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Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons. [ABSTRACT FROM AUTHOR]

Diatoms, efficient carbon capture organisms, contribute to 20% of global carbon fixation and 40% of ocean primary productivity, garnering significant attention to their growth. Despite their significance, the synthesis mechanism of polyamines (PAs), especially spermidine (Spd), which are crucial for growth in various organisms, remains unexplored in diatoms. This study reveals the vital role of Spd, synthesized through the spermidine synthase (SDS)-based pathway, in the growth of the diatom Phaeodactylum tricornutum. PtSDS1 and PtSDS2 in the P. tricornutum genome were confirmed as SDS enzymes through enzyme-substrate selectivity assays. Their distinct activities are governed primarily by the Y79 active site. Overexpression of a singular gene revealed that PtSDS1, PtSDS2, and PtSAMDC from the SDS-based synthesis pathway are all situated in the cytoplasm, with no significant impact on PA content or diatom growth. Co-overexpression of PtSDS1 and PtSAMDC proved essential for elevating Spd levels, indicating multifactorial regulation. Elevated Spd content promotes diatom growth, providing a foundation for exploring PA functions and regulation in diatoms. [ABSTRACT FROM AUTHOR]

Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons.

Diatoms, efficient carbon capture organisms, contribute to 20% of global carbon fixation and 40% of ocean primary productivity, garnering significant attention to their growth. Despite their significance, the synthesis mechanism of polyamines (PAs), especially spermidine (Spd), which are crucial for growth in various organisms, remains unexplored in diatoms. This study reveals the vital role of Spd, synthesized through the spermidine synthase (SDS)-based pathway, in the growth of the diatom Phaeodactylum tricornutum. PtSDS1 and PtSDS2 in the P. tricornutum genome were confirmed as SDS enzymes through enzyme-substrate selectivity assays. Their distinct activities are governed primarily by the Y79 active site. Overexpression of a singular gene revealed that PtSDS1, PtSDS2, and PtSAMDC from the SDS-based synthesis pathway are all situated in the cytoplasm, with no significant impact on PA content or diatom growth. Co-overexpression of PtSDS1 and PtSAMDC proved essential for elevating Spd levels, indicating multifactorial regulation. Elevated Spd content promotes diatom growth, providing a foundation for exploring PA functions and regulation in diatoms.

Spermidine synthase (SPDS) is a key enzyme in the polyamine anabolic pathway. SPDS genes help regulate plant response to environmental stresses, but their roles in pepper remain unclear. In this study, we identified and cloned a SPDS gene from pepper (Capsicum annuum L.), named CaSPDS (LOC107847831). Bioinformatics analysis indicated that CaSPDS contains two highly conserved domains: an SPDS tetramerisation domain and a spermine/SPDS domain. Quantitative reverse-transcription polymerase chain reaction results showed that CaSPDS was highly expressed in the stems, flowers, and mature fruits of pepper and was rapidly induced by cold stress. The function of CaSPDS in cold stress response was studied by silencing and overexpressing it in pepper and Arabidopsis, respectively. Cold injury was more serious and reactive oxygen species levels were greater in the CaSPDS-silenced seedlings than in the wild-type (WT) seedlings after cold treatment. Compared with the WT plants, the CaSPDS-overexpression Arabidopsis plants were more tolerant to cold stress and showed higher antioxidant enzyme activities, spermidine content, and cold-responsive gene (AtCOR15A, AtRD29A, AtCOR47, and AtKIN1) expression. These results indicate that CaSPDS plays important roles in cold stress response and is valuable in molecular breeding to enhance the cold tolerance of pepper. [ABSTRACT FROM AUTHOR]

Parasites of the genus Leishmania cause a variety of devastating and often fatal diseases in humans and domestic animals worldwide. The need for new therapeutic strategies is urgent because no vaccine is available, and treatment options are limited due to a lack of specificity and the emergence of drug resistance. Polyamines are metabolites that play a central role in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania. Numerous studies using a variety of inhibitors as well as gene deletion mutants have elucidated the pathway and routes of transport, revealing unique aspects of polyamine metabolism in Leishmania parasites. These studies have also shed light on the significance of polyamines for parasite proliferation, infectivity, and host–parasite interactions. This comprehensive review article focuses on the main polyamine biosynthetic enzymes: ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase, and it emphasizes recent discoveries that advance these enzymes as potential therapeutic targets against Leishmania parasites. [ABSTRACT FROM AUTHOR]

Spermidine synthase (SPDS) is a key enzyme in the polyamine anabolic pathway. SPDS genes help regulate plant response to environmental stresses, but their roles in pepper remain unclear. In this study, we identified and cloned a SPDS gene from pepper (Capsicum annuum L.), named CaSPDS (LOC107847831). Bioinformatics analysis indicated that CaSPDS contains two highly conserved domains: an SPDS tetramerisation domain and a spermine/SPDS domain. Quantitative reverse-transcription polymerase chain reaction results showed that CaSPDS was highly expressed in the stems, flowers, and mature fruits of pepper and was rapidly induced by cold stress. The function of CaSPDS in cold stress response was studied by silencing and overexpressing it in pepper and Arabidopsis, respectively. Cold injury was more serious and reactive oxygen species levels were greater in the CaSPDS-silenced seedlings than in the wild-type (WT) seedlings after cold treatment. Compared with the WT plants, the CaSPDS-overexpression Arabidopsis plants were more tolerant to cold stress and showed higher antioxidant enzyme activities, spermidine content, and cold-responsive gene (AtCOR15A, AtRD29A, AtCOR47, and AtKIN1) expression. These results indicate that CaSPDS plays important roles in cold stress response and is valuable in molecular breeding to enhance the cold tolerance of pepper.

Spermidine (SPD), as a signal molecule, participates in the response of plants to adversity stress. In the biosynthesis of SPD, spermidine synthase (SPDS) is the key enzyme that catalyzes the synthesis of putrescine into SPD. TWO cDNA sequence coding spermidine synthases have been cloned from mulberry (MnSPDS) in the present report. Sequence analysis revealed that their ORF were 1005 and 1188bp in length, encoding 334 and 395 amino acid protein, respectively. MnSPDS genes belonged to S-adenosyl-L-methionone, AdoMet-MTase, Class I superfamily. Phylogenetic analysis of the amino acid sequences encoded by the spermidine synthase gene from different species reveals that Morus notabilis C.K. Schneid. was closely connected to Arabidopsis thaliana. MnSPDS genes expression patterns treated under drought, pathogen treatment were investigated using quantitative PCR (RT-qPCR) in real-time. The level of mRNA showed a significant change in drought and pathogen treatment compared to the standard growth environment. Especially, the peak expression of MnSPDS2 gene was 3.2 times that of MnSPDS1gene under drought stress, however, for the treatment of Botrytis cinerea, the peak of transcript level of MnSPDS1 gene was 4.2 times that of MnSPDS2 gene. These findings provide useful reference information for the molecular basis of signal transduction in mulberry trees during stress responses. [ABSTRACT FROM AUTHOR]

Polyamines, such as spermidine (Spd) spermine (Spm) and their direct precursor, the diamine putrescine (Put) are vital and essential aliphatic amines which are also present in plants. Although ethylene and polyamines are also involved in fruit ripening, the genes coding them must also take part in other biosynthetic pathways. In the ethylene and polyamines play an important role in development of salt stress tolerance, and in responses for biotic and abiotic stresses. Exogenous application of all three main polyamines (Put, Spd, Spm) increase salt tolerance of plants, but, accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance. Nicotiana tabacum L. plants were grown in vitro on MS medium, the treatments were as follows: arginine (150 mg l -1), putrescine (10 mg l -1), spermidine (10 mg l -1). Proline, chlorophyll a, b and polyamine contents were measured. The obtained results show that the arginine decarboxylase and the spermidine synthase genes involved in polyamine metabolism, cannot be enhanced by exogenous addition of their precursor molecules. On the contrary, the spermine synthase gene has a positive effect to the lower-class forms of polyamines. [ABSTRACT FROM AUTHOR]

In plants, spermidine synthase (SPE) received detailed investigation due to its key roles in the biosynthesis of spermidine, a major form of polyamine. However, there is no report on the identification and characterisation of SPE in edible mushrooms. Here, a full-length cDNA of a gene encoding a potential SPE was isolated from fruiting bodies of Agaricus bisporus. Homology search, InterPro inquiry and multiple sequence alignment showed that it was a chimeric bifunctional gene (named as nSpe-Sdh) with a single transcript that could also encode saccharopine dehydrogenase (SDH). Furthermore, we found that the expression of nSPE-SDH mRNA and protein were both significantly up-regulated with the cap expansion of A. bisporus during storage. Once the protecting veil broken, both expression levels decreased to an initial storage level and remained relatively constant until the end of the storage, indicating the possible involvement of nSpe-Sdh in postharvest mushroom development. Besides, the up-regulation of the expression of nSPE-SDH mRNA and protein was both tissue-specially in cap tissues. To our knowledge, this is the first study focus on a specific gene for putative spermidine biosynthesis in edible mushrooms. Our present study provides valuable information for further functional characterisation of SPE-SDH proteins in postharvest mushroom physiology. [ABSTRACT FROM AUTHOR]

Black spot, caused by the fungus Alternaria alternata , is one of the postharvest diseases that cause significant economic losses. Polyamine metabolism in fungi plays key roles in cellular processes and pathogen-host interactions. However, little is known about their detailed regulatory mechanisms on pathogenicity of plant fungi. In this study, we characterized the spermidine synthase AaSPDS in A. alternata. Knocking out AaSPDS caused a total loss of vegetative growth and sporulation which was partially restored by exogenous spermidine (Spd), so supplying 0.5 mM exogenous Spd to medium for evaluating the characteristics of WT and Δ AaSPDS. The deletion strain showed albino colonies, sparse mycelial tips and significant decreased biomass accumulation and melanin production. Δ AaSPDS strain was also more sensitive to osmotic stress, oxidative stress and cell wall-perturbing agents. Interestingly, the expression level of AaSPDS was greatly up-regulated during infection structure differentiation stage of A. alternata , and appressorium formation of the AaSPDS deletion strain was completely blocked. In addition, deletion of the AaSPDS gene led to a significant reduction in pathogenicity to pear fruit, altenuene (ALT) mycotoxin accumulation and the activities of cellulase (CL), β-glucosidase (β-GC) and polygalacturonase (PG). The results presented in this study suggested that AaSPDS influences the pathogenicity of A. alternata by regulating infection structure formation, cell wall degrading enzyme (CWDE) activity and mycotoxin production. • The deletion of the AaSPDS resulted in a spermidine auxotrophic phenotype. • AaSPDS is required for infection structure differentiation of A. alternata. • Δ AaSPDS strain was more sensitive to external stress. • Decreased cell wall degrading enzyme activities were found in Δ AaSPDS strain. • AaSPDS is essential for pathogenicity of A. alternata. [ABSTRACT FROM AUTHOR]

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.

Polyamines are small organic cations that are important for several biological processes such as cell proliferation, cell cycle progression, and apoptosis. The dysregulation of intracellular polyamines is often associated with diseases such as cancer, diabetes, and developmental disorders. Although polyamine metabolism has been well studied, the effects of key enzymes in the polyamine pathway on lipid metabolism are not well understood. Here, we determined metabolic effects resulting from the absence of spermidine synthase (SpdS) and spermine synthase (Sms) in Drosophila. While SpdS mutants developed normally and accumulated triglycerides, Sms mutants had reduced viability and stored less triglyceride than the controls. Interestingly, when decreasing SpdS and Sms, specifically in the fat body, triglyceride storage increased. While there was no difference in triglycerides stored in heads, thoraxes and abdomen fat bodies, abdomen fat body DNA content increased, and protein/DNA decreased in both SpdS- and Sms-RNAi flies, suggesting that fat body-specific knockdown of SpdS and Sms causes the production of smaller fat body cells and triglycerides to accumulate in non-fat body tissues of the abdomen. Together, these data provide support for the role that polyamines play in the regulation of metabolism and can help enhance our understanding of polyamine function in metabolic diseases.

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N 1, N 7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii -infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum , suggesting that they exert their antimalarial activities by inhibition of spermidine synthase. [ABSTRACT FROM AUTHOR]

Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines.

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.

Spermidine belongs to a class of polycationic compounds known as polyamines. Polyamines are known to be involved in a wide range of biological processes but the exact role and contribution of different polyamines to these processes are still not clear. In the present study, we have tried to understand the contribution of triamine spermidine to the growth and development of tobacco by downregulating spermidine synthase gene (SPDS) using RNA interference. Down-regulatioin of SPDS gene resulted in decreased spermidine levels and a slight increase in the levels of its precursor, the diamine putrescine and the molecule downstream of Spd, the tetraamine spermine. While the vegetative growth of the transgenics remained largely unaffected, SPDS down-regulation resulted in smaller size of flowers, decreased pollen viability and seed setting, and a reduced and delayed seed germination. When subjected to abiotic stress, the transgenics showed an increased tolerance to salinity and drought conditions owing to a steady intracellular pool of putrescine and spermine. The results not only highlight the importance of spermidine in determining reproductive potential in plants but have also help delineate its function from that of putrescine and spermine. [ABSTRACT FROM AUTHOR]

Ethylene favors carposporogenesis in the red seaweed Grateloupia imbricata. Analyses of cystocarp development in vitro in thalli treated with ethylene suggest an interconnection between polyamine and ethylene biosynthesis pathways. Yet, little is known about molecular mechanisms underlying carposporogenesis. Here, we used droplet digital PCR to analyze genes encoding enzymes related to polyamine (Spermidine [Spd] synthase) and ethylene (ACC synthase) synthesis; a pivotal compound of both pathways (S‐adenosyl methionine synthase, SAMS); the gene that encodes amine oxidase, which is involved in polyamine degradation, and a candidate gene involved in seaweed reproduction (ornithine decarboxylase, ODC). In addition, we analyzed genes encoding proteins related to stress and reactive oxygen species, ascorbate peroxidase (APX), cytochrome P450 and WD 40. We characterized gene expression in fertilized and fertile thalli from G. imbricata that were exposed to ethylene for 15 min at two time points after treatment (1 and 7 d). The differential gene expression of SAMS, Spd synthase, ACC synthase, and cytochrome P450 was related to disclosure and development of cystocarps in fertilized thalli that transitioned from having no visible cystocarps at 1 d to developing cystocarps at 7 d. Likewise, cytochrome P450 was associated with cystocarp disclosure and maturation. In addition, amine oxidase and APX were involved in fine‐tuning polyamine and reactive oxygen species during carposporogenesis, respectively, whereas WD 40 did so in relation to ethylene signaling. Expression of the candidate gene ODC was increased when cystocarps were not visible (fertilized thalli, 1d), as previously described. This analysis suggests developmental stage‐specific roles for these genes during carposporogenesis. [ABSTRACT FROM AUTHOR]

The expression pattern of genes related to ethylene and polyamine biosynthesis were analyzed in octoploid strawberry fruits in different ripening phases. RNA was isolated from Fragaria x ananassa Duch cv. Asia and used to synthesize cDNA by reverse transcriptase enzyme, which was applied in qPCR with primers designed for S-adenosyl-L-methionine synthases (FaSAMS1-3), S-adenosyl-L-methionine decarboxylases (FaSAMDC1-3), arginine decarboxylase (FaADC), spermidine synthase (FaSPDS), 1-aminocyclopropane-1-carboxylate synthases (FaACS1-4) and 1-aminocyclopropane-1-carboxylate oxidase (FaACO1-4) gene sequences. Based on the comparison made between the expression patterns of ethylene- and polyaminerelated genes and those of the common precursor- and pathway shift-related genes, it appeared that with the exception of FaSAMS2, FaSAMDC1 and FaSAMDC2, the genes involved in polyamine biosynthesis showed decreasing level of expression as ripening proceeded. [ABSTRACT FROM AUTHOR]

Background and Aims Polyamines are small metabolites present in all living cells and play fundamental roles in numerous physiological events in plants. The aminopropyltransferases (APTs), spermidine synthase (SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5), are essential enzymes in the polyamine biosynthesis pathway. In angiosperms, SPMS has evolved from SPDS via gene duplication, whereas in gymnosperms APTs are mostly unexplored and no SPMS gene has been reported. The present study aimed to investigate the functional properties of the SPDS and ACL5 proteins of Scots pine (Pinus sylvestris L.) in order to elucidate the role and evolution of APTs in higher plants. Methods Germinating Scots pine seeds and seedlings were analysed for polyamines by high-performance liquid chromatography (HPLC) and the expression of PsSPDS and PsACL5 genes by in situ hybridization. Recombinant proteins of PsSPDS and PsACL5 were produced and investigated for functional properties. Also gene structures, promoter regions and phylogenetic relationships of PsSPDS and PsACL5 genes were analysed. Key Results Scots pine tissues were found to contain spermidine, spermine and thermospermine. PsSPDS enzyme catalysed synthesis of both spermidine and spermine. PsACL5 was found to produce thermospermine, and PsACL5 gene expression was localized in the developing procambium in embryos and tracheary elements in seedlings. Conclusions Contrary to previous views, our results demonstrate that SPMS activity is not a novel feature developed solely in the angiosperm lineage of seed plants but also exists as a secondary property in the Scots pine SPDS enzyme. The discovery of bifunctional SPDS from an evolutionarily old conifer reveals the missing link in the evolution of the polyamine biosynthesis pathway. The finding emphasizes the importance of pre-existing secondary functions in the evolution of new enzyme activities via gene duplication. Our results also associate PsACL5 with the development of vascular structures in Scots pine. [ABSTRACT FROM AUTHOR]

Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops. [ABSTRACT FROM AUTHOR]

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.

Helicobacter pylori is the primary pathogen associated to gastritis and gastric cancer. Growth of H. pylori depends on the availability of spermidine in vivo . Interestingly, the genome of H. pylori contains an incomplete set of genes for the classical pathway of spermidine biosynthesis. It is thus not clear whether some other genes remained in the pathway would have any functions in spermidine biosynthesis. Here, we study spermidine synthase, which is responsible for the final catalytic process in the classical route. Protein sequence alignment reveals that H. pylori SpeE ( Hp SpeE) lacks key residues for substrate binding. By using isothermal titration calorimetry, we show that purified recombinant Hp SpeE does not interact with the putative substrates putrescine and decarboxylated S -adenosylmethionine, and the product spermidine. High performance liquid chromatography analysis further demonstrates that Hp SpeE has no detectable in vitro enzymatic activity. Additionally, intracellular spermidine level in speE -null mutant strain is comparable to that in the wild type strain. Collectively, our results suggest that Hp SpeE is functionally distinct from spermidine production. H. pylori may instead employ the alternative pathway for spermidine synthesis which is dominantly exploited by other human gut microbes. [ABSTRACT FROM AUTHOR]

Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei , the inhibition of the synthesis of intermediate polyamine Putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescence imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion. [ABSTRACT FROM AUTHOR]

The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene ( Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds (OX- spds) and the spds knockout (Δ spds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Δ spds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX- spds showed a slightly higher growth than the wild type. The OX- spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX- spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress. [ABSTRACT FROM AUTHOR]

Ornithine decarboxylase ( Ld ODC), a key enzyme in polyamine biosynthesis in Leishmania donovani, catalyzes the conversion of ornithine to putrescine that is finally used for synthesis of spermidine and other polyamines. Inhibition of ornithine decarboxylase is likely to deplete the parasite trypanothione and may result in increased reactive oxygen species ( ROS). Sequence as well as structure of Ld ODC and human ODC shows significant difference; therefore, we have identified novel specific inhibitors of Ld ODC. These inhibitors are able to inhibit recombinant Ld ODC and decrease intracellular putrescine concentration showing target specificity. The Ki values of Ld ODC inhibition do not correlate with IC50 values in Leishmania promastigote possibly due to different stability/pharmacokinetics. These inhibitors, except compound M-5, have only minor effect on Leishmania promastigotes, and IC50 values are several folds higher as compared to Ki values. In case of compound M-5, IC50 value is less than Ki value indicating that the compound may have additional targets. Our studies suggest that the parasite resists these Ld ODC inhibitors by overexpression of spermidine synthase mRNA. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required. Enzymes in the polyamine-trypanothione pathway are promising targets for the treatment of Chagas disease. Spermidine synthase is a key enzyme in this pathway that catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. Fragment-based drug discovery was therefore conducted to identify novel, potent inhibitors of spermidine synthase from T. cruzi (TcSpdSyn). Here, crystal structures of TcSpdSyn in complex with dcSAM, trans-4-methylcyclohexylamine and hit compounds from fragment screening are reported. The structure of dcSAM complexed with TcSpdSyn indicates that dcSAM stabilizes the conformation of the `gatekeeping' loop to form the putrescine-binding pocket. The structures of fragments bound to TcSpdSyn revealed two fragment-binding sites: the putrescine-binding pocket and the dimer interface. The putrescine-binding pocket was extended by an induced-fit mechanism. The crystal structures indicate that the conformation of the dimer interface is required to stabilize the gatekeeping loop and that fragments binding to this interface inhibit TcSpdSyn by disrupting its conformation. These results suggest that utilizing the dynamic structural changes in TcSpdSyn that occur upon inhibitor binding will facilitate the development of more selective and potent inhibitors. [ABSTRACT FROM AUTHOR]

Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS , from the gentian plant, Gentiana triflora . Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants. [ABSTRACT FROM AUTHOR]

The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum ( PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum is the most pathogenic of the human malaria parasite species and a major cause of death in Africa. It's resistance to most of the current drugs accentuates the pressing need for new chemotherapies. Polyamine metabolism of the parasite is distinct from the human pathway making it an attractive target for chemotherapeutic development. Plasmodium falciparum spermidine synthase (PfSpdS) catalyzes the synthesis of spermidine and spermine. It is a major polyamine flux-determining enzyme and spermidine is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A). The most potent inhibitors of eukaryotic SpdS's are not specific for PfSpdS. Methods: 'Dynamic' receptor-based pharmacophore models were generated from published crystal structures of SpdS with different ligands. This approach takes into account the inherent flexibility of the active site, which reduces the entropic penalties associated with ligand binding. Four dynamic pharmacophore models were developed and two inhibitors, (1R,4R)-(N1-(3-aminopropyl)-trans-cyclohexane-1,4-diamine (compound 8) and an analogue, N-(3-aminopropyl)-cyclohexylamine (compound 9), were identified. Results: A crystal structure containing compound 8 was solved and confirmed the in silico prediction that its aminopropyl chain traverses the catalytic centre in the presence of the byproduct of catalysis, 5'-methylthioadenosine. The IC50 value of compound 9 is in the same range as that of the most potent inhibitors of PfSpdS, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and 4MCHA and 100-fold lower than that of compound 8. Compound 9 was originally identified as a mammalian spermine synthase inhibitor and does not inhibit mammalian SpdS. This implied that these two compounds bind in an orientation where their aminopropyl chains face the putrescine binding site in the presence of the substrate, decarboxylated S-adenosylmethionine. The higher binding affinity and lower receptor strain energy of compound 9 compared to compound 8 in the reversed orientation explained their different IC50 values. Conclusion: The specific inhibition of PfSpdS by compound 9 is enabled by its binding in the additional cavity normally occupied by spermidine when spermine is synthesized. This is the first time that a spermine synthase inhibitor is shown to inhibit PfSpdS, which provides new avenues to explore for the development of novel inhibitors of PfSpdS. [ABSTRACT FROM AUTHOR]

Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania . S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. [ABSTRACT FROM AUTHOR]

Key message: Polyamines can regulate the expression of antioxidant enzymes and impart plants tolerance to abiotic stresses. Abstract: A comparative analysis of polyamines, their biosynthetic enzymes at kinetic and at transcriptional level, and their role in regulating the induction of antioxidant defense enzymes under salt stress condition in two foxtail millet ( Setaria italica L.) cultivars, namely Prasad, a salt-tolerant, and Lepakshi, a salt-sensitive cultivar was conducted. Salt stress resulted in elevation of free polyamines due to increase in the activity of spermidine synthase and S-adenosyl methionine decarboxylase enzymes in cultivar Prasad compared to cultivar Lepakshi under different levels of NaCl stress. These enzyme activities were further confirmed at the transcript level via qRT-PCR analysis. The cultivar Prasad showed a greater decrease in diamine oxidase and polyamine oxidase activity, which results in the accumulation of polyamine pools over cultivar Lepakshi. Generation of free radicals, such as O and HO, was also analyzed quantitatively. A significant increase in O and HO in the cultivar Lepakshi compared with cultivar Prasad was recorded in overall pool sizes. Further, histochemical staining showed lesser accumulation of O and of HO in the leaves of cultivar Prasad than cultivar Lepakshi. Our results also suggest the ability of polyamine oxidation in regulating the induction of antioxidative defense enzymes, which involve in the elimination of toxic levels of O and HO, such as Mn-superoxide dismutase, catalase and ascorbate peroxidase. The contribution of polyamines in modulating antioxidative defense mechanism in NaCl stress tolerance is discussed. [ABSTRACT FROM AUTHOR]

It has previously been indicated that polyamines (PAs) are involved in the response to adverse environmental stresses in various plant species. Increased cellular PA levels or exogenous PA application can reduce the accumulation of reactive oxygen species (ROS) imposed by multiple abiotic stresses via increasing antioxidant enzyme activities. In this study, a novel spermidine synthase gene, designated as EsSPDS1 , was cloned and characterized from the halophytic plant Eutrema salsugineum . Analyses of tissue-specific expression profiles revealed that EsSPDS1 was expressed in almost all tested organs including leaves, stems, flowers, roots and siliques. The expression of EsSPDS1 was obviously induced by polyethylene glycol (PEG) and high salinity treatments. Furthermore, the EsSPDS1 transcripts rapidly accumulated upon exposure to abscisic acid (ABA) treatments, implying that this gene was involved in ABA-mediated abiotic stress response. Transgenic tobacco plants harboring EsSPDS1 exhibited enhanced drought tolerance relative to wild-type (WT) plants. These transgenic plants had lower levels of malondialdehyde (MDA), less ion leakage (IL) and reactive oxygen species (ROS). However, relative water content (RWC) and activities of antioxidant enzymes including SOD and CAT of the transgenic plants was remarkably higher than those of WT plants under drought stress conditions. Collectively, our data suggested that the activation of EsSPDS1 expression in tobacco plants conferred drought tolerance, which might be attributed to enhance the ability to scavenge ROS levels. [ABSTRACT FROM AUTHOR]

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. SPDS 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 Datura 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.

Abstract: Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150mM NaCl) for 7d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H2O2) and superoxide (O2 −) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1mM Spd concentrations, but a lower concentration (0.01mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress. [Copyright &y& Elsevier]

Abstract: Spermidine synthase (SPDS) catalyses the formation of spermidine, which is an essential polyamine and widespread in living organisms. Spermidine is formed from putrescine by transfer of an aminopropyl group from decarboxylated S-adenosylmethionine. Spermidine is also a precursor to further polyamines, such as spermine and thermospermine, most of which contribute to tolerance against drought and salinity in plants. Thermospermine is indispensible for vascular tissue growth. Plant spermidine synthases have been cloned from several angiosperms; organ-specific gene expression levels are known for Arabidopsis only. In this study, immunolocalisation of SPDS in potato (Solanum tuberosum) organs is presented. Polyclonal antibodies for SPDS from potato produced in rabbits were purified by affinity chromatography. Cross-reaction with potato putrescine N-methyltransferase was eliminated. Accumulation of SPDS protein in the phloem region of vascular tissues throughout the potato plant is demonstrated. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis. [ABSTRACT FROM AUTHOR]

Abstract: Protozoa Leishmania donovani (Ld) is the main cause of the endemic disease leishmaniasis. Spermidine synthase (SS), an important enzyme in the synthetic pathway of polyamines in Ld, is an essential element for the survival of this protozoan. Targeting SS may provide an important aid for the development of drugs against Ld. However, absence of tertiary structure of spermidine synthase of Leishmania donovani (LSS) limits the possibilities of structure based drug designing. Presence of the same enzyme in the host itself further challenges the drug development process. We modeled the tertiary structure of LSS using homology modeling approach making use of homologous X-ray crystallographic structure of spermidine synthase of Trypanosoma cruzi (TSS) (2.5Å resolution). The modeled structure was stabilized using Molecular Dynamics simulations. Based on active site structural differences between LSS and human spermidine synthase (HSS), we screened a large dataset of compounds against modeled protein using Glide virtual screen docking and selected two best inhibitors based on their docking scores (−10.04 and −13.11 respectively) with LSS and having least/no binding with the human enzyme. Finally Molecular Dynamics simulations were used to assess the dynamic stability of the ligand bound structures and to elaborate on the binding modes. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction. [Copyright &y& Elsevier]

Exogenous ethylene and some inhibitors of polyamine biosynthesis can induce mature-fruit abscission in olive, which could be associated with decreased nitric oxide production as a signaling molecule. Whether HO also plays a signaling role in mature-fruit abscission is unknown. The possible involvement of HO and polyamine in ethylene-induced mature-fruit abscission was examined in the abscission zone and adjacent cells of two olive cultivars. Endogenous HO showed an increase in the abscission zone during mature-fruit abscission, suggesting that accumulated HO may participate in abscission signaling. On the other hand, we followed the expression of two genes involved in the polyamine biosynthesis pathway during mature-fruit abscission and in response to ethylene or inhibitors of ethylene and polyamine. OeSAMDC1 and OeSPDS1 were expressed differentially within and between the abscission zones of the two cultivars. OeSAMDC1 showed slightly lower expression in association with mature-fruit abscission. Furthermore, our data show that exogenous ethylene or inhibitors of polyamine encourage the free putrescine pool and decrease the soluble-conjugated spermidine, spermine, homospermidine, and cadaverine in the olive abscission zone, while ethylene inhibition by CoCl increases these soluble conjugates, but does not affect free putrescine. Although the impact of these treatments on polyamine metabolism depends on the cultivar, the results confirm that the mature-fruit abscission may be accompanied by an inhibition of S-adenosyl methionine decarboxylase activity, and the promotion of putrescine synthesis in olive abscission zone, suggesting that endogenous putrescine may play a complementary role to ethylene in the normal course of mature-fruit abscission. [ABSTRACT FROM AUTHOR]

Polyamines are organic polycations that are involved in a wide range of cellular activities related to growth, development, and stress response in plants. Higher polyamines spermidine and spermine are synthesized in plants and animals by a class of enzymes called aminopropyltransferases that transfer aminopropyl moieties (derived from decarboxylated S-adenosylmethionine) to putrescine and spermidine to produce spermidine and spermine, respectively. The higher polyamines show a much tighter homeostatic regulation of their metabolism than the diamine putrescine in most plants; therefore, the aminopropyltransferases are of high significance. We present here a comprehensive summary of the current literature on plant aminopropyltransferases including their distribution, biochemical properties, genomic organization, pattern of expression during development, and their responses to abiotic stresses, and manipulation of their cellular activity through chemical inhibitors, mutations, and genetic engineering. This minireview complements several recent reviews on the overall biosynthetic pathway of polyamines and their physiological roles in plants and animals. It is concluded that (1) plants often have two copies of the common aminopropyltransferase genes which exhibit redundancy of function, (2) their genomic organization is highly conserved, (3) direct enzyme activity data on biochemical properties of these enzymes are scant, (4) often there is a poor correlation among transcripts, enzyme activity and cellular contents of the respective polyamine, and (5) transgenic work mostly confirms the tight regulation of cellular contents of spermidine and spermine. An understanding of expression and regulation of aminopropyltransferases at the metabolic level will help us in effective use of genetic engineering approaches for the improvement in nutritional value and stress responses of plants. [ABSTRACT FROM AUTHOR]

Abstract: Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to 1H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative 1H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit. [ABSTRACT FROM AUTHOR]

By means of an in silico analysis, we demonstrated that a previously described chimeric gene ( Spe-Sdh) encoding spermidine synthase, a key enzyme involved in the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity of this procedure was tested using different fungal species. As expected, positive results were obtained only with Basidiomycota species, whereas no amplification was achieved with species belonging to other fungal phyla. [ABSTRACT FROM AUTHOR]

Polyamines (PAs) are required for cell growth and cell division in eukaryotic and prokaryotic organisms. The present study is aimed at understanding the developmental regulation of PA biosynthesis and catabolism during flower opening and early fruit development in relation to fruit size and shape. Two full-length cDNA clones coding for S-adenosyl methionine decarboxylase (SAMDC) and spermidine synthase (SPDS) homologs, key steps in the PA biosynthesis pathway, in the stone-fruit of olive ( Olea europaea L.) were identified and the spatial and temporal organization of these genes were described. In olive flowers, OeSAMDC gene transcripts were highly expressed in ovary wall, placenta and ovules, while OeSPDS transcript was confined to the ovules of ovary at anthesis stage. A correlation was detected between the SAMDC enzyme activity/accumulation transcript and spermidine (Spd) and spermine (Spm) levels during flower opening, implying that the synthesis of decarboxylated SAM might be a rate-limiting step in Spd and Spm biosynthesis. OeSAMDC and OeSPDS transcripts were co-expressed in fruit mesocarp and exocarp at all developmental stages analyzed as well as in nucellus, integuments and inner epidermis tissues of fertilized ovules. In contrast, the OeSAMDC and OeSPDS genes had different expression patterns during early fruit development. The results provide novel data about localization of PA biosynthesis gene transcripts, indicating that transcript levels of PA biosynthesis genes are all highly regulated in a developmental and tissue-specific manner. The differences between the two olive cultivars in the fruit size in relation to the differences in the accumulation patterns of PAs are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterised, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains – a small N-terminal β-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity. [Copyright &y& Elsevier]

Abstract: Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM)-dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed. [Copyright &y& Elsevier]

Abstract: Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development. [Copyright &y& Elsevier]

To analyze the role of spermidine in cell growth and differentiation of Ustilago maydis, the gene encoding spermidine synthase (Spe) was isolated using PCR. We found that the enzyme is encoded by a chimeric bifunctional gene ( Spe-Sdh) that also encodes saccharopine dehydrogenase (Sdh), an enzyme involved in lysine biosynthesis. The gene contains a 5′ region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3′ region encoding Sdh, and directs the synthesis of a single transcript that hybridizes with 3′ or 5′ regions' probes of the gene. The gene could not be disrupted in a wild-type strain, but only in a mutant defective in the gene encoding ornithine decarboxylase ( Odc). Single spe-sdh mutants were isolated after sexual recombination in planta with a compatible wild-type strain. Mutants were auxotrophic for lysine and spermidine, but not for putrescine, and contained putrescine and spermidine, but not spermine. Putrescine in double mutants is probably synthesized from spermidine by the concerted action of polyamine acetyl transferase and polyamine oxidase. spe-sdh mutants were sensitive to stress, unable to carry out the yeast-to-mycelium dimorphic transition, and showed attenuated virulence to maize. These phenotypic alterations were reverted by complementation with the wild-type gene. [ABSTRACT FROM AUTHOR]