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FEBS Letters 581 (2007) 3081–3086

Putative synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Ju¨rgen M. Knott, Piero Ro¨mer, Manfred Sumper* Lehrstuhl Biochemie I, Universita¨t Regensburg, Universita¨tsstraße 31, D-93053 Regensburg, Germany

Received 3 April 2007; revised 23 May 2007; accepted 27 May 2007

Available online 6 June 2007

Edited by Miguel De la Rosa

Vicia sativa [9]. Diatoms are capable of synthesizing species- Abstract Polyamines are involved in many fundamental cellu- lar processes. Common polyamines are putrescine, specific sets of long-chain polyamines [10]. In vitro studies with and spermine. Spermine is synthesized by transfer of an amino- these long-chain polyamines revealed their role in the forma- propyl residue derived from decarboxylated S-adenosylmethio- tion of the silica-based cell walls [11–13]. As the genome of nine to spermidine. Thermospermine is an isomer of spermine Thalassiosira pseudonana has been sequenced, we have used and assumed to be synthesized by an analogous mechanism. this diatom species in order to investigate the biosynthesis of However, none of the recently described spermine synthases polyamines in more detail. was investigated for their possible activity as thermospermine Both, spermidine and spermine are synthesized by the same synthases. In this work, putative spermine synthases from the mechanism. An aminopropyl residue derived from decarboxyl- diatom Thalassiosira pseudonana and from Arabidopsis thaliana ated S-adenosylmethionine is transferred to putrescine if sper- could be identified as thermospermine synthases. These findings midine is formed or to spermidine if spermine is synthesized. may explain the previous result that two putative spermine syn- thase genes in Arabidopsis produce completely different pheno- The catalyzing these reactions are spermidine syn- types in knock-out experiments. Likely, part of putative thase and . These enzymes were found to ex- spermine synthases identifiable by sequence comparisons repre- hibit a high degree of sequence identity. sents in fact thermospermine synthases. Several spermidine synthases have been cloned and charac- 2007 Federation of European Biochemical Societies. Published terized meanwhile, e.g. from Nicotiana sylvestris [14] and Hel- by Elsevier B.V. All rights reserved. icobacter pylori [15]. In addition, the three-dimensional structures of the spermidine synthases from Thermotoga mari- Keywords: Thermospermine; Spermine; Diatom; Polyamine tima [16] and Caenorhabditis elegans [17] have been deter- synthesis; Arabidopsis thaliana mined. In these investigations, the binding domains for putrescine and decarboxylated S-adenosylmethionine were identified and found to be highly conserved with respect to their amino acid sequence and relative spatial arrangement. Spermine synthases have been cloned from Homo sapiens 1. Introduction [18] and from Saccharomyces cerevisiae [19]. Two sequences, Acl5 and Spms, were identified as spermine synthases in A. tha- Polyamines ubiquitously occur in all organisms. In general, liana [20,21]. Homologues of Acl5 and Spms could be cloned they are involved in many fundamental cellular processes like from Malus sylvestris var. domestica [22]. replication, transcription, membrane stabilization, and modu- The synthesis of thermospermine is likely to be mechanisti- lation of activities [1,2]. In plants, polyamines play an cally related to the formation of spermine. However, enzymes important role in development as well as responses to biotic synthesizing thermospermine are presently unknown. In the and abiotic stress [2–4]. present paper, we demonstrate for two enzymes closely related The most common polyamines are putrescine, spermidine to spermine synthases, one from the diatom T. pseudonana and and spermine. Putrescine and spermidine are found in pro- the other from A. thaliana, that their reaction product is ther- karyotes and eukaryotes. In contrast, spermine mainly occurs mospermine rather than spermine. As a consequence, some of in eucaryotes. Beside these commonly observed polyamines, the putative spermine synthases described so far may actually other polyamines have been found in living organisms as well. be thermospermine synthases. Thermospermine, an isomer of spermine (Fig. 1), was discov- ered for the first time in the thermophilic bacterium Thermus thermophilus [5]. Later, thermospermine could also be found 2. Materials and methods in the halophilic archaeum Halobacterium cutirubrum [6],in several species of Agrobacterium [7],inParacoccus denitrificans 2.1. Chemicals [8] as well as in leguminous seeds, e.g. from Pisum sativum and Spermidine and spermine were purchased from Roth, and 14C-sper- midine was purchased from GE Healthcare. Decarboxylated S-adeno- sylmethionine was prepared from S-adenosylmethionine (Fluka) according to [23]. The expression plasmid pET21a with SpeD (J02804) from E. coli was a kind gift from Prof. Herbert Tabor, Bethes- da. S-adenosylmethionine decarboxylase SpeD was expressed in E. coli *Corresponding author. strain Bl21(DE3). Synthetic thermospermine was a kind gift from Prof. E-mail address: [email protected] (M. Sumper). Armin Geyer, Marburg.

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.05.074 3082 J.M. Knott et al. / FEBS Letters 581 (2007) 3081–3086

were ruptured by vortexing with 1 vol. glass beads (0.3 mm diameter). A crude extract was obtained by centrifugation at 20000 · g for 10 min at 4 C. The complete extract obtained from 4 l-culture was used for a single enzyme assay, which was carried out according to the standard assay for the purified enzyme, but in a total volume of 800 ll.

2.7. Thin-layer chromatography The enzyme assays were analyzed by thin-layer chromatography (TLC) in the solvent system BAPF (butanol/acetic acid/pyridine/37% formaldehyde = 3/3/2/1) according to [24]. The silica gel TLC-plates (Merck) were dried and exposed to a phospho-imager screen (Fuji) Fig. 1. Structure of spermine and thermospermine. The putrescine for at least 2 h. The phospho-imager screen was analyzed on a Fuji moiety is highlighted. BAS Reader 1000. Unlabelled polyamines were detected by staining with 1% ninhydrin in isopropanol. 2.2. Cultures T. pseudonana clone CCMP1335 was cultivated in an artificial sea- 2.8. Mass spectrometry water medium. A. thaliana ecotype Columbia (Col-0) was a kind gift For analysis of the reaction products by MALDI-TOF, the enzyme from Dr. Erich Glawischnig, Technical University of Munich. cDNA assay was carried out with 20 nmol unlabeled spermidine in a total es- from S. cerevisiae strain W303 was obtained from Dr. Achim Griesen- say volume of 4 ml. The polyamines were purified by cation exchange beck, University of Regensburg. chromatography (High S, BioRad). The polyamines were eluted with 4M NH3. After neutralization with 3 M acetic acid and drying, the 2.3. Cloning of aminopropyl cDNAs polyamines were perethylated in 100 ll of 50 mM sodium phosphate The isolation of total RNA from a 100 ml culture of T. pseudonana buffer pH 7.0, 100 mM NaCNBH3, and 100 mM acetaldehyde. After 8 (1 · 10 cells/ml) was carried out with Tri-Reagent (Sigma) according 2 h at 25 C, the solution was extracted two times with 1 vol. CHCl3. to the manufacturer’s instruction. To synthesize single strand cDNA The organic phase was dried and analyzed by MALDI-TOF. with Superscript III-reverse transcriptase (Invitrogen), 1 lg of total RNA was used. To get a full size cDNA-clone of TPS_41289 cDNA, 50- and 30- RACE-PCR were performed. A full length amplification of TPS_41289-cDNA sequence was achieved using the 50-primer (50- 3. Results CAT ATG CAC ATC CAC AAC CTC CTT TTG-30) containing a restriction site for NdeI (underlined) and the 30-primer (50-CTC 3.1. Thermospermine synthesis in crude extracts from GAG ATA CAT GAA GAT TGG ATT GG-30) containing a restric- Thalassiosira pseudonana tion site for XhoI (underlined). The PCR reaction mixtures were dena- To investigate the polyamine biosynthesis in T. pseudonana, tured at 94 C for 20 s, annealed at 55 C for 20 s, and extended at 72 C for 90 s. For recombinant expression the full length cDNA enzyme assays were carried out using a crude cell extract pre- was cloned into the pET-20b expression vector (Novagen), which pared from 4 l culture (106 cells/ml). After incubation (60 min was transformed into E. coli strain Bl21 (DE3). at 32 C) with decarboxylated S-adenosylmethionine and Acl5 and Spms from A. thaliana and Spe4 from yeast were cloned 14C-spermidine, the reaction products were analyzed by from cDNA and expressed in E. coli strain SG 13009 in expression vec- tor pQE-UA (Qiagen). TLC. Surprisingly, spermidine was found to be converted to thermospermine. Spermine was not detectable in this reaction 2.4. Enzyme purification (Fig. 5A). For purification of the expressed enzymes, 50 ml of Luria–Bertani broth containing the antibiotica required for selection was inoculated 3.2. Cloning and expression of TPS_41289 with a single colony of the transformant. This culture was incubated Homologues of spermidine- and spermine synthases were at 37 C and 160 rpm until the optical density at 600 nm reached 0.6. Isopropylthiogalactopyranoside (IPTG) was added (1 mM), and the searched in the genome database of T. pseudonana (http://gen- culture was further incubated for 4 h. Bacteria were harvested by cen- ome.jgi-psf.org). This database contains 10 gene models for trifugation, suspended in 400 ll of 0.1 M Tris–HCl buffer pH 7.5 and putative spermidine and spermine synthases. The gene model ruptured by ultrasonication. After centrifugation at 20000 · g for for a putative spermine synthase (TPS_41289) with a length 10 min at 4 C, the crude extract was obtained. The crude extract was applied to a Ni-NTA-agarose column (Qiagen) and the 6·His- of 861 bp shared the highest sequence identity with other tag fusion-proteins were purified according to the instructions for na- spermine synthases and was therefore chosen for a detailed tive purification from the manufacturer. The protein concentration analysis. This sequence was cloned from T. pseudonana cDNA was measured by the Bradford protein assay (Sigma). Samples of re- and the start and stop codons were assigned by 30 and 50 combinant proteins were electro-blotted on a PVDF membrane and RACE-PCR analysis. In contrast to the proposed gene model, the N-terminal sequence was obtained by Edman degradation. The molecular mass of TPS_41289 was determined by gel filtration the complete TPS_41289 cDNA displays a length of 1263 bp on Superose 6 columne (GE Healthcare) equilibrated with 20 mM (Fig. 2). Tris–HCl (pH 7.5)/150 mM NaCl and a flowrate at 1 ml/ min. Catalase Comparison of various spermidine and spermine synthases (240 kDa), aldolase (160 kDa), bovine albumin (67 kDa) and ovalbu- detects two groups of aminopropyl (Fig. 3). Re- min (45 kDa) were used as standards. cently identified spermine synthases are distributed among the SPMS- and the ACL5-group. Spermine synthases in the 2.5. Enzyme assays with recombinant proteins The standard reaction mixture for spermine- and thermospermine syn- SPMS-group are closely related to spermidine synthases. That thase assays contained 0.1 M Tris–HCl, pH 7.5, 12 pmol (5500 dpm) means, spermidine synthases (SPDS) and SPMS-like enzymes 14C-spermidine, 1 nmol decarboxylated S-adenosylmethionine and 1 lg can be combined in a SPDS/SPMS-group. Aminopropyl trans- purified enzyme in a total volume of 200 ll. After incubation at 32 C ferases in the ACL5-group exhibit a relatively low sequence for 60 min, the reaction was terminated by heating at 95 C for 5 min. identity to the enzymes found in the SPDS/SPMS-group [21,22]. 2.6. Enzyme assay with a crude extract from T. pseudonana Four liter culture (106 cells/ml) was centrifuged at 10000 · g for Alignment of TPS_41289 with other aminopropyl transfer- 10 min. After suspension in 800 ll of 0.1 M Tris–HCl, pH 7.5, the cells ases clearly identifies TPS_41289 as a member of the ACL5- J.M. Knott et al. / FEBS Letters 581 (2007) 3081–3086 3083

Fig. 3. Phylogenetic tree of aminopropyl transferases (created with http://align.genome.jp/). The distance scale represents the evolutionary distance. Arabis gemmifera AgSPMS (AB076744), Arabidopsis thaliana AtACL5 (AF184093), AtSPDS1 (AJ251296), AtSPDS2 (AJ251297), AtSPMS (AY040013), Malus sylvestris var. domestica MdACL5-1 (AB204521), MdACL5-2 (AB204522), MdSPDS1 (AB072915), MdSPDS2a (AB072916), MdSPDS2b (AB072917), MdSPMS (AB204520), Saccharomyces cerevisiae ScSPE3 (U27519), ScSPE4 (AF067970). TPS: Thalassiosira pseudonana.

synthases. TPS_41289 shares 33% sequence identity with ACL5. The SPMS-group includes SPMS from A. thaliana, MdSPMS from apple and SPE4 from yeast, which were also identified as spermine synthases with a high sequence identity to the spermidine synthases SPDS1 and SPDS2 from A. thali- ana, MdSPDS1, MdSPDS2a and MdSPDS2b from apple and SPE3 from yeast (Fig. 3). The TPS_41289 cDNA was cloned into the expression-vec- tor pET 20b together with a C-terminal 6·His-tag sequence and expressed in E. coli strain Bl21. Affinity purification on Ni-NTA agarose results in a distinct 50 kDa protein band in SDS–PAGE (Fig. 4), which is in good agreement with the cal- culated mass for the His-tagged fusion protein of 48 kDa. By Edman degradation, the N-terminal sequence was found to be MHIHNLLLP matching the predicted amino acid sequence of the TPS_41289 protein (Fig. 2). The TPS_41289 protein dis- played a mass of nearly 200 kDa as shown by size exclusion gel filtration chromatography on a superose 6 column. This result indicates that the TPS_41289 protein forms a homotetramer in its native state.

Fig. 2. Nucleotide and protein sequence of TPS_41289. The under- 3.3. Enzyme assays with the recombinant TPS_41289 protein lined amino acids were confirmed by Edman degradation. The The enzymatic activity of TPS_41289 protein was investi- sequence of the gene model from the T. pseudonana genome database is indicated by asterisks. gated by the incubation of 1 lg of the purified enzyme with decarboxylated S-adenosylmethionine and 14C-spermidine as described in Section 2. The reaction products were analyzed group, including ACL5 from A. thaliana, MdACL5-1 and by thin-layer chromatography (TLC) together with unlabeled MdACL5-2 from apple as well as AgSPMS from Arabis spermidine, spermine and thermospermine as references. 14C- gemmifera, all of which were previously identified as spermine spermidine was converted to a product co-migrating with 3084 J.M. Knott et al. / FEBS Letters 581 (2007) 3081–3086

Fig. 4. Coomassie-stained SDS–PAGE of purified TPS_41289 protein. Lane 1: molecular marker, lane 2: purified TPS_41289 protein. thermospermine (Fig. 5B), while no reaction was detectable in a control assay with the heat denatured enzyme. This result was confirmed by mass spectrometry. After enzy- matic synthesis, the polyamine product was perethylated by reductive alkylation with acetaldehyde for MALDI-TOF anal- ysis. The enzymatically synthesized product displayed an m/z 371 corresponding to perethylated spermine or thermosper- mine. This material could be identified as perethylated thermo- spermine by fragmentation analysis. Diagnostic mass fragments are m/z 128 and 199 (Fig. 6), which are absent in the fragmentation spectrum from perethylated spermine. These results clearly demonstrate that TPS_41289 synthesizes Fig. 6. (A) MALDI-TOF fragmentation spectrum of the perethylated thermospermine rather than spermine. reaction product of the TPS_41289 enzyme. (B) Fragmentation spectrum of perethylated spermine.

3.4. Identification of ACL5 from Arabidopsis thaliana as expressed in E. coli. SPE4, ACL5 and SPMS were all described As can be seen in Fig. 3, the putative spermine synthase as spermine synthases in recent investigations [19–21]. ACL5 from A. thaliana displays the highest sequence identity Enzyme assays were carried out with the purified enzymes to TPS_41289. Therefore ACL5 could also act as a thermo- ACL5, SPMS and SPE4 using 14C-spermidine as a substrate. spermine synthase. To investigate this possibility, Acl5 as well After incubation at 32 C, the reaction products were analyzed as Spms from A. thaliana and Spe4 from yeast were cloned and by TLC. SPE4 from yeast and SPMS from A. thaliana indeed acted as spermine synthases. In contrast, the ACL5 product could be identified as thermospermine (Fig. 5B). ACL5 and TPS_41289 were found to synthesize thermosper- mine as the only product under different experimental condi- tions. Spermidine concentrations ranging from 0.05 lMto 5 lM and pH values between 6.4 and 11.0 did not result in the synthesis of other products than thermospermine.

4. Discussion

ACL5 from A. thaliana has recently been described as a spermine synthase [20]. However, it is difficult to separate the isomers spermine and thermospermine. The most efficient method is TLC analysis in the solvent system described in Fig. 5. Autoradiogram of a TLC analysis of enzyme assays for [24]. MALDI-TOF mass spectrometry confirmed the results aminopropyl transferase activity. (A) Crude extract from T. pseudo- nana. (B) Recombinant proteins, TPS_41289 (T. pseudonana), ACL5 obtained by TLC-analysis and proves ultimately, that ACL5 (A. thaliana), SPMS (A. thaliana) and SPE4 (yeast). The substrate 14C acts as a thermospermine synthases as it is found for spermidine was not completely converted to the reaction products. TPS_41289 from the diatom T. pseudonana. J.M. Knott et al. / FEBS Letters 581 (2007) 3081–3086 3085

By aligning aminopropyl transferases, sequences identified [2] Martin-Tanguy, J. (2000) Metabolism and function of polyamines as spermine synthases can be divided in two groups, denoted in plants: recent development (new approaches). Plant Growth in Fig. 3 as the ACL5-group and the SPMS-group. The ther- Regulat. 100, 675–688. [3] Bouchereau, A., Aziz, A., Larher, F. and Martin-Tanguy, J. mospermine synthases described in this work are members of (1999) Polyamines and environmental challenges: recent develop- the ACL5-group, whereas the spermine synthases SPMS from ment. Plant Sci. 165, 95–101. A. thaliana and SPE4 from yeast fit into the SPMS-group. This [4] Evans, P.T. and Malmberg, R.L. (1989) Do polyamines have roles suggests that other aminopropyl transferases within the ACL5- in plant development? Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 235–269. group, like MdACL5-1 and MdACL5-2 from apple, may also [5] Oshima, T. (1979) A new polyamine, thermospermine, 1,12- represent thermospermine synthases. diamino-4,8-diazadodecane, from an extreme thermophile. J. For spermidine synthases, the substrate binding sites were Biol. Chem. 254 (18), 8720–8722. identified by X-ray analysis [16,17]. By sequence comparison, [6] Carteni-Farina, M., Porcelli, M., Cacciapuoti, G., De Rosa, M., spermine as well as thermospermine synthases exhibit a sim- Gambacorta, A., Grant, W.D. and Ross, H.N.M. (1985) Poly- amines in halophilic archaebacteria. FEMS Microbiol. Lett. 28, ilar spatial arrangement of these binding sites. The conserved 323–327. amino acid sequence GGGDG constitutes the for [7] Hamana, K., Matsuzaki, S., Niitsu, M. and Samejima, K. (1989) decarboxylated S-adenosylmethionine in spermidine syn- Polyamine distribution and the potential to form novel polyam- thases [16], and this domain is also found in spermine ines in phytopathogenic agrobacteria. FEMS Microbiol. Lett. 65, 269–274. synthases of the SPMS-group. The aspartate residue in this [8] Hamana, K., Matsuzaki, S., Niitsu, M. and Samejima, K. (1990) sequence is responsible for an ionic interaction with the Synthesis of novel polyamines in Paracoccus, Rhodobacter and amino group of the aminopropyl moiety of decarboxylated Micrococcus. FEMS Microbiol. Lett. 67, 267–274. S-adenosylmethionine [17]. Interestingly, all aminopropyl [9] Hamana, K., Niitsu, M., Samejima, K. and Matsuzaki, S. (1991) transferases within the ACL5-group replace this aspartate Linear and branched pentaamines, hexaamines and heptaamines in seeds of Vicia sativa. Phytochemistry 30 (10), 3319–3322. (D) by a glutamate (E) resulting in the domain sequence [10] Kro¨ger, N., Deutzmann, R., Bergsdorf, C. and Sumper, M. (2000) G GGGE /L (Fig. 2). Species-specific polyamines from diatoms control silica morphol- Thermospermine synthases seem to be more widespread ogy. PNAS 97 (26), 14133–14138. in plants. Besides ACL5 from A. thaliana, and MdACL5-1 [11] Sumper, M. (2002) A phase separation model for the nanopat- terning of diatom biosilica. Science 295, 2430–2433. and -2 from apple, a number of other gene models were found [12] Sumper, M. and Brunner, E. (2006) Learning from diatoms: in the genome databases for Picea abies, Solanum tuberosum nature’s tools for the production of nanostructured silica. Adv. and Medicago truncatula which can be classified as members Funct. Mater. 16, 17–26. of the ACL5 family. Therefore, it is likely that all these genes [13] Sumper, M., Brunner, E. and Lehmann, G. (2005) Biomineral- encode thermospermine synthases rather than spermine syn- ization in diatoms: characterization of novel polyamines associ- ated with silica. FEBS Lett. 579, 3765–3769. thases. [14] Hashimoto, T., Tamaki, K., Suzuki, K. and Yamada, Y. (1998) In A. thaliana, acl5-1 and spms-l mutants were characterized Molecular cloning of plant spermidine synthases. Plant Cell lacking both the aminopropyl transferase activities assumed to Physiol. 39 (1), 73–79. synthesize spermine [20,25]. The acl5-1 mutant but not the [15] Lee, M.J., Huang, C.Y., Sun, Y.J. and Huang, H. (2005) Cloning and characterization of and its implication in spms-1 mutant resulted in a phenotype with severely reduced polyamine biosynthesis in Helicobacter pylori strain 26696. length of the inflorescent stems compared to the wild type Protein Exp. Purif. 43, 140–148. [25]. Our findings that these gene products synthesize different [16] Korolev, S., Ikeguchi, Y., Skarina, T., Beasley, S., Arrowsmith, compounds might explain these different phenotypes. Lack of C., Edwards, A., Joachimiak, A., Pegg, A.E. and Savchenko, A. the thermospermine synthase not only results in reduced length (2002) The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor. Nat. Struct. Biol. 9 (1), 27–31. of the inflorescence stem, but also in increased thickness of [17] Dufe, V.T., Lu¨ersen, K., Eschbach, M.L., Haider, N., Karlberg, veins in rosette leaves [26]. Thus, thermospermine seems to T., Walter, R.D. and Al-Karadaghi, S. (2005) Cloning, expres- play an important role in growth and development of the vas- sion, characterisation and three-dimensional structure determina- cular tissue in plants. tion of Caenorhabditis elegans spermidine synthase. FEBS Lett. 579, 6037–6043. In diatoms, extremely modified proteins denoted as silaffins [18] Korhonen, V.P., Halmekyto¨, M., Kauppinen, L., Myo¨ha¨nen, S., are involved in the control of silica biomineralization. A typi- Wahlfors, J., Keina¨nen, T., Hyvo¨nen, T., Alhonen, L., Eloranta, cal feature of these proteins is the covalent attachment of poly- T. and Ja¨nne, J. (1995) Molecular cloning of a cDNA encod- amine moieties to lysine residues. Attachment of two ing human spermine synthase. DNA Cell Biol. 14 (10), 841– aminopropyl units to lysine is a predominant type of modifica- 847. [19] Hamasaki-Katagiri, N., Katagiri, Y., Tabor, C.W. and Tabor, H. tion [27]. The biosynthetic pathway for this posttranslational (1998) Spermine is not essential for growth of Saccharomyces modification is as yet unknown, but thermospermine might cerevisiae: identification of the Spe4 gene (spermine synthase) act as the donor for this modification. and characterization of a spe4 deletion mutant. Gene 210, 195– 201. [20] Hanzawa, Y., Takahashi, T., Michael, A.J., Burtin, D., Long, D., Acknowledgements: We thank R. Deutzmann and E. Hochmuth for Pineiro, M., Coupland, G. and Komeda, Y. (2000) ACAULIS5, MALDI-TOF analysis and Edman degradation. This work was sup- an Arabidopsis gene required for stem elongation, encodes a ported by the Volkswagenstiftung and the Fonds der Chemischen spermine synthase. EMBO J. 19 (16), 4248–4256. Industrie. [21] Panicot, M., Minguet, E.G., Ferrando, A., Alcazar, R., Blazquez, M.A., Carbonell, J., Altabella, T., Koncz, C. and Tiburcio, A.F. (2002) A polyamine metabolon involving aminopropyl transferase complexes in arabidopsis. Plant Cell 14, 2539–2551. References [22] Kitashiba, H., Hao, Y.J., Honda, C. and Moriguchi, T. (2005) Two types of spermine synthase genes: MdACL5 and MdSPMS [1] Tabor, C.W. and Tabor, H. (1984) Polyamines. Annu. Rev. are differentially involved in apple fruit development and cell Biochem. 53, 749–790. growth. Gene 361, 101–111. 3086 J.M. Knott et al. / FEBS Letters 581 (2007) 3081–3086

[23] Zappia, V., Galletti, P., Oliva, A. and de Santis, A. (1977) New [26] Clay, N.K. and Nelson, T. (2005) Arabidopsis thickvein mutation methods for preparation and analysis of S-adenosyl-(50)-3-meth- affects vein thickness and organ vascularization, and resides in a ylthiopropylamine. Anal. Biochem. 79 (1-2), 535–543. provascular cell-specific spermine synthase involved in vein [24] Shirahata, A., Takeda, Y., Kawase, M. and Samejima, K. (1983) definition and in polar auxin transport. Plant Physiol. 138, 767– Detection of spermine and thermospermine by thin-layer chro- 777. matography. J. Chromatogr. 262, 451–454. [27] Wenzl, S., Deutzmann, R., Hett, R., Hochmuth, E. and Sumper, [25] Imai, A., Akiyama, T., Kato, T., Sato, S., Tabata, S., Yamamoto, M. (2004) Quaternary ammonium groups in silica associated K.T. and Takahashi, T. (2004) Spermine is not essential for proteins. Angew. Chem. Int. Ed. 43, 5933–5936. survival of Arabidopsis. FEBS Lett. 556, 148–152.