Polyamine Homeostasis Research Articles

Polyamines in Pollen: From Microsporogenesis to Fertilization.

The entire pollen life span is driven by polyamine (PA) homeostasis, achieved through fine regulation of their biosynthesis, oxidation, conjugation, compartmentalization, uptake, and release. The critical role of PAs, from microsporogenesis to pollen–pistil interaction during fertilization, is suggested by high and dynamic transcript levels of PA biosynthetic genes, as well as by the activities of the corresponding enzymes. Moreover, exogenous supply of PAs strongly affects pollen maturation and pollen tube elongation. A reduction of endogenous free PAs impacts pollen viability both in the early stages of pollen development and during fertilization. A number of studies have demonstrated that PAs largely function by modulating transcription, by structuring pollen cell wall, by modulating protein (mainly cytoskeletal) assembly as well as by modulating the level of reactive oxygen species. Both free low-molecular weight aliphatic PAs, and PAs conjugated to proteins and hydroxyl-cinnamic acids take part in these complex processes. Here, we review both historical and recent evidence regarding molecular events underlying the role of PAs during pollen development. In the concluding remarks, the outstanding issues and directions for future research that will further clarify our understanding of PA involvement during pollen life are outlined.

The Essential Role of Spermidine in Growth of Agrobacterium tumefaciens Is Determined by the 1,3-Diaminopropane Moiety.

The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of α-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine; however, exogenously supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.

Correlation between endogenous polyamines in human cardiac tissues and clinical parameters in patients with heart failure.

Polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy in experimental animals. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with cyclic adenosine monophosphate (cAMP) increases. The aim of the study was to establish the role of these in the human heart in living patients. For this, polyamines (by high performance liquid chromatography) and the activity of ODC and N(1)-acetylpolyamine oxidases (APAO) were determined in the right atrial appendage of 17 patients undergoing extracorporeal circulation to correlate with clinical parameters. There existed enzymatic activity associated with the homeostasis of polyamines. Left atria size was positively associated with ODC (r = 0.661, P = 0.027) and negatively with APAO-N(1) -acetylspermine (r = -0.769, P = 0.026), suggesting that increased levels of polyamines are associated with left atrial hemodynamic overload. Left ventricular ejection fraction (LVEF) and heart rate were positively associated with spermidine (r = 0.690, P = 0.003; r = 0.590, P = 0.021) and negatively with N(1)-acetylspermidine (r = -0.554, P = 0.032; r = -0.644, P = 0.018). LVEF was negatively correlated with cAMP levels (r = -0.835, P = 0.001) and with cAMP/ODC (r = -0.794, P = 0.011), cAMP/spermidine (r = -0.813, P = 0.001) and cAMP/spermine (r = -0.747, P = 0.003) ratios. Abnormal LVEF patients showed decreased ODC activity and spermidine, and increased N(1) -acetylspermidine, and cAMP. Spermine decreased in congestive heart failure patients. The trace amine isoamylamine negatively correlated with septal wall thickness (r = -0.634, P = 0.008) and was increased in cardiac heart failure. The results indicated that modifications in polyamine homeostasis might be associated with cardiac function and remodelling. Increased cAMP might have a deleterious effect on function. Further studies should confirm these findings and the involvement of polyamines in different stages of heart failure.

Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1.

Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation.

Structural basis of antizyme-mediated regulation of polyamine homeostasis

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Osmotic stress affects polyamine homeostasis and phenolic content in proembryogenic liquid cell cultures of Scots pine

Polyamines (PAs) are ubiquitous polycations involved in many physiological processes in plants, including somatic embryogenesis, cellular growth and stress reactions. In the present study, we focus on the consequences in PA metabolism caused by polyethylene glycol (PEG) in proembryogenic Scots pine (Pinus syl- vestris L.) liquid cultures. The growth and viability of the cell masses and changes in PA concentrations and phenolic secondary metabolites were investigated under control, 5 and 10 % PEG treatments. The effect of osmotic stress responses was investigated at the gene expression level including stress, cell division, programmed cell death and PA-related genes and PA metabolites. Moreover, the expression of ethylene and proline biosynthesis genes and phenylalanine ammonia lyase and stilbene synthase (psSTS) was analyzed. Under osmotic stress conditions, we found a consistent pattern of endogenous PAs in Scots pine proembryogenic cells. However, accumulation of free spermine (Spm) and methyl putrescine under osmotic stress might indicate their specific role in stress protection. Expression of polyamine oxidase was down-regulated under osmotic stress, suggesting the role of PA catabolism in regulation of Spm levels. Scots pine proliferating proembryogenic cells are in a developmentally undiffer- entiated stage where the content of secondary metabolites is generally low. However, in the present study the total content of phenolic compounds increased but the biosyn- thesis of phenylpropanoids and stilbenes, generally con- sidered as stress-protective molecules, was not affected by osmotic stress.

A role for antizyme inhibitor in cell proliferation.

The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.

Reciprocal osmotic challenges reveal mechanisms of divergence in phenotypic plasticity in the killifish Fundulus heteroclitus.

The killifish Fundulus heteroclitus is an estuarine species with broad physiological plasticity, enabling acclimation to diverse stressors. Previous work suggests that freshwater populations expanded their physiology to accommodate low salinity environments; however, it is unknown whether this compromises their tolerance to high salinity. We used a comparative approach to investigate the mechanisms of a derived freshwater phenotype and the fate of an ancestral euryhaline phenotype after invasion of a freshwater environment. We compared physiological and transcriptomic responses to high- and low-salinity stress in fresh and brackish water populations and found an enhanced plasticity to low salinity in the freshwater population coupled with a reduced ability to acclimate to high salinity. Transcriptomic data identified genes with a conserved common response, a conserved salinity-dependent response and responses associated with population divergence. Conserved common acclimation responses revealed stress responses and alterations in cell-cycle regulation as important mechanisms in the general osmotic response. Salinity-specific responses included the regulation of genes involved in ion transport, intracellular calcium, energetic processes and cellular remodeling. Genes diverged between populations were primarily those showing salinity-specific expression and included those regulating polyamine homeostasis and the cell cycle. Additionally, when populations were matched with their native salinity, expression patterns were consistent with the concept of 'transcriptomic resilience', suggesting local adaptation. These findings provide insight into the fate of a plastic phenotype after a shift in environmental salinity and help to reveal mechanisms allowing for euryhalinity.

Drought stress tolerance in grapevine involves activation of polyamine oxidation contributing to improved immune response and low susceptibility to Botrytis cinerea.

Environmental factors including drought stress may modulate plant immune responses and resistance to pathogens. However, the relationship between mechanisms of drought tolerance and resistance to pathogens remained unknown. In this study, the effects of drought stress on polyamine (PA) homeostasis and immune responses were investigated in two grapevine genotypes differing in their drought tolerance; Chardonnay (CHR), as sensitive and Meski (MSK), as tolerant. Under drought conditions, MSK plants showed the lowest leaf water loss and reduction of photosynthetic efficiency, and expressed a lower level of NCED2, a gene involved in abscisic acid biosynthesis, compared with CHR plants. The improved drought tolerance in MSK was also coincident with the highest change in free PAs and up-regulation of the genes encoding arginine decarboxylase (ADC), copper amine-oxidase (CuAO), and PA-oxidases (PAO) and their corresponding enzyme activities. MSK plants also accumulated the highest level of amino acids, including Arg, Glu, Gln, Pro, and GABA, emphasizing the participation of PA-related amino acid homeostasis in drought tolerance. Importantly, drought-tolerant plants also exhibited enhanced phytoalexin accumulation and up-regulation of PR genes, especially PR-2 and Chit4c, compared with the sensitive plants. This is consistent with a lower susceptibility of MSK than CHR to Botrytis cinerea. Data suggest a possible connection between water stress tolerance and immune response in grapevine. Pharmacological experiments revealed that under drought conditions CuAO and PAO pathways were involved in the regulation of photosynthetic efficiency, and also of immune response and resistance of grapevine to a subsequent pathogen attack. These results open new views to improve our understanding of crosstalk between drought tolerance mechanisms and immune response.

Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

Nanotechnology for cancer gene therapy is an emerging field. Nucleic acids, polyamine analogues and cytotoxic products of polyamine oxidation, generated insitu by an enzyme-catalyzed reaction, can be developed for nanotechnology-based cancer therapeutics with reduced systemic toxicity and improved therapeutic efficacy. Nucleic acid-based gene therapy approaches depend on the compaction of DNA/RNA to nanoparticles and polyamine analogues are excellent agents for the condensation of nucleic acids to nanoparticles. Polyamines and amine oxidases are found in higher levels in tumours compared to that of normal tissues. Therefore, the metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, can be targets for antineoplastic therapy since these naturally occurring alkylamines are essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. In particular, polyamine catabolism involves copper-containing amine oxidases. Several studies showed an important role of these enzymes in developmental and disease-related processes in animals through the control of polyamine homeostasis in response to normal cellular signals, drug treatment, and environmental and/or cellular stress. The production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these oxidases suggests a mechanism by which amine oxidases can be exploited as antineoplastic drug targets. The combination of bovine serum amine oxidase (BSAO) and polyamines prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. The findings described herein suggest that enzymatically formed cytotoxic agents activate stress signal transduction pathways, leading to apoptotic cell death. Consequently, superparamagnetic nanoparticles or other advanced nanosystem based on directed nucleic acid assemblies, polyamine-induced DNA condensation, and bovine serum amine oxidase may be proposed for futuristic anticancer therapy utilizing nucleic acids, polyamines and BSAO. BSAO based nanoparticles can be employed for the generation of cytotoxic polyamine metabolites.

Polyamines metabolism and breast cancer: state of the art and perspectives

Breast cancer (BC) is a common disease that generally occurs in women over the age of 50, and the risk is especially high for women over 60years of age. One of the major BC therapeutic problems is that tumors initially responsive to chemotherapeutic approaches can progress to more aggressive forms poorly responsive to therapies. Polyamines (PAs) are small polycationic alkylamines, naturally occurring and essential for normal cell growth and development in eukaryotes. The intracellular concentration of PA is maintained within strongly controlled contents, while a dysregulation occurs in BC cells. Polyamines facilitate the interactions of transcription factors, such as estrogen receptors with their specific response element, and are involved in the proliferation of ER-negative and highly invasive BC tumor cells. Since PA metabolism has a critical role in cell death and proliferation, it represents a potential target for intervention in BC. The goal of this study was to perform a literature search reviewing the association between PA metabolism and BC, and the current evidence supporting the BC treatment targeting PA metabolism. We here describe in vitro and in vivo models, as well as the clinical trials that have been utilized to unveil the relationship between PA metabolism and BC. Polyamine pathway is still an important target for the development of BC chemotherapy via enzyme inhibitors. Furthermore, a recent promising strategy in breast anticancer therapy is to exploit the self-regulatory nature of PA metabolism using PA analogs to affect PA homeostasis. Nowadays, antineoplastic compounds targeting the PA pathway with novel mechanisms are of great interest and high social impact for BC chemotherapy.

Amine Oxidase Copper-containing 1 (AOC1) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Identification of Polyamine Transporters in Plants: Paraquat Transport Provides Crucial Clues

Polyamine (PA) transport as well as PA biosynthesis, degradation and conjugation plays a vital role in the regulation of intracellular PA levels, which are essential for cell growth. Generally, PA uptake activity is elevated in rapidly proliferating cells. Previous studies showed that PA uptake in plant cells occurred via energy-dependent, protein-mediated transport systems. Numerous lines of evidence suggest that paraquat (PQ), one of the most widely used herbicides, is transported by the PA transport system in diverse organisms including plants. The PA/PQ transport interactions are proposed to be due to specific structural similarities between PA and PQ. The understanding of PA transport mechanisms has progressed in parallel with that of PQ transport, but the molecular identity of the plant PA/PQ transporter has remained an enigma. Recently, independent studies identified the L-type amino acid transporter (LAT) family transmembrane proteins as transporters of both PA and PQ. Arabidopsis LAT family proteins showed different subcellular localization properties, which suggested that these transporters were involved in intracellular PA trafficking and PA uptake across the plasma membrane. The identification of plant PA transporters is an important step in understanding the mechanism of PA homeostasis in plant cells. In this review, we highlight recent advances in the study of PA transport systems that are linked to the understanding of PQ translocation.

The roles of polyamines during the lifespan of plants: from development to stress

Compelling evidence indicates that free polyamines (PAs) (mainly putrescine, spermidine, spermine, and its isomer thermospermine), some PA conjugates to hydroxycinnamic acids, and the products of PA oxidation (hydrogen peroxide and γ-aminobutyric acid) are required for different processes in plant development and participate in abiotic and biotic stress responses. A tight regulation of PA homeostasis is required, since depletion or overaccumulation of PAs can be detrimental for cell viability in many organisms. In plants, homeostasis is achieved by modulation of PA biosynthesis, conjugation, catabolism, and transport. However, recent data indicate that such mechanisms are not mere modulators of PA pools but actively participate in PA functions. Examples are found in the spermidine-dependent eiF5A hypusination required for cell division, PA hydroxycinnamic acid conjugates required for pollen development, and the involvement of thermospermine in cell specification. Recent advances also point to implications of PA transport in stress tolerance, PA-dependent transcriptional and translational modulation of genes and transcripts, and posttranslational modifications of proteins. Overall, the molecular mechanisms identified suggest that PAs are intricately coordinated and/or mediate different stress and developmental pathways during the lifespan of plants.

Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias

The metabolism of polyamines, the cationic small molecules essential for cell proliferation and differentiation, is altered in cancer cells and can be exploited in cancer diagnosis and therapy. Spermidine/spermine N(1)-acetyltransferase (SSAT), which regulates intracellular levels of polyamines by catabolizing spermidine and spermine, has a controversial role in the development of cancers. In this study, the polyamine metabolism and function of SSAT were characterized in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia patient samples. Also, mice overexpressing SSAT and having a myeloproliferative phenotype were analyzed for their response to decitabine and histone deacetylase inhibitor trichostatin A. The presence of epigenetic factors in the bone marrow cells of SSAT mice was analyzed. Elevated levels of spermidine and spermine, as well as increased activity of SSAT, were detected in AML, CML, and acute lymphoid leukemia patients compared with the controls. However, we found SSAT activity to be associated with white blood cell count only in AML and CML patients. Decitabine treatment brought the peripheral blood and bone marrow cell counts of SSAT mice to the level of wild-type mice. Spermidine/spermine N(1)-acetyltransferase mice had increased histone methylation and an increased level of histone deacetylase 1 in their bone marrow cells. The study suggests that SSAT influences the development of myeloid malignancies, and epigenetic factors partly contribute to the SSAT overexpression-induced myeloproliferative disease in mice.

Ethanol-induced impairment of polyamine homeostasis – A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome

IntroductionPolyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. MethodsCD-1 mice received two intraperitoneal injections of 3g/kg ethanol at 4h intervals 8.75days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5dpc and polyamine concentrations were determined. ResultsNo measurable quantity of polyamines could be detected in the embryo head at 9.5dpc, 12h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. DiscussionOur results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.

Abscisic acid, H2O2 and nitric oxide interactions mediated cold‐induced S‐adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up‐regulating polyamine oxidation

S-adenosylmethionine synthetase (SAMS) is the key enzyme catalysing the formation of S-adenosylmethionine (SAM), a precursor of polyamines and ethylene. To investigate the potential role of SAMS in cold tolerance, we isolated MfSAMS1 from the cold-tolerant germplasm Medicago sativa subsp. falcata and analysed the association of SAM-derived polyamines with cold tolerance. The expression of MfSAMS1 in leaves was greatly induced by cold, abscisic acid (ABA), H2O2 and nitric oxide (NO). Our data revealed that ABA, H2O2 and NO interactions mediated the cold-induced MfSAMS1 expression and cold acclimation in falcata. SAM, putrescine, spermidine and spermine levels, ethylene production and polyamine oxidation were sequentially altered in response to cold, indicating that SAMS-derived SAM is preferentially used in polyamine synthesis and homeostasis during cold acclimation. Antioxidant enzyme activities were also induced in response to cold and showed correlation with polyamine oxidation. Overexpression of MfSAMS1 in tobacco resulted in elevated SAM levels, but polyamine levels and ethylene production in the transgenic plants were not significantly changed. Compared to the wild type, transgenic plants had increased levels of apoplastic H2O2, higher transcript levels of genes involved in polyamine synthesis and oxidation, and higher activities of polyamine oxidation and antioxidant enzymes. The results showed that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2 O2 -induced antioxidant protection, as a result enhanced tolerance to freezing and chilling stress in transgenic plants. This is the first report demonstrating that SAMS plays an important role in plant tolerance to cold via up-regulating polyamine oxidation.

UORF, a regulatory mechanism of the Arabidopsis polyamine oxidase 2

The translational efficiency of an mRNA can be modulated by elements located in the 5'-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.

Assay of Ornithine Decarboxylase and Spermidine/Spermine N1-acetyltransferase Activities

The polyamines, spermidine (Spd) and spermine, and their diamine precursor putrescine, are important regulators of various cellular functions, such as proliferation and differentiation. Polyamine homeostasis is tightly regulated on the level of uptake, excretion, biosynthesis, interconversion and terminal catabolism. The rate-controlling enzymes of polyamine biosynthesis and interconversion are ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT), respectively. Here, we describe a protocol to assay ODC (Jänne and Williams-Ashman, 1971) and SSAT (Libby, 1978) activities from cell or tissue samples.

The Genetic Reprogramming of Polyamine Homeostasis During the Functional Assembly, Maturation, and Senescence-Specific Decline of the Photosynthetic Apparatus in Hordeum vulgare

Polyamines (PAs) are ubiquitous aliphatic amines important and, in many cases, essential for plant growth, abiotic stress response, and tolerance. Here we provide evidence for genetic reprogramming of PA homeostasis that occurs during the de-etiolation, maturation, and senescence of the primary leaf in Hordeum vulgare (barley). We analyzed expression levels of key genes in the anabolic and catabolic branches of PA metabolism throughout the life cycle of etioplasts, at all steps of the functional assembly of the photosynthetic apparatus, and during leaf senescence. The changes in the total PAs titer of the leaf were followed throughout the different developmental stages. Furthermore, we align all three stages of the photosynthetic performance (rapid light-dependent de-etiolation, phase of optimal efficiency, and senescence-induced deterioration) with the changes in PA homeostasis. Finally, we focus on two phases during aging (early and late senescence) and we present their bioenergetic (for example, PSII maximal efficiency, ATPase conductivity) and genetic profiles, with emphasis on sensitive parameters that describe this process for the photosynthetic apparatus and PA metabolism, respectively. In conclusion, the fine tuning of PA homeostasis is regulated by the simultaneous genetic reprogramming of the anabolic and catabolic branches of PA metabolism and adjusts all the developmental changes from de-etiolation to maturation and senescence.