations. The enhanced expression of the ySAMdc gene resulted in increased conversion of putrescine into higher polyamines and thus to ripening-specific accumulation of spermidine and spermine. This led to an increase in lycopene, prolonged vine life, and enhanced fruit juice quality. Lycopene levels in cultivated tomatoes are generally low, and increasing them in the fruit enhances its nutrient value. Furthermore, the rates of ethylene production in the transgenic tomato fruit were consistently higher than those in the nontransgenic control fruit. These data show that polyamine and ethylene biosynthesis pathways can act simultaneously in ripening tomato fruit. Taken together, these results provide the first direct evidence for a physiological role of polyamines and demonstrate an approach to improving nutritional quality, juice quality, and vine life of tomato fruit.     

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

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.     

Suppression of TNF-alpha production by S-adenosylmethionine in human mononuclear leukocytes is not mediated by polyamines

Endotoxin-induced cytokine production is an important mechanism in the development of several types of liver damage. Methionine, some of its precursors and metabolites were reported to have protective effects against such injury. The aim of this study was to investigate whether methionine, its precursors or metabolites [phosphatidylcholine, choline, betaine, S-adenosylmethionine (SAM)] have a modulating effect on tumor necrosis factor alpha (TNF-alpha) production by endotoxin-stimulated human mononuclear leukocytes and whether SAM-dependent polyamines (spermidine, spermine) are mediators of SAM-induced inhibition of TNF-alpha synthesis. Methionine and betaine had a moderate stimulatory effect on TNF-alpha production, whereas phosphatidylcholine (ID(50) 5.4 mM), SAM (ID(50) 131 microM), spermidine (ID(50) 4.5 microM) and spermine (ID(50) 3.9 microM) had a predominantly inhibitory effect. Putrescine did not alter TNF-alpha release. Inhibitors of polyamine synthesis that blocked either putrescine (difluoromethylornithine) or spermine (CGP48664A) production did not affect TNF-alpha synthesis. Endotoxin stimulation of leukocytes did not alter the intracellular levels of polyamines. In addition, supplementation with SAM did not change the intracellular concentration of either polyamine measured. We conclude that phosphatidylcholine-induced immunosuppression is not caused by methionine and polyamines are not involved in SAM-induced inhibition of TNF-alpha production. The limitation of TNF-alpha release by spermidine is specific and is not due to its conversion into spermine.     

Molecular Genetic Analyses of Plant Polyamines

Polyamines are small, positively charged aliphatic amines that play a variety of roles in plant physiology. Putrescine, spermidine, and spermine are usually what are collectively meant by the term polyamines, although plants also have a variety of other related compounds and secondary product conjugates to polyamines. Organisms synthesize putrescine, spermidine, and spermine by pathways leading from ornithine, arginine, and SAM, with three of the important enzymes being amino acid decarboxylases. There has been recent progress in understanding plant polyamines, both their function and the regulation of their synthesis, as a result of molecular genetic investigations. The cDNAs for many of the key enzymes have been cloned and se-quenced, and studies on regulation of the enzymes have begun. Mutational and transgenic approaches are being used to perturb the pathway. Some of the phenotypes observed suggest interactions between polyamines and either ethylene or cytokinin, consistent with some of the correlations observed many years previously by polyamine physiologists. These studies, while still in their early stages, should improve our understanding of polyamine synthesis, but difficult problems remain to be solved before we can answer the question: What are the biological functions and associated mechanisms of action of polyamines?     

Enhancement of berberine production by spermidine in Thalictrum minus cell suspension cultures

Berberine production in cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. was significantly enhanced by administration of spermidine, whereas other polyamines such as cadaverine, putrescine and spermine were ineffective. The results of experiments indicated that spermidine causes an increase of ethylene generation which is closely associated with activation of berberine biosynthesis.