Free Polyamine Levels Research Articles

Enriched endogenous free Spd and Spm in alfalfa (Medicago sativa L.) under drought stress enhance drought tolerance by inhibiting H2O2 production to increase antioxidant enzyme activity

Drought stress is a major factor limiting agricultural development, and exogenous polyamines (PAs) can increase plant drought resistance by enhancing antioxidant activity, but few studies have examined whether endogenous PAs enhance the plant antioxidant system. Here, to investigate the effects of endogenous PAs on the antioxidant system of alfalfa under drought stress and the underlying mechanisms, two alfalfa cultivars, Longzhong (drought resistant) and Gannong No. 3 (drought sensitive), were used as test materials, and their seedlings were treated with polyethylene glycol (PEG-6000) for 8 days at −1.2 MPa to simulate drought stress. The levels of free PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)], hydrogen peroxide (H2O2), malondialdehyde (MDA), key PA metabolism enzyme [arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO)] activities, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)] activities were measured. These physiological indicators were used for correlation analysis to investigate the relationship between PA metabolism and the antioxidant enzyme system. The results showed that PA synthesis in alfalfa under drought stress was dominated by the ADC pathway. Spd and Spm played an important role in improving drought tolerance. The high levels of ADC and SAMDC activities were facilitated by the conversion of Put to Spd and Spm. H2O2 generation by oxidative decomposition of PAs was mainly dependent on the oxidative decomposition of DAO but not PAO. Low DAO activity favored low H2O2 production. Spd, Spm, ADC, ODC and SAMDC were positively correlated with the antioxidant enzymes SOD, CAT and POD in both cultivars under drought. Therefore, we concluded that high ADC and SAMDC activities in alfalfa promoted the conversion of Put to Spd and Spm, leading to high accumulation of Spd and Spm and low Put accumulation. Low Put levels led to low H2O2 production through low DAO activity, and low H2O2 levels induced the expression of antioxidant enzyme-encoding genes to improve antioxidant enzyme activity and reduce MDA accumulation and thereby enhanced drought resistance in alfalfa.

L-Aminoguanidine Induces Imbalance of ROS/RNS Homeostasis and Polyamine Catabolism of Tomato Roots after Short-Term Salt Exposure.

Polyamine (PA) catabolism mediated by amine oxidases is an important process involved in fine-tuning PA homeostasis and related mechanisms during salt stress. The significance of these amine oxidases in short-term responses to salt stress is, however, not well understood. In the present study, the effects of L-aminoguanidine (AG) on tomato roots treated with short-term salt stress induced by NaCl were studied. AG is usually used as a copper amine oxidase (CuAO or DAO) inhibitor. In our study, other alterations of PA catabolism, such as reduced polyamine oxidase (PAO), were also observed in AG-treated plants. Salt stress led to an increase in the reactive oxygen and nitrogen species in tomato root apices, evidenced by in situ fluorescent staining and an increase in free PA levels. Such alterations were alleviated by AG treatment, showing the possible antioxidant effect of AG in tomato roots exposed to salt stress. PA catabolic enzyme activities decreased, while the imbalance of hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) concentrations displayed a dependence on stress intensity. These changes suggest that AG-mediated inhibition could dramatically rearrange PA catabolism and related reactive species backgrounds, especially the NO-related mechanisms. More studies are, however, needed to decipher the precise mode of action of AG in plants exposed to stress treatments.

Polyamines and ethylene interact in mediating the effect of nitrogen rates on synthesis of amino acids in rice grains

AbstractNitrogen (N) is one of the essential factors influencing amino acid synthesis in rice (Oryza sativa L.) grains. Polyamines (PAs) and ethylene (ETH) play vital roles in grain amino acid biosynthesis of rice. However, knowledge is limited about whether and how PAs and ETH mediate the effect of N rate on amino acid biosynthesis. Two rice cultivars were used with three N rates of low (LN), medium (MN), and high (HN) in the study. Levels of free PAs and ETH, activities/gene expressions of key enzymes related to amino acid synthesis in grains, and the amino acid contents in milled rice were investigated. Free spermidine (Spd) and spermine (Spm) concentrations, especially ratios of free PAs to 1‐aminocylopropane‐1‐carboxylic acid (ACC), and total amino acids (TAAs), essential amino acids (EAAs), and non‐essential amino acids (NEAAs) contents were increased with N rate, while ethylene evolution rate (EER) and ACC concentration were reduced with N increases. There was relatively little difference in the above parameters between MN and HN. Levels of free Spd and Spm, and their ratios to ACC exhibited significant and positive correlations with the activities/gene expressions of key enzymes related to amino acid biosynthesis, and TAAs, NEAAs, and EAAs contents, whereas ACC and EER levels had significantly negative correlations with those parameters. Exogenous Spd, Spm, or AVG (an ETH synthesis inhibitor) increased the enzymatic activities and gene expressions, whereas MGBG (a Spd or Spm synthesis inhibitor) or ethephon (an ETH‐releasing agent) exerted the opposite effect. These results demonstrated that free PAs and ETH antagonistically interact in mediating the effect of N rate on grain amino acid synthesis of rice. Higher ratios of free PAs to ACC could promote synthesis of amino acids in grains of rice.

Conjugated Polyamines in Root Plasma Membrane Enhanced the Tolerance of Plum Seedling to Osmotic Stress by Stabilizing Membrane Structure and Therefore Elevating H+-ATPase Activity.

Polyamines are small positively charged molecules in plants and play important functions in many biological processes under various environmental stresses. One of the most confounding problems relating to polyamines (PAs) in stresses is the lack of understanding of the mechanisms underlying their function(s). Furthermore, a limited number of studies have addressed this issue at the sub-cellular level, especially in tree plants under drought stress. Therefore, in this research, by simulating natural drought stress with polyethylene glycol (PEG) osmotic stress, the relationship between the levels of conjugated polyamines and the activity of H+-ATPase in the plasma membrane was elucidated with the roots of two plum (Prunus salicina L.) cultivars, which were different in drought tolerance, as experimental materials. Furthermore, free PA levels and the activities of S-adenosylmethionine decarboxylase (SAMDC) and transglutaminase (TGase), which were closely associated with the levels of free and conjugated PAs, were also detected. Results showed that under osmotic stress, the increases of the levels of non-covalently conjugated (non-CC) spermidine (Spd) and spermine (Spm), covalently conjugated (CC) putrescine (Put) and Spd in the plasma membrane of drought-tolerant Ganli No. 5 were more significant than those of drought-sensitive Suli No. 3, indicating that these conjugated PAs might be involved in the tolerance of plum seedlings to stress. Furthermore, the conjugated PAs were closely correlated with plum seedling growth, water retention capacity, plasma membrane damage degree, and hydrogen (H+)-ATPase activity in the plasma membrane. To get more complementary pieces of evidence, we subjected plum seedlings to combined treatments of PEG and exogenous PA (Spd and Spm), and an inhibitor of SAMDC [methylglyoxal-bis (guanylhydrazone), (MGBG)] or TGase (o-phenanthroline). These results collectively suggested that non-CC Spd and Spm, CC Put and Spd in plasma membrane might function in enhancing the tolerance of plum seedlings to osmotic stress by stabilizing membrane structure and therefore elevating H+-ATPase activity.

Ethylene is a local modulator of jasmonate-dependent phenolamide accumulation during Manduca sexta herbivory in Nicotiana attenuata.

Rapid reconfigurations of interconnected phytohormone signalling networks allow plants to tune their physiology to constantly varying ecological conditions. During insect herbivory, most of the induced changes in defence-related leaf metabolites are controlled by jasmonate (JA) signalling, which, in the wild tobacco Nicotiana attenuata, recruits MYB8, a transcription factor controlling the accumulation of phenolic-polyamine conjugates (phenolamides). In this and other plant species, herbivory also locally triggers ethylene (ET) production but the outcome of the JA-ET cross-talk at the level of secondary metabolism regulation has remained only superficially investigated. Here, we analysed local and systemic herbivory-induced changes by mass spectrometry-based metabolomics in leaves of transgenic plants impaired in JA, ET and MYB8 signalling. Parsing deregulations in this factorial data-set identified a network of JA/MYB8-dependent phenolamides for which impairment of ET signalling attenuated their accumulation only in locally damaged leaves. Further experiments revealed that ET, albeit biochemically interrelated to polyamine metabolism via the intermediate S-adenosylmethionine, does not alter the free polyamine levels, but instead significantly modulates phenolamide levels with marginal modulations of transcript levels. The work identifies ET as a local modulator of phenolamide accumulations and provides a metabolomics data-platform with which to mine associations among herbivory-induced signalling and specialized metabolites in N. attenuata.

Changes in polyamine pattern mediates sex differentiation and unisexual flower development in monoecious cucumber (Cucumis sativus L.)

Changes in the levels of polyamines are associated with fundamental physiological processes such as embryogenesis, induction of flowering, fruit development and ripening, senescence, and responses to environmental stresses, but the role of polyamines in sex differentiation and unisexual flower development has not been deeply studied. To extend the knowledge on the regulatory mechanisms of flowering in monoecious plant (producing unisexual flowers), we investigated the morphogenesis and free polyamine levels in Cucumis sativus during sex differentiation and unisexual flower development in vitro using histocytological and biochemical methods. As shown in our study, floral development in vitro was undisturbed and flowers of both sexes were produced. Sex differentiation relied on preventing the development of generative organs of the opposite sex, as we observed carpel repression in male flowers and stamen repression in female flowers. Pollen viability was negatively correlated with female flower development on the same node. Biochemical analysis revealed increased accumulation of aliphatic amines (tri, tetra-amines) in generative (flower buds and flowers) compare to vegetative (axillary buds and leaves) organs. Undifferentiated floral buds contained elevated levels of agmatine, cadaverine, spermidine and spermine. Sex differentiation was associated with significantly decreased levels of agmatine and cadaverine. Our results showed that female flowers contained higher levels of total polyamine than male flowers. The increased level of cadaverine was associated with macrogametogenesis and female flower maturation. Putrescine was important for male flower development. Such results support the hypothesis that aliphatic amines are involved in unisexual flower development.

CsICE1 Functions in Cold Tolerance by Regulating Polyamine levels May through Interacting with Arginine Decarboxylase in the Tea Tree

Background: The identification of C-repeat binding factor (CBF), and the characterization as an inducer of CBF Expression 1 (ICE1), and a major activator for C-repeat binding factor, were important breakthroughs in the cold signaling network. Methods: In the present study, the full length cDNA of ICE1 was isolated from the tea tree (Camellia sinensis). CsICE1 protein was located in the cell nucleus as revealed by subcellular localization analysis. To investigate the biological functions of CsICE1, a transgenic line fused with the CsICE1 gene in Arabidopsis thaliana (arabidopsis) was generated by the floral dip method. Results: The CsICE1 was expressed differentially in various tea tree tissues, mostly in buds and leaves, and the transcript level of CsICE1 was increased after 1 h and peaked at 2 h under cold treatment. Transcription activity assay indicated that the spermine synthase (SPMS) and arginine decarboxylase (ADC) genes were possible targets of CsICE1. In addition, the values of net photosynthetic rate, transpiration rate, stomatal conductance in transgenic lines declined by less extent than wild-type plants under low temperatures. Furthermore, transcript levels of ADC genes in the transgenic lines had no apparent alteration under normal growth conditions but substantially increased under cold conditions, consistent of changes in free polyamine levels. Taken together, these results demonstrated that CsICE1 plays a positive role in cold tolerance, which may be due to the modulation of polyamine levels through interacting with CsADC.

Spermidine Enhanced Free Polyamine Levels and Expression of Polyamine Biosynthesis Enzyme Gene in Rice Spikelets under Heat Tolerance before Heading

High temperatures (HT) before heading strongly inhibit the development of spikelets in rice. Spermidine (Spd) can improve rice’s resistance to HT stress; however, the mechanism underlying this effect has not been elucidated. This study investigated several parameters, including yield, superoxide anion (O2.-), protective enzyme activities, and polyamine content, in a heat-sensitive genotype, Shuanggui 1. The yield and yield components decreased dramatically when subjected to HT stress, while this reduction could be partially recovered by exogenous Spd. Spd also slowed the generation rate of O2.- and increased protective enzyme, superoxide dismutase (SOD) and catalase (CAT) activities both under normal and high temperatures, which suggested that Spd may participate in the antioxidant system. Furthermore, genes involved in polyamine synthesis were analyzed. The results show that HT before heading significantly increased the expression of arginine decarboxylase OsADC1, Spd synthase OsSPDS1 and OsSPDS3 and had little effect on the expression of the S-adenosylmethionine decarboxylase OsSAMDC2 and ornithine decarboxylase OsODC1. In addition, exogenous Spd considerably reduced the expression of OsSAMDC2, OsSPDS1 and OsSPDS3 under HT but not the expression of OsADC1. The above mentioned results indicate that the exogenous Spd could help young rice spikelets to resist HT stress by reducing the expression of OsSAMDC2, OsSPDS1 and OsSPDS3, resulting in higher levels of endogenous Spd and Spm, which were also positively correlated with yield. In conclusion, the adverse effect of HT stress on young spikelets seems to be alleviated by increasing the amounts of Spd and Spm, which provides guidance for adaptation to heat stress during rice production.

Polyamine-producing actinobacteria enhance biomass production and seed yield in Salicornia bigelovii

Thirty-five actinobacterial isolates obtained from Salicornia bigelovii rhizosphere in the United Arab Emirates were evaluated for their potential to produce polyamines and for their abilities to be rhizosphere-competent. The highly polyamine-producing Actinoplanes deccanensis UAE1 and Streptomyces euryhalinus UAE1 were selected, and their inoculation impacts on S. bigelovii were assessed under greenhouse conditions. Application of any of the two isolates significantly increased the length and dry weight of shoot and root tissues, photosynthetic pigments, and seed yields compared to control plants, suggesting that A. deccanensis and S. euryhalinus can promote S. bigelovii growth. The application of these isolates resulted in significant increases in the levels of endogenous free polyamines and plant growth regulators (auxins, gibberellic acid, and cytokinins), but a concomitant reduction in abscisic acid in S. bigelovii-tested tissues. Specifically, we found that putrescine, spermidine, and spermine produced by S. euryhalinus were the major contributors to plant growth parameters, but to a lesser extent by A. deccanensis. Both A. deccanensis and S. euryhalinus were, however, not capable of producing significant levels of other plant growth regulators in vitro. Plant yield was higher in the presence of S. euryhalinus than with A. deccanensis, suggesting relative superiority of S. euryhalinus as a rhizosphere-competent isolate compared to the nonrhizosphere-competent isolate A. deccanensis. This is the first study reporting the production of polyamines by marine actinobacteria and demonstrating the potential of polyamine-producing actinobacteria to enhance growth of halophytic plants through the increase in the endogenous levels of free polyamines and other plant growth regulators.

Comparative analysis of overexpressed Fragaria vesca S-adenosyl-l-methionine synthase (FvSAMS) and decarboxylase (FvSAMDC) during salt stress in transgenic Nicotiana benthamiana

We investigated the effect of overexpressing Fragaria vesca L. cv. Rügen S-adenosyl-l-methionine synthase (FvSAMS) and decarboxylase (FvSAMDC) genes on control and salt stressed Nicotiana benthamiana Domin plants. According to previous studies the overproduction of both proteins enhances the abiotic stress tolerance of plants, but the two enzymes have not yet been studied in one experimental system. We found that the transgenic plants subjected to long-term salt stress displayed higher levels of tolerance than the wild type (WT). In contrast to several earlier studies no antagonistic effect between ethylene and polyamine biosynthesis was observed in our experimental system. Overexpression of FvSAMDC had higher impact on the plant physiological parameters both in control and salt stress conditions, than that of FvSAMS. Based on the data measured in the FvSAMDC lines there appears to be a positive correlation between the free polyamine levels and the proline content as well as the amount of ethylene, while there is a negative correlation between the free polyamine levels and the lignin content in the plants exposed to salt stress. The transformation vectors contained the CaMV35S promoter, the coding sequence of FvSAMS and FvSAMDC fused with synthetic green fluorescent protein (sGFP). We detected the subcellular localization of both enzymes and examined the possible stress induced changes in their distribution. In the case of FvSAMS::sGFP nuclear, nucleolar, cytoplasmic (near to the plasmalemma), plastid membrane, whereas in FvSAMDC::sGFP nuclear and homogenous cytoplasmic localization was detected. Therefore, SAM is assumed to be produced in situ for numerous biochemical reactions.

Differential effects of azinphos-methyl and chlorpyrifos on polyamine oxidative metabolism during the embryonic development of Rhinella arenarum and its relation to oxidative stress

The organophosphorus pesticides azinphos-methyl (AZM) and chlorpyrifos (CPF) exert their toxic action by inhibition of acetylcholinesterase, but non-target processes such as polyamine metabolism can also be affected. Our objective was to evaluate the effects of different concentrations of AZM (0.5-, 2- and 9 mg L−1) and CPF (0.5- and 1 mg L−1) on polyamine oxidative metabolism along Rhinella arenarum embryonic development and to explore its relationship to oxidative stress. Free and conjugated polyamines were measured by HPLC. The activity of spermine oxidase (SMOX), N1-acetylpolyamine oxidase (PAOX) and diamine oxidase (DAO) were measured through kinetic spectrofluorometry. Free putrescine and spermine were significantly increased in open mouth embryos exposed to AZM. Free polyamine levels were not affected by CPF exposure. In embryos exposed to AZM, DAO was increased in tail bud stage and SMOX was increased in open mouth stage, while embryos exposed to CPF showed an increase of PAOX activity in tail bud stage and a decrease of DAO and SMOX activity in open mouth stage. Polyamine levels and oxidative degradation enzymes respond differently if R. arenarum embryos are exposed to AZM or CPF, despite that both insecticides belong to the same chemical family. The early increase of DAO and PAOX would play a protective role to guarantee the normal progression of embryonic development. The increased production of reactive species might contribute to an oxidative stress situation generated by exposure to the insecticides and to the alteration of the antioxidant defense system.In tail bud stage embryos, PAOX and SMOX were positively correlated to acetylcholinesterase activity and reduced glutathione levels (GSH), and negatively correlated to the antioxidant enzymes catalase (CAT) and glutathione S-transferase (GST). In complete operculum embryos, a negative correlation between antioxidant parameters and polyamine levels and polyamine oxidative metabolism was observed, except for SMOX, which showed a low positive correlation with CAT and GSH and a negative correlation to PAOX and DAO. We suggest the use of DAO and PAOX as biomarkers of exposure to AZM and CPF, respectively, as they respond earlier than the classical biomarker acetylcholinesterase.

Fruit Architecture in Polyamine-Rich Tomato Germplasm Is Determined via a Medley of Cell Cycle, Cell Expansion, and Fruit Shape Genes.

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE, and those of CDKB2, CYCB2, KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A/B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.

Diverse response of three sweetpotato cultivars to abiotic stresses and adjustment of free polyamine levels

THREE SWEETPOTATO cultivars, Abees, Mogamba and Santo Amaro, were subjected to moderate (-0.5MPa) and severe drought stress (-2.5MPa) or to salt treatments up to 200mM NaCl, respectively. Patterns of leaf polyamine contents were analyzed to verify whether these metabolites could be used as early traits of tolerance. Physiological parameters like leaf relative water content, total chlorophyll content, leaf electron conductance, stomata resistance and transpiration rate were used to indicate stress conditions of the plants and growth parameters were applied to identify the level of tolerance among the used cultivars. In all three cultivars, drought led to an increase of putrescine (Put) and decrease of spermidine (Spd) concentrations while spermine (Spm) was decreased only in the Santo Amaro cultivar. The most tolerant cultivar was Mogamba followed by Santo Amaro and Abees. However, this descending order was not reflected by the specific changes in polyamine levels but it is altered under drought as well as under salinity in both sensitive and tolerant cultivars. More investigations are needed to figure out if polyamines behave as a response of stress and/or a protective mechanism.

Biostimulant properties of cyanobacterial hydrolysate related to polyamines

Developing new and natural sources of plant growth promotors is essential to ensure the safe and sustainable production of vegetables for human consumption. In recent years, the potential of microalgae as plant biostimulants has been investigated. Arthrospira platensis (Spirulina platensis) biomass is a recognized protein source and its enzymatic hydrolysis contains molecules such as polyamines with potential to promote plant growth. Therefore, the aim of the present study was to investigate the biostimulant properties of hydrolyzed biomass of A. platensis. Bioassays were performed to determine auxin-like and cytokinin-like bioactivity of the hydrolysates. In addition, its effect on lettuce seedling growth was investigated and an organic system field trail performed where yield and free polyamine levels in leaves quantified. The hydrolysates had a cytokinin-like effect in the bioassay. Foliar applications promoted the growth of lettuce seedlings with the 4-h reaction hydrolysate (Sph4) being the most effective at promoting growth and increasing the spermine content by 64% in the lettuce leaves. The polyamine concentration was also compared in non-hydrolyzed A. platensis and Sph4. Hydrolysis resulted in a 34% increase in spermine content. It was concluded that Sph4 is a natural plant growth promoter that can be used as a raw material for biostimulants, and spermine could be an active compound and a metabolic indicator of Sph4 bioactivity.

Exogenous applications of Polyamines modulate drought responses in wheat through osmolytes accumulation, increasing free polyamine levels and regulation of polyamine biosynthetic genes

Polyamines (PAs) can improve drought stress tolerance in plants; however, very limited information is available on the mechanism of action of exogenous application by different methods under drought stress in wheat. The present study investigates the mechanism through which seed priming and foliar spraying with PAs protect wheat plants from drought stress. 10 days old wheat seedlings were exposed to drought stress by withholding water alone or with 100 μM PAs solutions (putrescine, Put; spermine, Spm; and mixture of Put and Spm for 10 h seed-priming or three foliar sprays during withholding water. Drought stress impaired the wheat growth and altered the osmoprotectants, endogenous PAs levels, PAs biosynthetic genes expression and weight of 1000 grains compared to the corresponding control values. Exogenously applied PAs improved cell water status, accumulated osmoprotectants and PAs and up-regulated PAs biosynthetic genes, ADC, arginine decarboxylase; DHS, deoxyhypusine synthase; ODC, ornithine decarboxylase and SAMDC, S-adenosyl methionine decarboxylase. Put significantly regulate the endogenous PAs by both methods of application, however, Spm and mixture of Put and Spm could positively regulate the endogenous PAs and the biosynthetic gene expression by foliar spraying rather than seed priming. The data provide evidence that maintenance of water economy through stabilized cellular structure is an important strategy of drought tolerance by PAs in wheat.

Effects of medium-wave ultraviolet radiation on levels and spectrum of polyamines in leaves and roots of wild-growing plants

The work continues serial studies on short-term effects of medium-wave ultraviolet radiation (UV-B) at 12.5 kJ/m2 on plants. Special attention is paid to the rapid response of the antioxidant system. Free and conjugated forms of putrescine polyamines (putrescine, spermine, and spermidine), as well as those of cadaverine, are recognized to be constituents of the antioxidant system. These compounds were analyzed in plants 24 h after UV-B irradiation. Thellungiella salsuginea (Pallas) O.E.Schulz, Salvia officinalis L, Plantago major L., and Geum urbanum L. grown in aquatic culture under phytotron conditions were examined. The results support the hypothesis that putrescine plays the chief role in the plant defense response against medium-wave ultraviolet irradiation. Three of four plants manifested an increase in the content of this polyamine in leaves. It is the change that determines the enhanced total level of free polyamines. We failed to reveal a general tendency in dynamics of levels of conjugated forms of spermine, spermidine, and cadaverine; only conjugates of putrescine demonstrated a distinct increase. This study allows a conclusion that contributions of particular polyamines to the protective response primarily depend on the species to which the investigated plant belongs. It is likely that conjugated polyamines can be reserved as a pool necessary for rapid recovery of free polyamine levels.

Abstract 5828: Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma

Abstract The highly conserved and unique post-translational hypusine modification is essential for the activation of a single cellular protein called eukaryotic initiation factor 5A (eIF5A). Hypusination of eIF5A represents an essential mechanism in the control of cell proliferation and has been linked to cancer. The hypusine modification of eIF5A requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) and, importantly, uniquely depends on the polyamine spermidine as a selective substrate. Polyamines including spermidine are frequently dysregulated in neoplastic disease and are critical for cell proliferation. Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. We pioneered the repurposing of the irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO) for neuroblastoma (NB), a devastating pediatric cancer of the sympathetic nervous system. Our preclinical work was confirmed by other groups and spurred two consortia (NMTRC and NANT) to test DFMO in independent phase I/II NB clinical trials. The oncogene MYCN is commonly amplified in high-risk NB patients and predicts poor prognosis. MYCN directly activates ODC (and eIF5A) and drives NB tumor progression, presumably through the activation of polyamines, but the precise molecular mechanisms are not fully understood. Since spermidine is the only molecule that can activate eI5FA through hypusine modification, we hypothesized that the polyamine-hypusine nexus provides a rational target site to identify new drug combinations that trigger synergistic anti-tumor effects. In this study we found that DFMO combined with DHPS inhibitor GC7 induced synergistic drug reactions in NB at concentrations that are not lethal on their own. While each drug alone at higher concentrations induced p27/Rb-mediated G1/S cell cycle arrest, the combination of both drugs resulted in the induction of programmed cell death (apoptosis). The intracellular free polyamine levels (putrescine, spermidine, and spermine) were quantified by C-18 RP-HPLC and responded directly to drug treatments, suggesting specific drug targeting effects. This dual-pronged drug combination approach that specifically targets the blockade of the unique polyamine/spermidine-dependent hypusine-eIF5A signaling circuit known to be involved in cell proliferation might lead to more effective DFMO treatment options by reducing high DFMO doses currently needed while achieving better therapeutic outcomes in NB. Citation Format: Andre S. Bachmann, Raid El-Khawaja, Chad Schultz. Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5828. doi:10.1158/1538-7445.AM2017-5828

Nitric oxide-polyamines cross-talk during dormancy release and germination of apple embryos

Nitric oxide (NO) and polyamines (PAs) belong to plant growth and development regulators. These compounds play a key role in numerous physiological processes e.g. seed germination. Based on the suggestion of overlapping of NO and PAs biosynthetic pathways, we demonstrated a cross-talk of NO and PAs in regulation of embryonic dormancy release. The aim of the work was to investigate an impact of PAs (Put, Spd and Spm) or NO short-term fumigation on nitrite, urea, Arg and ornithine (Orn) content, NO synthase-like (NOS-like) and arginase activity in axes of apple (Malus domestica Borkh.) embryos during dormancy alleviation and at the stage of termination of germination sensu stricto. NO, Put/Spd induced dormancy breakage and germination of apple embryos corresponded to stimulation of urea cycle and high free Arg pool in seedlings roots. After two days of the culture Put and Spd stimulated Arg dependent NO formation, inhibition of which was observed after Spm application. Put or Spd application as well as NO short-term pretreatment of apple embryos influenced level of ubiquitin-conjugated proteins. Higher abundance of such modified proteins correlated well to the declined content of nitrated proteins, suggesting their important role in regulation of embryo germination. NO led to stimulation of embryos germination by increasing level of free PAs (mostly Put). While transcriptomic approach showed down regulation of Spm synthesis and up-regulation of Spm degradation by NO, confirming negative role of Spm over-accumulation in embryo dormancy removal. Our data clearly indicate positive relationship of NO-Put/Spd acting as dormancy removing factors.

Physiological and biochemical mechanisms of spermine-induced cadmium stress tolerance in mung bean (Vigna radiata L.) seedlings.

The role of exogenous spermine (0.25 mM Spm, a type of polyamine (PA) in reducing Cd uptake and alleviating Cd toxicity (containing 1 and 1.5mM CdCl2 in the growing media) effects was studied in the mung bean (Vigna radiata L. cv. BARI Mung-2) plant. Exogenously applied Spm reduced Cd content, accumulation, and translocation in different plant parts. Increasing phytochelatin content, exogenous Spm reduced Cd accumulation and translocation. Spm application reduced the Cd-induced oxidative damage which was reflected from the reduction of H2O2 content, O2•- generation rate, lipoxygenase (LOX) activity, and lipid peroxidation level and also reflected from the reduction of spots of H2O2 and O2•- from mung bean leaves (compared to control treatment). Spm pretreatment increased non-enzymatic antioxidant contents (ascorbate, AsA, and glutathione, GSH) and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) which reduced oxidative stress. The cytotoxicity of methylglyoxal (MG) is also reduced by exogenous Spm because it enhanced glyoxalase system enzymes and components. Through osmoregulation, Spm maintained a better water status of Cd-affected mung bean seedlings. Spm prevented the chl damage and increased its content. Exogenous Spm also modulated the endogenous free PAs level which might have the roles in improving physiological processes including antioxidant capacity, osmoregulation, and Cd and MG detoxification capacity. The overall Spm-induced tolerance of mung bean seedlings to Cd toxicity was reflected through improved growth of mung bean seedlings.

The miR396b of Poncirus trifoliata Functions in Cold Tolerance by Regulating ACC Oxidase Gene Expression and Modulating Ethylene-Polyamine Homeostasis.

MicroRNAs (miRNAs) are non-coding regulatory molecules that play important roles in a variety of biological processes. Although a number of cold-responsive miRNAs have been computationally identified, functions and mechanisms of most miRNAs are not well understood. Herein, the function of trifoliate orange [Poncirus trifoliata (L.) Raf.] miRNA396b (ptr-miR396b) in cold tolerance and its potential regulatory module were investigated. Compared with the wild type (WT), transgenic lemon (Citrus limon) plants overexpressing ptr-MIR396b, the precursor of ptr-miR396b, displayed enhanced cold tolerance. Ptr-miR396b was experimentally confirmed to guide the cleavage of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). The overexpressing lines exhibited a reduction in ACO transcript levels and ethylene content compared with the WT, and the expression pattern of ACO was opposite to that of ptr-miR396b in response to cold stress. In addition, the transgenic lines exhibited higher levels of free polyamines and mRNA abundance of polyamine biosynthetic genes than WT plants under cold treatment, consistent with reduced reactive oxygen species (ROS) accumulation in the former. Taken together, this study demonstrates that ptr-miR396b positively regulates cold tolerance through reducing ACO transcript levels, thereby repressing ethylene synthesis and simultaneously promoting polyamine synthesis, leading to enhanced ROS scavenging. Identification of the ptr-miR396b-ACO regulatory module provides new insights into the molecular mechanism underlying the reduction of ethylene production under cold.