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1.  Interaction of Brassinosteroids and Polyamines Enhances Copper Stress Tolerance in Raphanus Sativus  
Journal of Experimental Botany  2012;63(15):5659-5675.
Brassinosteroids (BRs) and polyamines (PAs) regulate various responses to abiotic stress, but their involvement in the regulation of copper (Cu) homeostasis in plants exposed to toxic levels of Cu is poorly understood. This study provides an analysis of the effects of exogenously applied BRs and PAs on radish (Raphanus sativus) plants exposed to toxic concentrations of Cu. The interaction of 24-epibrassinolide (EBR, an active BR) and spermidine (Spd, an active PA) on gene expression and the physiology of radish plants resulted in enhanced tolerance to Cu stress. Results indicated that the combined application of EBR and Spd modulated the expression of genes encoding PA enzymes and genes that impact the metabolism of indole-3-acetic acid (IAA) and abscisic acid (ABA) resulting in enhanced Cu stress tolerance. Altered expression of genes implicated in Cu homeostasis appeared to be the main effect of EBR and Spd leading to Cu stress alleviation in radish. Ion leakage, in vivo imaging of H2O2, comet assay, and improved tolerance of Cu-sensitive yeast strains provided further evidence for the ability of EBR and Spd to improve Cu tolerance significantly. The study indicates that co-application of EBR and Spd is an effective approach for Cu detoxification and the maintenance of Cu homeostasis in plants. Therefore, the use of these compounds in agricultural production systems should be explored.
PMCID: PMC3444278  PMID: 22915739
Abscisic acid; brassinosteroids; comet assay; copper transporters; Cu homeostasis; Cu-sensitive yeast; indole-3-acetic acid; oxidative stress; polyamines
2.  24-epibrassinolide induced antioxidative defense system of Brassica juncea L. under Zn metal stress 
The present study deals with the effects of 24-epibrassinolide on growth, lipid peroxidation, antioxidative enzyme activities, non-enzymatic antioxidants and protein content in 30 days old leaves of Brassica juncea (var. PBR 91) under zinc metal stress in field conditions. Surface sterilized seeds of B. juncea were given pre-soaking treatments of 24-EBL (10−10, 10−8 and 10−6 M) for 8 h. Different concentrations of zinc metal in the form of ZnSO4.7H2O (0, 0.5, 1.0, 1.5 and 2.0 mM) were added in the soil kept in experimental pots. Seeds soaked in 24-EBL for 8 h were sown in the earthern pots containing different concentrations of Zn metal. After 30 days of sowing, the plants were analyzed for growth parameters in terms of shoot length and number of leaves. Thereafter, leaves were excised and content of proteins, non-enzymatic antioxidants, malondialdehyde (MDA) and the activities of antioxidative enzymes (superoxide dismutase (SOD) (EC catalase (CAT) (EC, ascorbate peroxidase (APOX) (EC, guaiacol peroxidase (POD) (EC glutathione reductase (GR) (EC, monodehydroascorbate reductase (MDHAR) (EC and dehydroascorbate reductase (DHAR) (EC were analyzed. It was observed that the growth of plants was inhibited under Zn metal stress. However, 24-EBL seed-presoaking treatment improved the plant growth in terms of increase in shoot length. 24-EBL also mitigated the toxicity of Zn metal by increasing the number of leaves. The activities of antioxidative enzymes (SOD, CAT, POD, GR, APOX, MDHAR and DHAR) and contents of proteins and glutathione were also enhanced in leaves of plants treated with 24-EBL alone, 10−8 M concentration being the most effective. The activities of antioxidative enzymes also increased in leaves of B. juncea plants by the application 24-EBL supplemented Zn metal solutions. Similarly, the content of proteins and glutathione increased considerably in leaves of B. juncea plants treated with 24-EBL, whereas the level of MDA content decreased in 24-EBL treated plants as compared to untreated control plants thereby revealing stress-protective properties of the brassinolide.
PMCID: PMC3550670  PMID: 23572978
Antioxidative enzymes; Brassica juncea; 24-epibrassinolide; Zn toxicity
3.  The Photoprotective Role of Spermidine in Tomato Seedlings under Salinity-Alkalinity Stress 
PLoS ONE  2014;9(10):e110855.
Polyamines are small, ubiquitous, nitrogenous compounds that scavenge reactive oxygen species and stabilize the structure and function of the photosynthetic apparatus in response to abiotic stresses. Molecular details underlying polyamine-mediated photoprotective mechanisms are not completely resolved. This study investigated the role of spermidine (Spd) in the structure and function of the photosynthetic apparatus. Tomato seedlings were subjected to salinity-alkalinity stress with and without foliar application of Spd, and photosynthetic and morphological parameters were analyzed. Leaf dry weight and net photosynthetic rate were reduced by salinity-alkalinity stress. Salinity-alkalinity stress reduced photochemical quenching parameters, including maximum photochemistry efficiency of photosystem II, quantum yield of linear electron flux, and coefficient of photochemical quenching (qP). Salinity-alkalinity stress elevated nonphotochemical quenching parameters, including the de-epoxidation state of the xanthophyll cycle and nonphotochemical quenching (NPQ). Microscopic analysis revealed that salinity-alkalinity stress disrupted the internal lamellar system of granal and stromal thylakoids. Exogenous Spd alleviated the stress-induced reduction of leaf dry weight, net photosynthetic rate, and qP parameters. The NPQ parameters increased by salinity-alkalinity stress were also alleviated by Spd. Seedlings treated with exogenous Spd had higher zeaxanthin (Z) contents than those without Spd under salinity-alkalinity stress. The chloroplast ultrastructure had a more ordered arrangement in seedlings treated with exogenous Spd than in those without Spd under salinity-alkalinity stress. These results indicate that exogenous Spd can alleviate the growth inhibition and thylakoid membrane photodamage caused by salinity-alkalinity stress. The Spd-induced accumulation of Z also may have an important role in stabilizing the photosynthetic apparatus.
PMCID: PMC4207769  PMID: 25340351
4.  Effects of 24-epibrassinolide and 28-homobrassinolide on the growth and antioxidant enzyme activities in the seedlings of Brassica juncea L. 
The present paper deals with the effects of two active forms of brassinosteroids (BRs) as epibrassinosteroid (24-EBL) and homobrassinosteroid (28-HBL) on percentage germination, growth in the form of shoot length, activities of auxinase (IAAO), polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APOX) in 10 day old seedlings of Brassica juncea L. (RCM 619) under field conditions. Exogenous application of 240-EBL and 28-HBL significantly ameliorate the total protein content as compared to untreated control seedlings. 10−8 M 28-HBL helps in enhancing the PPO activity very significantly, as compared to all other concentrations of EBL and HBL and also to that of untreated control. Similar trend was observed in IAAO activity. It was observed that all the concentrations of EBL were unable to enhance the APOX activity as compared to untreated control seedlings but 10−8 M HBL significantly ameliorates APOX activity. CAT and SOD activities ameliorate significantly with exogenous application of EBL and HBL. Out of two active forms of BRs, 28-HBL was more effective at germination stage in scavenging the free radicals, which are produced in greater amount during germination from basic metabolic processes, whereas 28-EBL was effective in the initial growth of seedlings in the form of increase in shoot length.
PMCID: PMC3550350  PMID: 23572944
Brassinosteroids; Brassica juncea; Antioxidant enzymes; Polyphenol oxidase; Auxinase; Total Proteins
5.  Characterization of transgenic mice with overexpression of spermidine synthase 
Amino Acids  2011;42(2-3):495-505.
A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) in order to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to 9-fold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5 to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.
PMCID: PMC3245749  PMID: 21809077
polyamine; aminopropyltransferase; transgenic mice; S-adenosylmethionine decarboxylase; spermidine; spermine
6.  Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots 
Journal of Experimental Botany  2013;64(14):4271-4287.
MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.
PMCID: PMC3808317  PMID: 24014874
Cadmium stress; degradome; high-throughput sequencing; microRNAs; Raphanus sativus; transcriptome.
7.  The Relationship between Polyamines and Hormones in the Regulation of Wheat Grain Filling 
PLoS ONE  2013;8(10):e78196.
The grain weight of wheat is strongly influenced by filling. Polyamines (PA) are involved in regulating plant growth. However, the effects of PA on wheat grain filling and its mechanism of action are unclear. The objective of the present study was to investigate the relationship between PAs and hormones in the regulation of wheat grain filling. Three PAs, spermidine (Spd), spermine (Spm), and putrescine (Put), were exogenously applied, and the grain filling characteristics and changes in endogenous PA and hormones, i.e., indole-3-acetic acid (IAA), zeatin (Z) + zeatin riboside (ZR), abscisic acid (ABA), ethylene (ETH) and gibberellin 1+4 (GAs), were quantified during wheat grain filling. Exogenous applications of Spd and Spm significantly increased the grain filling rate and weight, but exogenous Put had no significant effects on these measures. Exogenous Spd and Spm significantly increased the endogenous Spd, Spm, Z+ZR, ABA, and IAA contents and significantly decreased ETH evolution in grains. The endogenous Spd, Spm and Z+ZR contents were positively and significantly correlated with the grain filling rate and weight of wheat, and the endogenous ETH evolution was negatively and significantly correlated with the wheat grain filling rate and weight. Based upon these results, we concluded that PAs were involved in the balance of hormones that regulated the grain filling of wheat.
PMCID: PMC3812141  PMID: 24205154
8.  Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought 
Soil salinity affects a large proportion of rural area and limits agricultural productivity. To investigate differential adaptation to soil salinity, we studied salt tolerance of 18 varieties of Oryza sativa using a hydroponic culture system. Based on visual inspection and photosynthetic parameters, cultivars were classified according to their tolerance level. Additionally, biomass parameters were correlated with salt tolerance. Polyamines have frequently been demonstrated to be involved in plant stress responses and therefore soluble leaf polyamines were measured. Under salinity, putrescine (Put) content was unchanged or increased in tolerant, while dropped in sensitive cultivars. Spermidine (Spd) content was unchanged at lower NaCl concentrations in all, while reduced at 100 mM NaCl in sensitive cultivars. Spermine (Spm) content was increased in all cultivars. A comparison with data from 21 cultivars under long-term, moderate drought stress revealed an increase of Spm under both stress conditions. While Spm became the most prominent polyamine under drought, levels of all three polyamines were relatively similar under salt stress. Put levels were reduced under both, drought and salt stress, while changes in Spd were different under drought (decrease) or salt (unchanged) conditions. Regulation of polyamine metabolism at the transcript level during exposure to salinity was studied for genes encoding enzymes involved in the biosynthesis of polyamines and compared to expression under drought stress. Based on expression profiles, investigated genes were divided into generally stress-induced genes (ADC2, SPD/SPM2, SPD/SPM3), one generally stress-repressed gene (ADC1), constitutively expressed genes (CPA1, CPA2, CPA4, SAMDC1, SPD/SPM1), specifically drought-induced genes (SAMDC2, AIH), one specifically drought-repressed gene (CPA3) and one specifically salt-stress repressed gene (SAMDC4), revealing both overlapping and specific stress responses under these conditions.
PMCID: PMC4021140  PMID: 24847340
polyamines; salt stress; drought stress; gene expression; rice; natural variety
9.  Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A 
Amino Acids  2011;42(2-3):685-695.
The polyamines, putrescine, spermidine and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report a novel series of mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) and their role in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9–18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine-modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.
PMCID: PMC3223563  PMID: 21861168
polyamine; methylated spermidine; cell growth; deoxyhypusine synthase; hypusine; eIF5A
10.  Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence▿†  
Infection and Immunity  2009;77(12):5418-5427.
Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.
PMCID: PMC2786486  PMID: 19752030
11.  Polyamine metabolic canalization in response to drought stress in Arabidopsis and the resurrection plant Craterostigma plantagineum 
Plant Signaling & Behavior  2011;6(2):243-250.
In this work, we have studied the transcriptional profiles of polyamine biosynthetic genes and analyzed polyamine metabolic fluxes during a gradual drought acclimation response in Arabidopsis thaliana and the resurrection plant Craterostigma plantagineum. The analysis of free putrescine, spermidine and spermine titers in Arabidopsis arginine decarboxylase (adc1–3, adc2–3), spermidine synthase (spds1–2, spds2–3) and spermine synthase (spms-2) mutants during drought stress, combined with the quantitative expression of the entire polyamine biosynthetic pathway in the wild-type, has revealed a strong metabolic canalization of putrescine to spermine induced by drought. Such canalization requires spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) activities and, intriguingly, does not lead to spermine accumulation but to a progressive reduction in spermidine and spermine pools in the wild-type. Our results suggest the participation of the polyamine back-conversion pathway during the drought stress response rather than the terminal catabolism of spermine. The putrescine to spermine canalization coupled to the spermine to putrescine back-conversion confers an effective polyamine recycling-loop during drought acclimation. Putrescine to spermine canalization has also been revealed in the desiccation tolerant plant C. plantagineum, which conversely to Arabidopsis, accumulates high spermine levels which associate with drought tolerance. Our results provide a new insight to the polyamine homeostasis mechanisms during drought stress acclimation in Arabidopsis and resurrection plants.
PMCID: PMC3121985  PMID: 21330782
Arabidopsis; Craterostigma plantagineum; drought; polyamines; polyamine oxidase; abiotic stress
12.  Spermidine affects the transcriptome responses to high temperature stress in ripening tomato fruit* #  
Objective: High temperature adversely affects quality and yield of tomato fruit. Polyamine can alleviate heat injury in plants. This study is aimed to investigate the effects of polyamine and high temperature on transcriptional profiles in ripening tomato fruit. Methods: An Affymetrix tomato microarray was used to evaluate changes in gene expression in response to exogenous spermidine (Spd, 1 mmol/L) and high temperature (33/27 °C) treatments in tomato fruits at mature green stage. Results: Of the 10 101 tomato probe sets represented on the array, 127 loci were differentially expressed in high temperature-treated fruits, compared with those under normal conditions, functionally characterized by their involvement in signal transduction, defense responses, oxidation reduction, and hormone responses. However, only 34 genes were up-regulated in Spd-treated fruits as compared with non-treated fruits, which were involved in primary metabolism, signal transduction, hormone responses, transcription factors, and stress responses. Meanwhile, 55 genes involved in energy metabolism, cell wall metabolism, and photosynthesis were down-regulated in Spd-treated fruits. Conclusions: Our results demonstrated that Spd might play an important role in regulation of tomato fruit response to high temperature during ripening stage.
PMCID: PMC3323944  PMID: 22467370
Solanum lycopersicum L.; Spermidine; High temperature; Microarray; Gene expression
13.  Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells 
Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death.
PMCID: PMC3747394  PMID: 23983896
14.  Does polyamine catabolism influence root development and xylem differentiation under stress conditions? 
Plant Signaling & Behavior  2011;6(11):1844-1847.
Amine oxidases (AOs) oxidize polyamines (PAs) to aldehydes, simultaneously producing the removed amine moiety and hydrogen peroxide (H2O2). AOs, which include copper-containing amine oxidases (CuAOs) and flavin-containing amine oxidases (PAOs), are stress-inducible enzymes involved in both PA homeostasis and H2O2 production. Here, we suggest that H2O2 derived from PAO-mediated PA catabolism has a role in inducing root xylem differentiation during plant stress responses, whereas its involvement in this event during plant development under physiological conditions is not suitably supported by the currently available data. Moreover, we show that spermidine (Spd) supply leads to a higher induction of cell death in wild-type (WT) tobacco (Nicotiana tabacum) plants as compared to tobacco plants over-expressing maize (Zea mays) PAO (S-ZmPAO) in the cell wall, in apparent contradiction with the already reported results obtained by the analysis of the corresponding WT and S-ZmPAO Spd-untreated plants. Considering this last observation, we propose that PAs  diversely affect plant development and stress responses depending on the expression levels of AOs, which in turn may lead to different plant responses by altering the PAs/H2O2 balance.
PMCID: PMC3329365  PMID: 22057326
copper amine oxidase; hydrogen peroxide; PCD; polyamine; polyamine oxidase; xylem
15.  Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels 
BMC Genomics  2010;11:235.
Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.
Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.
This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
PMCID: PMC2867828  PMID: 20385001
16.  Transcriptome Profiling of Radish (Raphanus sativus L.) Root and Identification of Genes Involved in Response to Lead (Pb) Stress with Next Generation Sequencing 
PLoS ONE  2013;8(6):e66539.
Lead (Pb), one of the most toxic heavy metals, can be absorbed and accumulated by plant roots and then enter the food chain resulting in potential health risks for human beings. The radish (Raphanus sativus L.) is an important root vegetable crop with fleshy taproots as the edible parts. Little is known about the mechanism by which radishes respond to Pb stress at the molecular level. In this study, Next Generation Sequencing (NGS)–based RNA-seq technology was employed to characterize the de novo transcriptome of radish roots and identify differentially expressed genes (DEGs) during Pb stress. A total of 68,940 assembled unique transcripts including 33,337 unigenes were obtained from radish root cDNA samples. Based on the assembled de novo transcriptome, 4,614 DEGs were detected between the two libraries of untreated (CK) and Pb-treated (Pb1000) roots. Gene Ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Pb stress are predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to heavy metal Pb. Identification of potential DEGs involved in responses to Pb stress significantly reflected alterations in major biological processes and metabolic pathways. The molecular basis of the response to Pb stress in radishes was comprehensively characterized. Useful information and new insights were provided for investigating the molecular regulation mechanism of heavy metal Pb accumulation and tolerance in root vegetable crops.
PMCID: PMC3688795  PMID: 23840502
17.  Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes 
PLoS ONE  2014;9(11):e112893.
The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O2 uptake rates, P700 photooxidation and the accumulation of superoxide anions (O2−) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O2− generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O2− generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.
PMCID: PMC4242612  PMID: 25420109
18.  ACME encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines 
Molecular microbiology  2011;82(1):9-20.
Polyamines, including spermine (Spm) and spermidine (Spd), are aliphatic cations that are reportedly synthesized by all living organisms. They exert pleiotropic effects on cells and are required for efficient nucleic acid and protein synthesis. Here, we report that the human pathogen Staphylococcus aureus lacks identifiable polyamine biosynthetic genes, and consequently produces no Spm/Spd or their precursor compounds putrescine and agmatine. Moreover, while supplementing defined medium with polyamines generally enhances bacterial growth, Spm and Spd exert bactericidal effects on S. aureus at physiologic concentrations. Small colony variants specifically lacking menaquinone biosynthesis arose after prolonged Spm exposure and exhibited reduced polyamine-sensitivity. However, other respiratory-defective mutants were no less susceptible to Spm implying menaquinone itself rather than general respiration is required for full Spm-toxicity. Polyamine hypersensitivity distinguishes S. aureus from other bacteria and is exhibited by all tested strains save those belonging to the USA-300 group of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA). We identified one gene within the USA-300-specific Arginine Catabolic Mobile Element (ACME) encoding a Spm/Spd N-acetyltransferase that is necessary and sufficient for polyamine resistance. S. aureus encounters significant polyamine levels during infection, however the acquisition of ACME encoded speG allows USA-300 clones to circumvent polyamine-hypersensitivity, a peculiar trait of S. aureus.
PMCID: PMC3183340  PMID: 21902734
19.  28-Homobrassinolide Alters Protein Content and Activities of Glutathione-S-Transferase and Polyphenol Oxidase in Raphanus Sativus L. Plants Under Heavy Metal Stress 
Toxicology International  2014;21(1):44-50.
The application of brassinosteroids (BRs), the plant steroidal hormones, results in an increased tolerance toward stress and thus helps improving the yield of crop plants. The present study was carried out to investigate the effect of 28-homobrassinolide (28-HBL) on the protein content as well as activities of antioxidant enzymes viz., glutathione-s-transferase (GST) and polyphenol oxidase (PPO) in radish plants grown under Cadmium (Cd) and Mercury (Hg) metal stress.
Materials and Methods:
Shoots of 60 and 90 days old radish plants, grown under Cd and Hg metal stress (0, 0.5, 1.0, 1.5 mM) and given the presowing treatment of 28-HBL (0, 10-7, 10-9, 10-11 M) to seeds for 8 h, were analyzed for protein content and GST and PPO enzyme activities.
Protein content showed decrease in plants given Cd and Hg metal treatment alone, while treatment with 28-HBL enhanced the protein content, suggesting its stress protective role. An increase in the activity of antioxidative enzymes was also observed in plants stressed with heavy metals as well as in those supplemented with 28-HBL.
In the present investigation, the activity of antioxidative enzymes was found to increase due to metal stress and a further increase was noticed in plants given both metal and 28-HBL treatment, suggesting the stress protective role of 28-HBL via modulating the antioxidative enzymes.
PMCID: PMC3989914  PMID: 24748734
28-HBL; glutathione-s-transferase; polyphenol oxidase; Raphanus sativus L.
20.  Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the bauRABCD Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1 ▿  
Journal of Bacteriology  2011;193(15):3923-3930.
Pseudomonas aeruginosa and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them, pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 (polyamine utilization) encode enzymes homologous to Escherichia coli PuuABCD of the γ-glutamylation pathway in converting Put into GABA. A series of unmarked pauA mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of pauA knockouts to abolish utilization of different polyamines and support the importance of γ-glutamylation for polyamine catabolism in P. aeruginosa. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the pauA3B2 operon and the bauABCD operon (β-alanine utilization). Mutation analysis led to the conclusion that pauA3B2 participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that bauABCD are essential for growth on β-alanine derived from DAP (or Spd) catabolism via the γ-glutamylation pathway. Measurements of the pauA3-lacZ and bauA-lacZ expression indicated that these two promoters were differentially induced by Spd, DAP, and β-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the pauA3 and bauA promoters was abolished in the bauR mutant. The recombinant BauR protein was purified to demonstrate its interactions with the pauA3 and bauA regulatory regions in vitro. In summary, the present study support that the γ-glutamylation pathway for polyamine utilization is evolutionarily conserved in E. coli and Pseudomonas spp. and is further expanded in Pseudomonas to accommodate a more diverse metabolic capacity in this group of microorganisms.
PMCID: PMC3147493  PMID: 21622750
21.  Histatin 5-Spermidine Conjugates Have Enhanced Fungicidal Activity and Efficacy as a Topical Therapeutic for Oral Candidiasis 
Oropharyngeal candidiasis (OPC) is caused by the opportunistic fungi Candida albicans and is prevalent in immunocompromised patients, individuals with dry mouth, or patients with prolonged antibiotic therapies that reduce oral commensal bacteria. Human salivary histatins, including histatin 5 (Hst 5), are small cationic proteins that are the major source of fungicidal activity of saliva. However, Hsts are rapidly degraded in vivo, limiting their usefulness as therapeutic agents despite their lack of toxicity. We constructed a conjugate peptide using spermidine (Spd) linked to the active fragment of Hst 5 (Hst 54–15), based upon our findings that C. albicans spermidine transporters are required for Hst 5 uptake and fungicidal activity. We found that Hst 54–15-Spd was significantly more effective in killing C. albicans and Candida glabrata than Hst 5 alone in both planktonic and biofilm growth and that Hst 54–15-Spd retained high activity in both serum and saliva. Hst 54–15-Spd was not bactericidal against streptococcal oral commensal bacteria and had no hemolytic activity. We tested the effectiveness of Hst 54–15-Spd in vivo by topical application to tongue surfaces of immunocompromised mice with OPC. Mice treated with Hst 54–15-Spd had significant clearance of candidal tongue lesions macroscopically, which was confirmed by a 3- to 5-log fold reduction of C. albicans colonies recovered from tongue tissues. Hst 54–15-Spd conjugates are a new class of peptide-based drugs with high selectivity for fungi and potential as topical therapeutic agents for oral candidiasis.
PMCID: PMC3910849  PMID: 24247141
22.  Effects of extended exposure to cadmium and subsequent recovery period on growth, antioxidant status and polyamine pattern in in vitro cultured carnation 
The effect of different doses of Cd (0.05, 0.1 and 0.2 mM) and subsequent period in a Cd-free medium on growth, the antioxidant status and the polyamine (PA) pattern was studied using in vitro cultured nodal segments of carnation. The Cd within the tissues increased in parallel with its concentration in the culture medium, inhibited growth, altered the concentration of some minerals and decreased the levels of pigments and the total antioxidants. However, the concentration of ascorbate (Asc) + dehydroascorbate (DHA) and the Asc redox status remained unaffected, and malondialdehyde (MDA) increased only with 0.2 mM Cd. Cd also affected PA metabolism, decreasing the total PA concentration and disturbing the relative predominance of each PA fraction. Cd exposure increased the total putrescine (Put)/(spermidine (Spd) + spermine (Spm)) ratio, and an opposite pattern was recorded during the phase in Cd-free medium. Regarding individual amines, Cd induced significant changes mainly in the free Put levels. Our results suggest that Cd produces oxidative stress and that PA (especially free Put and the total Put/(Spd+Spm) ratio), are good indicators of the stress caused by Cd.
PMCID: PMC3550592  PMID: 23573026
Cadmium; Carnation; Dianthus caryophyllus; Oxidative stress; Polyamines; Antioxidants
23.  Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase 
Putrescine N-methyltransferases (PMTs) are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-l-methionine (SAM) as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs), which are ubiquitous enzymes of polyamine metabolism. SPDSs use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in D. stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.
PMCID: PMC3725402  PMID: 23908659
spermidine synthase; putrescine N-methyltransferase; evolution; Datura stramonium; Arabidopsis thaliana; homology modeling
24.  Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells 
PLoS ONE  2012;7(10):e46907.
Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.
PMCID: PMC3466176  PMID: 23056524
25.  Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways 
Cell Death & Disease  2012;3(10):e401-.
The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.
PMCID: PMC3481127  PMID: 23059820
Drosophila melanogaster; spermidine; paraquat; oxidative stress; activity; starvation

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