Polyamines produced by an extreme thermophile are essential for cell growth at high temperature.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis.

Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3'-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2, in comparison to other pathways such as TCA cycle-, amino acid- metabolisms and photosynthesis. The cell wall pectin metabolism, cell wall proteins and degradation process were highly modulated. Intriguingly Fe-deficiency responsive genes and drought stress-induced genes were also up-regulated, suggesting the importance of thermospermi'ne flux on regulation of gene network. Histological observation showed that the vascular system of the joint part between stem and leaves was structurally dissociated, indicating its involvement in vascular maintenance. Endogenous increase in T-Spm and reduction in H2O2 contents were found in mutant grown in T-Spm containing media. The results indicate that T-Spm homeostasis by a fine tuned balance of its synthesis and catabolism is important for maintaining gene regulation network and the vascular system in plants.

The C-terminal flexible region of branched-chain polyamine synthase facilitates substrate specificity and catalysis.

Branched-chain polyamine synthase (BpsA) catalyzes sequential aminopropyl transfer from the donor, decarboxylated S-adenosylmethionine (dcSAM), to the acceptor, linear-chain polyamine, resulting in the production of a quaternary-branched polyamine via tertiary branched polyamine intermediates. Here, we analyzed the catalytic properties and X-ray crystal structure of Tth-BpsA from Thermus thermophilus and compared them with those of Tk-BpsA from Thermococcus kodakarensis, which revealed differences in acceptor substrate specificity and C-terminal structure between these two enzymes. To investigate the role of the C-terminal flexible region in acceptor recognition, a region (QDEEATTY) in Tth-BpsA was replaced with that in Tk-BpsA (YDDEESSTT) to create chimeric Tth-BpsA C9, which showed a severe reduction in catalytic efficiency toward N4 -aminopropylnorspermidine, but not toward N4 -aminopropylspermidine, mimicking Tk-BpsA substrate specificity. Tth-BpsA C9 Tyr346 and Thr354 contributed to discrimination between tertiary branched-chain polyamine substrates, suggesting that the C-terminal region of BpsA recognizes acceptor substrates. Liquid chromatography-tandem mass spectrometry analysis on a Tk-BpsA reaction mixture with dcSAM revealed two aminopropyl groups bound to two of five aspartate/glutamate residues (Glu339 , Asp342 , Asp343 , Glu344 , and Glu345 ) in the C-terminal flexible region. Mutating each of these five amino acid residues to asparagine/glutamine resulted in a slight decrease in activity. The quadruple mutant D342N/D343N/E344Q/E345Q exhibited a severe reduction in catalytic efficiency, suggesting that these aspartate/glutamate residues function to receive aminopropyl chains. In addition, the X-ray crystal structure of the Tk-BpsA ternary complex bound to N4 -bis(aminopropyl)spermidine revealed that Asp126 and Glu259 interacted with the aminopropyl moiety in N4 -aminopropylspermidine.

Identification of seven polyamine oxidase genes in tomato (Solanum lycopersicum L.) and their expression profiles under physiological and various stress conditions.

Polyamines (PAs) are implicated in developmental processes and stress responses of plants. Polyamine oxidases (PAOs), flavin adenine dinucleotide-dependent enzymes that function in PA catabolism, play a critical role. Even though PAO gene families of Arabidopsis and rice have been intensely characterized and their expression in response to developmental and environmental changes has been investigated, little is known about PAOs in tomato (Solanum lycopersicum). We found seven PAO genes in S. lycopersicum and named them SlPAO1∼7. Plant PAOs form four clades in phylogenetic analysis, of which SlPAO1 belongs to clade-I, SlPAO6 and SlPAO7 to clade-III, and the residual four (SlPAO2∼5) to clade-IV, while none belongs to clade-II. All the clade-IV members in tomato also retain the putative peroxisomal-targeting signals in their carboxy termini, suggesting their peroxisome localization. SlPAO1 to SlPAO5 genes consist of 10 exons and 9 introns, while SlPAO6 and SlPAO7 are intronless genes. To address the individual roles of SlPAOs, we analyzed their expression in various tissues and during flowering and fruit development. The expression of SlPAO2∼4 was constitutively high, while that of the other SlPAO members was relatively lower. We further analyzed the expressional changes of SlPAOs upon abiotic stresses, oxidative stresses, phytohormone application, and PA application. Based on the data obtained, we discuss the distinctive roles of SlPAOs.

Identification of Branched-Chain Polyamines in Hyperthermophiles.

Thermophiles are organisms that grow optimally at temperatures higher than 55 °C. They contain two types of unusual longer/branched-chain polyamines in addition to common polyamines such as spermidine and putrescine. These unusual polyamines contribute to the survival of hyperthermophiles at high temperatures. Recently, the novel aminopropyltransferase BpsA was found to be responsible for the biosynthesis of branched-chain polyamines in the hyperthermophilic archaeon Thermococcus kodakarensis, which contains N 4-bis(aminopropyl)spermidine as the major polyamine. This compound is synthesized by the sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine via the bifunctional catalytic action of BpsA. In this chapter, methods for the extraction and identification of branched-chain polyamines are presented, along with methods for the production and characterization of recombinant T. kodakarensis BpsA as a model aminopropyltransferase.

Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile-specific branched-chain polyamine.

Branched-chain polyamines are found exclusively in thermophilic bacteria and Euryarchaeota and play essential roles in survival at high temperatures. In the present study, kinetic analyses of a branched-chain polyamine synthase from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-BpsA) were conducted, showing that N4 -bis(aminopropyl)spermidine was produced by sequential additions of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine, through bifunctional catalytic action. Tk-BpsA catalyzed the aminopropylation of the linear-chain polyamines spermidine, spermine, norspermidine, and the tertiary-branched polyamines N4 -aminopropylspermidine and N4 -aminopropylnorspermidine, but not of short-chain diamines, putrescine, and cadaverine, suggesting that Tk-BpsA does not catalyze the aminopropylation of primary amino groups of diamines. X-ray structural analyses of Tk-BpsA in the presence or absence of the substrates spermidine and dcSAM revealed that a large, negatively charged cavity is responsible for the binding of branched-chain substrates. The binding is different from that in the active site of linear polyamine spermidine/spermine synthases, and loop-closures occur upon the binding of spermidine. Based on structural analyses, further kinetic studies were carried out for various mutants, revealing that Asp159, positioned between the reactive secondary amino group of the substrate polyamine and a sulfur atom of the product 5'-methylthioadenosine and in a Gly-Asp-Asp-Asp motif, functions as a catalytic center, with reactions proceeding via a ping-pong mechanism. Our study provides a novel aminopropyltransfer reaction mechanism, distinct from the SN 2 displacement mechanism found in other known linear spermidine/spermine synthases. DATABASE: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with PDB codes 5XNF for apo-Tk-BpsA, 5XNH for the binary complex, and 5XNC for the ternary complex.

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis.

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N4-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N4-bis(aminopropyl)-N1-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N4-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

KEY MESSAGE: Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

The polyamine spermine induces the unfolded protein response via the MAPK cascade in Arabidopsis.

In Arabidopsis three basic region leucine zipper (bZIP) transcription factor genes, bZIP17, bZIP28, and bZIP60, play crucial roles in the unfolded protein response (UPR). Previously we found that bZIP60 is one of the spermine-induced genes. Consequently we further investigated the response of all the three bZIP genes to spermine. Expression of bZIP17, bZIP28, and bZIP60, and also their target genes was activated by spermine application as well as in plants with elevated endogenous spermine levels. Furthermore, spermine activated the splicing of the bZIP60 transcript mediated by the ribonuclease activity of inositol-requiring enzyme 1 and also recruited bZIP17 and bZIP60 proteins from endoplasmic reticulum to nucleus. We therefore propose that spermine is a novel UPR inducer. Moreover, induction of UPR by spermine required calcium-influx to the cytoplasm and the genes for mitogen-activated protein kinase kinase 9 (MKK9), mitogen-activated protein kinase 3 (MPK3) and MPK6. The result indicates that spermine-induced UPR is mediated by the MKK9-MPK3/MPK6 cascade in Arabidopsis.

The polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike that of angiosperms, back-converts thermospermine to norspermidine.

In the phylogeny of plant polyamine oxidases (PAOs), clade III members from angiosperms, such as Arabidopsis thaliana PAO5 and Oryza sativa PAO1, prefer spermine and thermospermine as substrates and back-convert both of these substrates to spermidine in vitro. A clade III representative of lycophytes, SelPAO5 from Selaginella lepidophylla, also prefers spermine and thermospermine but instead back-converts these substrates to spermidine and norspermidine, respectively. This finding indicates that the clade III PAOs of lycophytes and angiosperms oxidize thermospermine at different carbon positions. We discuss the physiological significance of this difference.

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity.

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.

Arabidopsis mutant plants with diverse defects in polyamine metabolism show unequal sensitivity to exogenous cadaverine probably based on their spermine content.

Arabidopsis plants do not synthesize the polyamine cadaverine, a five carbon-chain diamine and structural analog of putrescine. Mutants defective in polyamine metabolic genes were exposed to exogenous cadaverine. Spermine-deficient spms mutant grew well while a T-DNA insertion mutant (pao4-1) of polyamine oxidase (PAO) 4 was severely inhibited in root growth compared to wild type (WT) or other pao loss-of-function mutants. To understand the molecular basis of this phenomenon, polyamine contents of WT, spms and pao4-1 plants treated with cadaverine were analyzed. Putrescine contents increased in all the three plants, and spermidine contents decreased in WT and pao4-1 but not in spms. Spermine contents increased in WT and pao4-1. As there were good correlations between putrescine (or spermine) contents and the degree of root growth inhibition, effects of exogenously added putrescine and spermine were examined. Spermine mimicked the original phenomenon, whereas high levels of putrescine evenly inhibited root growth, suggesting that cadaverine-induced spermine accumulation may explain the phenomenon. We also tested growth response of cadaverine-treated WT and pao4-1 plants to NaCl and found that spermine-accumulated pao4-1 plant was not NaCl tolerant. Based on the results, the effect of cadaverine on Arabidopsis growth and the role of PAO during NaCl stress are discussed.

Identification of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles.

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers.

Polyamine oxidase (PAO), which requires FAD as a cofactor, functions in polyamine catabolism. Plant PAOs are classified into two groups based on their reaction modes. The terminal catabolism (TC) reaction always produces 1,3-diaminopropane (DAP), H2O2, and the respective aldehydes, while the back-conversion (BC) reaction produces spermidine (Spd) from tetraamines, spermine (Spm) and thermospermine (T-Spm) and/or putrescine from Spd, along with 3-aminopropanal and H2O2. The Oryza sativa genome contains seven PAO-encoded genes termed OsPAO1-OsPAO7. To date, we have characterized four OsPAO genes. The products of these genes, i.e. OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze BC-type reactions. Whereas OsPAO1 remains in the cytoplasm, the other three PAOs localize to peroxisomes. Here, we examined OsPAO7 and its gene product. OsPAO7 shows high identity to maize ZmPAO1, the best characterized plant PAO having TC-type activity. OsPAO7 seems to remain in a peripheral layer of the plant cell with the aid of its predicted signal peptide and transmembrane domain. Recombinant OsPAO7 prefers Spm and Spd as substrates, and it produces DAP from both substrates in a time-dependent manner, indicating that OsPAO7 is the first TC-type enzyme identified in O. sativa. The results clearly show that two types of PAOs co-exist in O. sativa. Furthermore, OsPAO7 is specifically expressed in anthers, with an expressional peak at the bicellular pollen stage. The physiological function of OsPAO7 in anthers is discussed.

Thermospermine modulates expression of auxin-related genes in Arabidopsis.

Thermospermine, a structural isomer of spermine, is widely distributed in the plant kingdom and has been shown to play a role in repressing xylem differentiation by studies of its deficient mutant, acaulis5 (acl5), in Arabidopsis. Our results of microarray and real-time PCR analyses revealed that, in addition to a number of genes involved in xylem differentiation, genes related to auxin signaling were up-regulated in acl5 seedlings. These genes include MONOPTEROS, an auxin response factor gene, which acts as a master switch for auxin-dependent procambium formation, and its target genes. Their expression was reduced by exogenous treatment with thermospermine or by transgenic induction of the ACL5 gene. We examined the effect of synthetic polyamines on the expression of these auxin-related genes and on the vascular phenotype of acl5, and found that tetramines containing the NC3NC3N chain could mimic the effect of thermospermine but longer polyamines containing the same chain had little or no such effect. We also found that thermospermine had an inhibitory effect on lateral root formation in wild-type seedlings and it was mimicked by synthetic tetramines with the NC3NC3N chain. These results suggest the importance of the NC3NC3N chain of thermospermine in its action in modulating auxin signaling.

Oryza sativa polyamine oxidase 1 back-converts tetraamines, spermine and thermospermine, to spermidine.

KEY MESSAGE: Oryza sativa polyamine oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism. Rice (Oryza sativa) contains seven genes encoding polyamine oxidases (PAOs), termed OsPAO1 to OsPAO7, based on their chromosomal number and gene ID number. We previously showed that three of these members, OsPAO3, OsPAO4 and OsPAO5, are abundantly expressed, that their products localize to peroxisomes and that they catalyze the polyamine back-conversion reaction. Here, we have focused on OsPAO1. The OsPAO1 gene product shares a high level of identity with those of Arabidopsis PAO5 and Brassica juncea PAO. Expression of OsPAO1 appears to be quite low under physiological conditions, but is markedly induced in rice roots by spermine (Spm) or T-Spm treatment. Consistent with the above finding, the recombinant OsPAO1 prefers T-Spm as a substrate at pH 6.0 and Spm at pH 8.5 and, in both cases, back-converts these tetraamines to spermidine, but not to putrescine. OsPAO1 localizes to the cytoplasm of onion epidermal cells. Differing in subcellular localization, four out of seven rice PAOs, OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze back-conversion reactions of PAs. Based on the results, we discuss the catabolic path(s) of PAs in rice plant.

POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5.

POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5.