©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Polyamine 4-Aminobutylcadaverine
OCCURRENCE AND ITS BIOSYNTHESIS IN ROOT NODULES OF ADZUKI BEAN PLANT VIGNA ANGULARIS(*)

Shinsuke Fujihara (1)(§), Hiroto Abe (2), Tadakatsu Yoneyama (1) (2)

From the (1) Plant Nutrition and Diagnosis Laboratory, National Agriculture Research Center, Kannondai 3-1-1, Tsukuba 305, Japan and (2) Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Root nodules of adzuki bean plant ( Vigna angularis) contained a novel polyamine. The chemical structure of the new polyamine was determined to be NH(CH)NH(CH)NH(4-aminobutylcadaverine) based on gas chromatography-mass spectrometry. The occurrence of 4-aminobutylcadaverine was specific to the root nodules, since the unusual triamine was not detected in other organs of the adzuki bean plant. Bacteroids, isolated from root nodules, contained both sym-homospermidine and 4-aminobutylcadaverine, whereas the plant cytosol fraction contained large quantities of putrescine and cadaverine. A cell-free extract of bacteroids showed the ability to form this triamine from putrescine and cadaverine under the presence of NADand K. 1,3-Diaminopropane and NADH were inhibitory for the synthesis of both sym-homospermidine and 4-aminobutylcadaverine. [1,4-N]Putrescine was incorporated not only into sym-homospermidine but also into 4-aminobutylcadaverine by the cell-free extract of bacteroids when incubated with excess cadaverine. Analysis of the fragment ion peaks in the N-enriched 4-aminobutylcadaverine indicated the transfer of a aminobutyl moiety to the amino terminus of cadaverine. These results suggest that, in adzuki bean, 4-aminobutylcadaverine is formed through the action of homospermidine synthase in nodule bacteroids under a cadaverine-rich environment.


INTRODUCTION

The polyamines, putrescine (Put),() spermidine (Spd), and spermine (Spm) are ubiquitously found in higher cells. Their physiological and biochemical significance in the process of cell growth and differentiation has been repeatedly reviewed (1, 2, 3) . During the last two decades, a number of unusual polyamines including long chain and branched polyamines have been found in a variety of organisms (4, 5, 6, 7) . Recently, we investigated the distributions of polyamines in various root or stem nodules collected from a variety of leguminous and nonleguminous plants (8) . During the course of the analyses of polyamines by high performance liquid chromatography (HPLC), we encountered the presence of several unknown peaks on the elution chromatograms of the extracts from various Bradyrhizobium-infected legume root nodules. These compounds, like polyamines, were highly cationic in nature, being strongly adsorbed on a Dowex 50W-X8 column, and one of these organic cations was found to be specifically present in root nodules of adzuki bean ( Vigna angularis). From the structural analysis based on gas chromatography-mass spectrometry (GC-MS), it was identified as a new polyamine, 4-aminobutylcadaverine (4-ABcad), which has not yet been recognized in any of the living systems so far. We describe here the data showing a natural occurrence of 4-ABcad in adzuki bean and provide the evidence that bacteroids, isolated from root nodules, or Bradyrhizobium japonicum, which establishes a symbiotic relationship with adzuki bean plant, has an ability to synthesize this unusual polyamine under the cadaverine (Cad)-rich environment. Based on the results from in vitro experiment using cell-free extract of isolated bacteroids and from a N tracer experiment, a possible mechanism for the biosynthesis of 4-ABcad in adzuki bean root nodule has also been presented.


MATERIALS AND METHODS

Plants and Microbial Growth

Seeds of adzuki bean plant ( V. angularis cv. Erimoshoudzu, cv. Kamuidainagon, cv. Tsurugi-3, and var. nipponennsis ACC1238) were obtained from Hokkaido Prefectural Tokachi Agricultural Experiment Station, Japan. The seeds were sown and grown in a field of National Agriculture Research Center at Tsukuba, Japan. Plants were harvested during the vegetative to early reproductive stage, separated into leaf, stem, root, and nodule, and stored below 30 °C until use. B. japonicum A1017 (MAFF303005), obtained from the Laboratory of Soil General Microbiology (National Institute of Agro-Environmental Sciences, Tsukuba, Japan), was transferred into a liquid medium (pH 6.4) containing 0.06% yeast extract (Difco), 0.5% KHPO, 0.02% MgSO7HO, 0.01% NaCl, and 0.5% sucrose, and grown aerobically in the presence or absence of 1 mM Cad or 2 mM lysine. The cells were harvested at the early stationary phase of growth. Nodule formation on the roots of adzuki bean with B. japonicum A1017 was ascertained by the inoculation test under sterile growth condition.

Preparation of Bacteroids and Plant Cytosol Fraction from Root Nodules

Frozen nodules were gently crushed with a pestle and mortar in a cold grinding medium (9) . After removal of nodule debris by passage through four layers of cotton gauze, the filtrate was centrifuged at 300 g for 4 min. The resulting supernatant fluid was centrifuged at 5,000 g for 10 min. The pellet was washed twice with the grinding medium to give the final bacteroid fraction. The supernatant was centrifuged at 16,000 g for 30 min, and the resulting clear supernatant was referred to as the plant cytosol fraction. Protein content in each fraction was determined by the method of Bradford (10) using bovine serum albumin as a standard.

Analysis of Polyamines by HPLC, Capillary GC, and GC-MS

Polyamines in plant tissues or bacterial cells were extracted with 0.5 M perchloric acid and purified as described previously (11) . Quantitative analysis of polyamines was performed with HPLC (Shimadzu LC-9A) equipped with a reversed phase column (Shim-pack CLC-ODS, 6 150 mm) after derivatization with benzoyl chloride (12) . Diaminohexane was used as an internal standard. Polyamines, after derivatization with pentafluoropropionic (PFP) anhydride (13) , were also analyzed with acapillary gas chromatography (Shimadzu GC-9A) fitted with a split injector and a flame ionization detector. For the structural analysis of the unknown compound and for the detection of N incorporation into the molecules, GC-MS was performed using a quadrapole GC-mass spectrometer (Shimadzu QP-5000) equipped with a fused silica capillary column (CBJ1-M30-025; 30 m 0.25 mm inside diameter, 0.25 µm film thickness). Electron impact mass spectra were obtained under the following GC-MS conditions: injection mode and temperature, splitless (sampling time 0.5 min) at 300 °C; initial column temperature, 50 °C (2 min); temperature program, 5 °C min; final column temperature, 270 °C (2 min); carrier gas flow rate, helium 1.1 ml min; ion source temperature, 300 °C; ionizing energy, 70 eV.

Assay of Homospd Synthase and 4-ABcad Producing Activity

Cell-free extract of bacteroids and plant cytosol fraction were used for the detection of Homospd synthase and 4-ABcad producing activities. The bacteroids were suspended in 10 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 20 mM 2-mercaptoethanol, and 0.05% heparin sodium, sonicated in an ice bath, and centrifuged at 16,000 g for 30 min. For complete removal of the cellular polyamines, the supernatant obtained was dialyzed against 500 volumes of 10 mM sodium phosphate buffer, pH 7.0, containing 0.5 mM EDTA and 10 mM 2-mercaptoethanol with two changes for 10-16 h. The plant cytosol fraction was also dialyzed against the same buffer. The reaction mixture generally contained the following constituents in a final volume of 1.0 ml: 50 mM Tris-HCl, pH 8.7, 50 mM KCl, 2 mM dithiothreitol, 0.5 mM NAD, and dialyzed cell-free extract of bacteroids or plant cytosol fraction (0.9-2.3 mg of protein). The reaction was started by the addition of the substrate (Put or Cad, or both compounds) to the reaction mixture at 30 °C and terminated by the addition of 0.1 ml of 20% PCA. After centrifugation, an aliquot of the acid extract was subjected to the quantitative analysis of Homospd and 4-ABcad by HPLC. The reaction mixture without substrate was also incubated at 30 °C as a blank.

N Experiment

Cell-free extract of bacteroids was prepared from 20 g (fresh weight) of frozen nodules, dialyzed as above, and served for the N experiment. [1,4-N]Put (99% N), in which two terminal amino groups of Put were labeled with N, and Cad were added to the reaction mixture as substrates at final concentrations of 0.5 mM and 5 mM, respectively. The reaction mixture (final volume 20 ml) contained 50 mM Tris-HCl, pH 8.7, 50 mM KCl, 5 mM NAD, 2 mM dithiothreitol, and a dialyzed cell-free extract of bacteroids equivalent to 8.66 mg of protein. The reaction medium was incubated at 30 °C with gentle shaking for 15 h, and the reaction was terminated by the addition of 2 ml of 20% PCA. After precipitation of the protein by centrifugation, the supernatant was neutralized with 10 M KOH and applied to a Dowex 50W-X8 column (Hform, 200-400 mesh, 0.9 4 cm). After washing the column with 100 ml of distilled water and 50 ml of 0.5 M HCl, the polyamines were eluted with 15 ml of 6 M HCl, evaporated to dryness, converted to their volatile derivatives by reaction with PFP reagent (13) , and subjected to the GC-MS analysis.

Chemicals

Homospd and 3-aminopropylcadaverine (3-APcad) were chemically synthesized according to the method of Okada et al. (14) . Authentic 4-ABcad and 5-aminopentylcadaverine (5-APcad) were the generous gift of Dr. S. Yamamoto (Okayama University, Japan). [1,4-N]Put, NH(CH)NH, was chemically synthesized from potassium [N]phthalimide (99% N; Shoko, Tokyo) and 1,4-dibromobutane as starting materials, and its hydrochloride salt was crystallized from 90% ethanol as described previously in detail (15) . Other polyamines and organic and inorganic chemicals were obtained from commercial sources.


RESULTS

Occurrence of Unknown Compound X in Root Nodules of Adzuki Bean

Root nodules of adzuki bean ( V. angularis) contained large quantities of Put, Cad, and Homospd besides small quantities of Spd and Spm. Fig. 1shows a typical elution chromatogram of nodule polyamines in HPLC analysis. Generally, two or more unknown peaks, which could not be assigned to any of the standard polyamines, appeared between Put and Spm on the elution chromatograms. These compounds were resistant to acid hydrolysis, since each peak height was not changed even though the acid extracts of root nodules were hydrolyzed with 6 M HCl at 100 °C prior to the derivatization procedure. Among these peaks, the peak posterior to Homospd (denoted as X in the figure) was detected only in adzuki bean plant while other peaks were found in the chromatograms of many other legume root nodules as well. shows distribution of polyamines in leaf, stem, root, and nodule separated from various adzuki bean plants (one variety and three cultivars). The peak X in adzuki bean was found to occur specifically in root nodules and the unusual peaks were not detected in other organs such as leaf, stem, or root. A wild type adzuki bean plant (var. nipponennsis ACC1238), which is a putative wild ancestor of the cultivated adzuki bean, showed apparently distinct morphology from other adzuki bean cultivars, e.g. grain size and leaf and stem shape. A characteristic pattern of polyamine composition was observed in the root nodules of this wild progenitor species of adzuki bean, showing a very low Cad content. The peak X was not detected in the root nodules of the wild type adzuki bean.


Figure 1: Elution profiles of polyamines in adzuki bean root nodules ( A) and standard polyamines ( B). Root nodules were collected from a field-grown adzuki bean plant ( V. angularis cv. Erimoshoudzu) at 46 days after sowing. Polyamines were extracted from the freeze-dried sample with 0.5 M PCA, purified with Dowex 50W-X8 column, derivatized with benzoylchloride, and subjected to the HPLC analysis. Diaminohexane was used as an internal standard. Peak X in Panel A denotes unknown compound. Peaks in Panel B: 1, Put; 2, Cad; 3, internal standard (diaminohexane); 4, Spd; 5, Homospd; 6, Spm.



Chemical Structure of the Unknown Compound X

To determine the chemical structure, the unknown compound X in the nodule acid extract was separated from other polyamines by ion-exchange chromatography, and the pooled fraction containing compound X was then subjected to the HPLC, capillary GC, and GC-MS analysis. Both in HPLC and capillary GC analysis, the retention time of compound X coincided with that of authentic 4-ABcad, NH(CH)NH(CH)NH: the relative retention time against the internal standard (diaminohexane) was 1.42 in HPLC and 2.01 in capillary GC. The evidence of identity was further established by GC-MS. Fig. 2shows the data comparing the mass spectrum of the PFP derivative of compound X with those of chemically synthesized 4-ABcad and Homospd. Under electron impact, the mass fragmentation pattern of the compound X (Fig. 2 C) was identical with that of authentic 4-ABcad (Fig. 2 B). Very weak molecular ion (M) of PFP derivative of the compound X or synthetic 4-ABcad was obtained at m/e 611 while that of Homospd (Fig. 2 A) was at m/e 597. The loss of CFand CFCO from the parent molecules gave intense fragment ions both at m/e 492 and m/e 464, respectively. A characteristic fragment ion peak of 4-ABcad, which was not obtained in the mass fragment of Homospd, was observed at m/e 407, due to the presence of NH(CH)NHCH- moiety in the molecules.


Figure 2: Mass spectra of PFP derivatives of Homospd ( A), 4-ABcad ( B) and unknown compound X ( C). Chemically synthesized Homospd and 4-ABcad and unknown compound X were converted to their volatile derivatives with PFP anhydride as described in Flores and Galston (12). Chemical structures and possible split regions assigned from the fragment ion peaks are also shown.



Localization of 4-ABcad within Root Nodule Tissues

In order to know the localization of this unusual triamine within the root nodule tissues, root nodules harvested from the 64-day-old adzuki bean plant were divided into two fractions, bacteroids (endosymbionts) and a plant cytosol fraction. Bacteroids contained 4-ABcad besides small quantities of Put and Cad and a large quantity of Homospd, while plant cytosol fraction contained large quantities of Put and Cad (). Since both Spd and Spm, characteristic polyamines of the host plant, were not detected in the bacteroid fraction, contamination of plant cells to the bacteroid fraction seems unlikely. By contrast, small quantities of 4-ABcad and Homospd were found also in plant cytosol fraction, possibly due to the leakage from bacteroids or rupture of bacteroid cells during the fractionation procedure, since bacteroids, having a grossly swollen cell structure within root nodules, are fragile as compared with cultured rhizobial cells (16) .

4-ABcad Formation in B. japonicum

To examine whether the endosymbiont of adzuki bean root nodules participates in the formation of 4-ABcad, the ability of B. japonicum A1017, which forms root nodules on the roots of adzuki bean plant, to produce this unusual polyamine was investigated. It was found that 4-ABcad was detected only in the cells grown in the culture supplemented Cad (data not shown). Addition of lysine into the growth medium was not effective on the production of Cad nor 4-ABcad, suggesting the lack or very low activity of lysine decarboxylase in B. japonicum A1017.

Biosynthesis of 4-ABcad by Cell-free Extract of Bacteroids

To confirm the formation of 4-ABcad by bacteroids, an in vitro experiment was carried out using a cell-free extract of bacteroid cells, in which cellular polyamines were completely removed by dialysis (I). Addition of Put to the reaction mixture including NADand Kresulted in the production of Homospd. Cad was inhibitive for the synthesis of Homospd, but 4-ABcad was formed under the presence of both Put and Cad if Cad was present sufficiently against Put. The peak correspondent to 5-APcad was not found during this experiment even if Cad was used as a sole substrate. Removal of NADor Kfrom the reaction mixture significantly reduced the formation not only of Homospd but also of 4-ABcad. Furthermore, addition of NADH or 1,3-diaminopropane, which inhibited Homospd formation, showed strong repression of 4-ABcad formation as well, implying the involvement of Homospd synthase on the synthesis of 4-ABcad. The polyamines having aminopropyl group in the molecules, such as Spd, norspermidine (Norspd), and 3-APcad, were not produced by the addition of diaminopropane with Put and Cad. The same experiment was also done with dialyzed plant cytosol fraction, but neither Homospd nor 4-ABcad was produced by the plant cytosol fraction. Incorporation of [N]Put into 4-ABcad-In order to know the molecular mechanism for biosynthesis of 4-ABcad in nodule bacteroids, a N tracer experiment was also carried out (Fig. 3). [1,4-N]Put was added into the dialyzed cell-free extract of bacteroids with excess amount of Cad. The reaction mixture including NADand Kwas incubated overnight to obtain sufficient amount of the reaction products necessary for the analysis of GC-MS. Fig. 3A shows the total ion monitoring of the sample after derivatization of polyamines with PFP reagent. Both Homospd and 4-ABcad were detected as reaction products, and no other products appeared on the elution chromatogram. The positions of N incorporated into the reaction products were examined by comparing their fragment ion peaks under electron impact with those of the authentic samples without N shown in Fig. 2. In Homospd, relatively intense fragment ion peaks observed at m/e 450 and m/e 478 (Fig. 2 A) shifted at m/e 453 and m/e 481, respectively (Fig. 3 B), indicating that N was incorporated into all of the positions of amino and imino groups in the Homospd molecules. On the other hand, in 4-ABcad, intense fragment ion peaks at m/e 464 and m/e 492, due to the loss of CFor CFCO from the parent molecules (Fig. 2, B or C), shifted at m/e 465 and m/e 493, respectively (Fig. 3 C). The ion peak at m/e 393, derived from -CHNH(CH)NHgroup, also showed a shift by 1 mass unit ( m/e 394), whereas the ion peak at m/e 407, derived from NH(CH)NHCH- group, remained at m/e 407. The data strongly suggest that 4-ABcad is formed through the transfer of the aminobutyl moiety to the amino terminus of Cad and not through the transfer of aminopentyl moiety to the amino terminus of Put.


Figure 3: Incorporation of [N]Put into Homospd and 4-ABcad by cell-free extract of bacteroids. [1,4-N]Put (99% N) and Cad were added at final concentrations of 0.5 mM and 5 mM, respectively, to the reaction mixture including 50 mM Tris-HCl, pH 8.7, 50 mM KCl, 5 mM NAD, 2 mM dithiothreitol, and dialyzed cell-free extract of bacteroids (8.66 mg of protein) in a final volume of 20 ml and incubated at 30 °C with gentle shaking. After 15 h, polyamines were extracted, purified, derivatized, and subjected to the GC-MS analysis. Total ion monitoring of the sample during separation with capillary GC column ( A), mass spectra of PFP derivatives of the reaction products Homospd ( B), and 4-ABcad ( C) are shown.




DISCUSSION

Since the occurrence of unusual polyamines in the extreme thermophile, Thermus thermophilus, and their possible functional roles in the thermostability of such microorganism has been reported (17, 18, 19, 20) , many researchers have attempted to find novel polyamines in various living systems. As a result of a broad survey, a number of uncommon polyamines including long chain and branched polyamines have been found from a variety of natural sources (see the tables of Refs. 5 or 7). With regard to triamines, except for commonly found Spd, only three polyamines have been known to occur naturally, i.e. Norspd, Homospd, and 3-APcad. The present investigation adds a new triamine, 4-ABcad, to the list of natural polyamines. In contrast to Norspd or Homospd, which show relatively widespread distributions from microorganisms to higher plants and animals, 3-APcad has been recognized only in specific cells, such as polyamine-dependent mutants of Esherichia coli (21) or Neurospora crassa (22) , and polyamine-depleted animal cells pretreated with an inhibitor of polyamine biosynthesis (23, 24) . As is the case for 3-APcad, the occurrence of 4-ABcad appears to be very specific, since this triamine was detected exclusively in adzuki bean root nodules and not in other root or stem nodules tested (22 species of legumes and five species of non-legumes). The adzuki bean plant was characterized by its high Cad content within the root nodules differing from other legumes (8) . In the wild progenitor species of adzuki bean (ACC 1238), in which root nodules contained very low Cad, 4-ABcad was not present (). Together with the fact that B. japonicum A1017 cells produced 4-ABcad only in the growth medium supplemented with Cad (data not shown), a Cad-rich environment is found to be necessary for the production of this novel polyamine.

In E. coli, Cad is inducibly formed (25) when grown on medium containing lysine, a precursor for Cad synthesis. B. japonicum A1017, however, showed no ability to form Cad even though lysine was added at 2 mM into the growth medium. Our previous study (11, 26) , and analytical data of other investigators (27) also indicated that growing cells from various Bradyrhizobium and Rhizobium species produced little Cad as compared with Put and Homospd, implying the lack or very low activity of lysine decarboxylase in Rhizobiaceae. The data of systematic analyses of polyamine pattern in proteobacteria (28) are of special interest because Cad was rarely detected in the groups containing Homospd as a major polyamine. These facts probably explain why 4-ABcad has not been detected so far in spite of the extensive survey of microbial polyamines. In this regard, 4-ABcad is considered to be an unusual polyamine occurring in rhizobial cells under a specific environment.

Legume root nodules are specially differentiated complex tissues, which consist of bacteroids (symbiotic form of rhizobial cells) and surrounding host legume cells as a result of plant-microbe symbiotic associations (29) . After invasion into the host plant cells, rhizobia differentiate to become bacteroids which can fix atmospheric nitrogen (16, 29) . In adzuki bean root nodules, large quantities of Cad were detected in host plant cells but not in bacteroids, whereas most of the Homospd and 4-ABcad were localized in bacteroids (). The results from in vitro experiment (I) suggested that Homospd synthase, detected in bacteroids but not in plant cytosol fraction, was a responsible enzyme for 4-ABcad synthesis since 1) NADand K, necessary for the full activity of Homospd synthase (30, 31, 32, 33) , were required for the synthesis of 4-ABcad as well, and 2) diaminopropane and NADH, potent inhibitors of Homospd synthase (30, 32, 33) , repressed 4-ABcad formation.

A variety of leguminous plants usually contain copper-containing diamine oxidases which can oxidize both Put and Cad (34, 35, 36, 37, 38) . Aminoaldehydes are generally known to occur as an intermediate in the oxidation process of polyamines (39) , and the enzymes related to polyamine catabolism have been detected in various living systems (40, 41) . These facts offer an another possibility that aminoaldehydes resulting from the oxidative deamination of Put and Cad by diamine oxidase might have also been utilized for 4-ABcad synthesis via the Shiff-base formation between aminoaldehydes and amino-terminal of diamines. The involvement of diamine oxidase in the synthesis of 4-ABcad, however, seems unlikely because a N experiment (Fig. 3) clearly indicated that the transfer of aminopentyl group, produced by Cad oxidation, to the amino terminus of Put molecules did not occur. We recently detected the activity of ornithine decarboxylase in bacteroids (data not shown), implying Put formation from ornithine as a substrate for Homospd synthase within bacteroid tissues. In conclusion, as to the occurrence of 4-ABcad within the adzuki bean root nodules, the present data suggest that this novel polyamine is formed through a reaction sequence mediated by Homospd synthase in bacteroid tissues utilizing Cad supplied from the host legume cells as illustrated in Fig. 4. The recent report which describes enzymatic formation of 4-ABcad by a purified Homospd synthase from Acinetobacter tartarogenes (33) might support the present hypothesis. It is of interest why and how the Cad is produced abundantly in adzuki bean root nodules differing from other legume nodules. Together with the physiological traits of adzuki bean, these are the problems to be elucidated in the future.


Figure 4: Possible mechanism for the biosynthesis of 4-ABcad in adzuki bean root nodules. 4-ABcad is synthesized from putrescine and cadaverine supplied from host plant cells under the presence of NADby the action of Homospd synthase in bacteroid cells. NADH, produced by the first step reaction (Put oxidation), is recycled for the subsequent reduction of the putative intermediate into 4-ABcad.



  
Table: Polyamine composition in various organs of field-grown adzuki bean plants


  
Table: Polyamines in bacteroids and plant cytosol fraction separated from root nodules

Bacteroids and plant cytosol fraction were separated from the root nodules harvested from 67 days-old adzuki bean plant as described under ``Materials and Methods.'' An aliquot of each fraction was extracted with 0.5 M PCA and analyzed for polyamines. Another aliquot was served for the determination of protein content.


  
Table: Synthesis of Homospd and 4-ABcad by cell-free extract of bacteroids

The cell-free extract of bacteroids, which was thoroughly dialyzed for removal of cellular polyamines, was employed for this assay. The complete reaction mixture contained 50 mM Tris-HCl buffer, pH 8.7, 50 mM KCl, 0.5 mM NAD, 2 mM dithiothreitol, and dialyzed cell-free extract of bacteroids (2.3 mg of protein) in a final volume of 1.0 ml. The reaction was started by the addition of substrate (Put or Cad, or both), and incubated at 30 °C for 2 h. Quantitative analyses of the reaction products were performed with HPLC. The averages of duplicate determinations are shown.



FOOTNOTES

*
This study was supported by a research grant from the Bio-Media Program of the Ministry of Agriculture, Forestry and Fisheries of Japan (BMP 94-V-1-(3)-10). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Shikoku National Agricultural Experiment Station, Senyu-cho 1-3, Zentsuji, 765 Japan. Tel.: 81-877-62-0800; Fax: 81-877-63-1683.

The abbreviations used are: Put, putrescine; Cad, cadaverine; Spd, spermidine; Spm, spermine; Homospd, sym-homospermidine; 4-ABcad, 4-aminobutylcadaverine; 3-APcad, 3-aminopropylcadaverine; 5-APcad, 5-aminopentylcadaverine; Norspd, norspermidine; HPLC, high performance liquid chromatography; GC, gas chromatography; MS, mass spectrometry; PFP, pentafluoropropionic; PCA, perchloric acid.


ACKNOWLEDGEMENTS

We thank Dr. S. Yamamoto (Okayama University, Japan) for kindly providing authentic 4-ABcad and 5-APcad, and S. Hiradate (NIAES, Japan) for his assistance in GC-MS analysis. We are also grateful to Dr. Mark Taylor (Scottish Crop Research Institute, UK) for helpful comments and critical reading of the manuscript.


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