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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.

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.

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]