1-N-acetylspermidine in roots of maize seedlings

ELSEVIER

Plant Science 111 (IY96)

1-IV-Acetylspermidine Marina

18

13Y

in roots of maize seedlings

de Agazio”%*, Stefano Grego”, Luciano Cellai’, Simona

Rrccibcd

133

Massimo Zacchini”, Fabrizio Rizea-Savud, Luigi Silvestro”

July 1906: reciscd IO Scptemher

1996: accepted

I6

De Cesare”.

September IYYh

Abstract The presence of l-iY-acetylspermidine was revealed in root tissues of Z-d-old maize seedlings by inhibiting in vivo polyamine oxidase activity with acetylspermine added to the growth solution. The identity of I-.V-~etylspermidine “C-spermidine added to I mM spermidine in presence or absence of I was confirmed by HPLC-mass spectrometry. Results indicate that acetyhpermidine is not mM acetylspermine did not lead to the synthesis of ‘T-acetylspermidine. involved in the spermidine-to-putrescine interconversion pathway and exogenous spermidine cannot be the substrate for the acetylation process. It is suggested that acetylation occurs in a subcellular compartment not permeable to exogenous spermidine. Copyright 0 1996 Elsevier Science Ireland Ltd &,~~IIw~/.v: I -,Y-acetylspermidine;

Polyamines:

Polyamine

interconversion

pathway:

Roots:

Zea

mays

1. Introduction

.~hh~l,r.nrliclrr.\:ABAI. acctylspcrmidint: P40. Spm

polyaminc

;,-amj,,obutyraldehyde: Ac-spm. acetylspermine; PA. oxydase: Put. putrescine; Spd.

Ac-spd. polyamines spermidine;

spermine.

* <‘orresponding 0064497:

author.

Tel.:

+ 39 6 90671534;

f;lr: + 39 6

e-mail deagiwnser\.icmat.mlib.cnr.it

0167.9452.96,$15.00

Copyright

1’11 SOlhX-Y452(96)045’~-2

‘Cl 1996 Else&r

Science Ireland

Polyamines (PA) are ubiquitous polycations present in near-millimolar concentrations in most living cells. In mammalian cells metabolism of PA is highly regulated and their level is closely linked to the rate of cell growth [I]. Putrescine (put) is formed via ornithine decarboxylation by ornithine decarboxy&e and higher polyamines are formed from Ltd. All rights t-cwrced

putrescine by the addition of aminopropyl groups from S-adenosylmethionine. The addition of a single aminopropyl group to put yields spermidine (spd), and the transfer of a second aminopropyl group to spermidine yields spermine (spm) [2]. The interconversion of spm into spd, and of spd into put, can occur in animal cells, but it does not take place by reversal of the aminopropyl transferase reactions responsible for their synthesis [3]. The acetylation is an integral step in polyamine interconversion reactions as first proposed in the early 1980s by Seiler and co-workers [4]. The interconversion is mediated by two enzymes, spermidine/spermine 1-N-acetyltransferase (SAT) and polyamine oxidase (PAO) (Fig. 1). The acetylated polyamines are rapidly degraded by PA0 yielding spermidine or putrescine, respectively, and acetylaminopropanal. Detailed studies of the specificity, kinetics and active site of SAT revealed that the enzyme forms only 1-N-acetylspermidine; 8N-acetyl spermidine is known to be formed from spd only by a nuclear acetyl transferase [l]. In general, the SAT activity is much lower than that of polyamine oxidase and the cellular concentration of acetylated polyamine derivatives is very low because of their rapid degradation [5]. The inhibition of PA0 by specific inhibitors, or administration of exogenous PA induced the accumulation of I-N-acetylspermidine [3,6]. In higher plants, polyamines are involved in many processes such as response to stress [7,8], senescence [9], regulation of cell cycle [lO,ll], embryogenesis in tissue culture [I?] and floral initiation [ 131. Put is formed directly by decarboxylation of L-ornithine in a reaction catalyzed by ornithine decarboxylase. Alternatively. the decarboxylation of L-arginine by arginine decarboxylase leads to put, via agmatine and N-carbamoylputrescine as intermediates. Higher polyamines are synthesized from put by addition of aminopropyl groups as described for animal cells. Spd-to-put interconversion has been recently suggested in tobacco thin-layer explants [l4], and in chloroplast of Hrliuntkus tubema [15]. In maize seedlings, a dramatic increase in the level of endogenous put is observed in the primary roots after exposure to 1 mM spd [ 16,171. When 1 mM 13C spermidine was added to the growth medium,

endogenous put contained 44% of the total “C detected in the dansylated fraction. These results substantiate the existence of an interconversion pathway producing put from spd, but the biochemical steps remain unknown in plants. Acetylated polyamines have never been found in plant tissues, but a PA acetylating activity has been reported in chloroplasts of H~~litrntln~stuhwuwr [15]. It is possible that the acetylspermidine level. under physiological conditions, is too low to be detected. possibly due to a rapid turnover as reported in animal tissues [l8]. The goals of OUI paper are: (i) to reveal the presence of endogenous ac-spd in an extract of maize roots; (ii) to ascertain if acetylation is an integral step in polyamine interconversion reactions.

NHz(CQ) WWCH2)4WCH2) 3NHz spermine

acetyltransferase n

V CH$ONH(CHz)

~NH(CH~)LJNH(CH~)

3NHz

acetylspermme

polyamine + 02,+

oxidase CH3CONH(CHz)

Hz0

2CHO

+ Hz@

acetylaminopropanal

W(CK!)

3NH(W’)dW srxrmidlne

Nl -acetyltransferase

d CH3CONH(CH2)

sNH(CH2)

4NHz

Nl -acetylspermidme

polyamme + 02.

oxidase

CH$ONH(CHz)

2010

+

HZO;I

acetylaminopropanal

+ Hz0

NQ(CH2)4NHz putresane

Fig. I. Polyamine

interconversion

pathway

in animal cells.

2. Materials and methods

2.3. ‘T-Spermidine

-7.I. Plunt muteri&

Two-day-old maize seedlings were transferred to a fresh growth solution (20 seedlings of each sample in 45 ml of 0.5 mM CaSO, solution), containing 1 mM spd labelled with 5 x lo5 dpmi ml “C-spd, (Amersham, specific activity 3.7 GBq/ m mol) with or without 1 mM ac-spm. PA were extracted, dansylated and separated by TLC as reported above. Bands of 1 cm each were scraped off and radioactivity measured in a Packard tricarb liquid scintillation counter.

Maize (Zeu mu_tqs L., hybrid line Plenus V 5 16) by Dekalb Center caryopses were supplied (Chiarano. Italy). Seeds were continuously rinsed overnight under tap water and then germinated on wet filter paper in a 0.5 mM CaSO, solution, at 27°C in the dark in a controlled growth chamber. After 2 days, seedlings were transferred to a fresh solution in the presence or absence of 1 mM ac-spm for 18 h (unless otherwise stated). After 18 h. control and treated primary roots were harvested and analysed. Ac-spm and the other PA were purchased by Sigma. .?._?. Anulvsis of Polyumine content PA were extracted by grinding 1 cm apical segments of primary roots from control or treated maize seedlings in 5 volumes of ice-cold 5% (w/v) HClO, using a prechilled mortar and pestle. The homogenates were kept in ice for 1 h and then centrifuged at 15,000 x g for 10 min and the supernatants are set aside for detection of free PA. To a 0.18 ml aliquot of HClO, extract 0.35 ml dansyl-chloride (5 mg/ml acetone solution) were added in the presence of an excess of Na,CO, to adjust the pH to pH 7.0. After overnight incubation in the dark at 26°C. excess dansyl chloride was converted to dansyl proline by a 30 min incubation with 0.09 ml L-proline (100 mg/ml aqueous solution); dansylated PA were then extracted in 0.35 ml ethylacetate. Aliquots of ethylacetate extracts were spotted on TLC plates precoated with 0.25 mm silica-gel with fluorescent indicator F-254 (Merck) and co-chromatographed with dansylated PA standards processed in the same way. Plates were developed with chloroform: triethylamine: H,O (16:4:1 v/v/v). The spots. visualized by UV fluorescence, were identified by comparing to standards and their relative fluorescence intensities were measured by Shimadzu CS 9000 flying-spot scanner with fluorimetry attachment.

experiments

Aliquots of polyamines extracted from roots of 18 h ac-spm + spd-treated maize seedlings were dansylated and injected into a Perkin Elmer Series 3B liquid chromatograph equipped with a Waters U6-K injection valve, a Perkin Elmer LC-95 UV recorder and a Perkin Elmer CC 12 integrator. The HPLC column was a RP 18 Lichrocart Merck, 150 x 4 mm, mesh 5 pm. The solvent system was acetonitrile/methano1/50 mM sodium phosphate, pH 3.5 (1: 1:l v!viv) at a flow rate of 1 m1;‘min and absorbance of the eluate was monitored at 360 nm. The presence of different polyamines and their acetyl derivatives is referred to standards both as retention volumes and by the addition of internal reference compounds. The presence of dansyl-ac-spd in the extracts was confirmed by injecting an aliquot of the extract into the HPLC system. using the same column as above, interfaced with a Perkin-Elmer SCIEX API III ion spray mass spectrometer. -3.5. Characterkltion

of’ ~iu~2.Yl’l-uc~~t~‘lspernlir~iinc

standurds Twenty mg of commercial standards of I-NAc-spd and 8-N-ac-spd purchased from Sigma (USA) are separately derivatised by the previously described dansylchloride conjugation. The ethylacetate phases were recovered and dried under vacuum (N, flux), then resuspended in 5 ml chloroform and chromatographed on a silica gel column (2.5 x 10 cm. 60 pm particle size) using

146

M. de Agazio rr al. /Plant Science IZI (1996) 143-149

absolute chloroform as eluant for cleaning standards from additional impurities and then chloroform:methanol (95:5) as standard eluent. The two standard eluates (l-N- and 8-N-ac-spd) are dried under vacuum (NZ flux). The final solutions were filtered through 45 pm teflon syringe filters and a 10 ~1 aliquot of each standard was separately injected in a HPLC system connected to a UV spectrophotometer set at 360 nm. The two acpolyamine derivatives were identified by retention volumes with respect to other polyamine-derivative standards and to each other. HPLC-MS analysis was carried on also for the standards. 2.6. Polyanzine

oxidase

activity

amu)’

Root segments were homogenized in a mortar at 4°C with 3 volumes of 0.5 M phosphate (K+ salt, pH 7). The suspension was filtered through a nylon cloth and centrifuged at 15 000 x g for 10 min. Polyamine oxidase (PAO) activity is determined in the supernatant using an oxygraph equipped with a Clark electrode (Hansatech, King’s Lynn, Norfolk, UK) as previously described [19]. A unit of enzymatic activity represents the amount of enzyme catalysing the oxidation of 1 pmol substrate/min. Data reported in the tables and figures refer to single typical experiments. At least three series of independent experiments were carried out giving reproducible results. Variability among the experiments did not exceed 10%.

3. Results and discussion 3.1. Identification

of acetylspemidine

Two-days-old maize seedlings were treated for 18h with 1 mM ac-spm, a non-competitive inhibitor of in vitro PA0 activity in maize shoots [20], added alone or in combination with 1 mM spd, to allow acetylspermidine to accumulate, as reported in animal cells where ac-spd was detected by inhibiting PA0 activity or administration of exogenous PA [3,6]. TLC analysis of PA extracted from roots, treated with ac-spm + or - spd, and then dansylated, revealed the appearance of a

Fig. 2. HPLC analysis of dansylated polyamines. Polyamines were extracted from roots of Z-day-old maize seedlings treated for 18 h with I mM acetylspermine+ I mM spermidine and dansylated. The peaks corresponding to AcSPD = I-X-acetyl4,8-N’,N”-didansylspermidine; DAP = 1,3-fV,N’-didansyl-diaminopropane; PUT = I ,4-N.N’-didansylputrescine; AcSPM = I-N-acetyl-4.8.1 I tridansylspermine and SPD = 1,4,8-N.N’,.V”tridansylspermidine, were identified by adding to the mixture aliquots of the corresponding synthetic standards.

spot with the same Rf as that of dansylated commercial I-N-ac-spd while it was completely absent from control roots. The HPLC chromatogram of the dansylated extract consisted of several peaks, one of which having the same retention volume (9.6 ml) as a synthetic standard of I-N-acetyl,4,8-N’,N”-didansylspermidine (Fig. 2). In order to confirm the identity of the two metabolites, the dansylated extract was analysed by HPLC-MS using the same column and solvent system utilised for HPLC analysis, but replacing the phosphate buffer with an ammonium acetate buffer at the same concentration. The substitution of the phosphate salt with the volatile ammonium acetate did not affect the chromatographic profile of the dansylated extract (data not shown). The MS analysis, carried out in the MS-MS mode, clearly revealed that the peak having the same retention volume as the standard of 1-N-acetyl4,8-N’,N”-didansylspermidine, (MW: 654), gave also the same MS spectrum (Fig. 3a). In order to assess whether the dansylated extract also con-

M. de Agazio

rt al., Plant Scierxe

l-71 (1996) 143-149

147

0 b

362

O-N-AcetyC1,4-N.N’-di-dansylspermidine

[Wl:NOl+ 11.l I

7s

E

[C~H,oNOl+

403 1X

I’GH~IN@~SZ+~I+ (I?

I

541

I

I/

l-N-Acclyl-~,D-N,N’-di-d,,nsylsperlnidinc

100

E mz CL

Fig. 3. MS-MS spectrum of (a) the HPLC peak corresponding to I-N-acetyl-4.8-N’,IV”-didansylspermidine present in the dansylated polyamine extract of Z-day-old maize seedlings treated for 18 h with 1 mM acetylspermine + I mM spermidine. The spectrum is identical to that of its synthetic standard and different from the spectrum (b) of the synthetic standard of 8..Y-acetyl-1,4-Y’..~“‘-didansylspermidine. _

tained 8-N-acetyl-1,4-N’,N”-didansylspermidine, this compound was synthesised under standard conditions. The HPLC analysis revealed a slightly lower retention volume than for the I-N-acetyl4.8-N’,N”-didansylspermidine standard. but the two compounds are indistinguishable from one another when injected as a mixture. Therefore the presence of 8-N-acetyl-4,8-N’,N”-didansylspermidine in the extract could not be excluded on the basis of the HPLC analysis only. However, in the comparison of the MS-MS spectra of both acetyldidansyl derivatives, I-N-acetyl-4,8-N’,N”-didansylspermidine was fragmented leading to a couple of peaks of m/z 100 (C,H,,,NO+ ) and 555 (C2,H,,N,0&+ + l), (Fig. 3a), while the 8-Nacetyl-4,8-N’,N”-didansylspermidine gave a corresponding couple of m/z 114 (C,H,,NO + ) and 541 (C27H3,N404S+ + l), (Fig. 3b). The presence of the two couples of peaks, i.e. (m/z 100 + 555 and

114 + 541) respectively in combination was due to the primary fragmentation occurring at N-4 in both molecules, so that the fragmentation pattern can be assumed as diagnostic for the presence of each of these compounds. Since the latter peaks are absent in the MS-MS-HPLC analysis of the extract (Fig. 3a), the presence of 8-N-acetyl,4,8N’,N”-didansylspermidine in the extract can be reasonably excluded. 3.2. Ace@ spermidine, PA0 utirit), und spd-to-put interconversion itz maize root In Table 1 the changes in the PA pattern induced in roots by the treatment of maize seedlings for 18 h with or without 1 mM spd or 1 mM ac-spm added alone or in combination are reported. Ac-spd is detectable in ac-spm and acspm + spd treated roots with a higher amount in

M. de Agazio et al.

148 Table I Effect of treatment

with spermidine+acetyl

Treatment

Put

Control Spd Ac-Spm Ac-Spm + Spd

336 873 235 689

I26 + 62 * 1% f 42

spermine

1Plant Science 121 (1996) 143-149

on endogenous

polyamine

content

Spd

ABA1

Ac-Spd

Ac-Spm

371 + 28 506 + 44 310+31 389 + 33

124+ 11 216 k 17 19*3 125 + 10

nd nd 106i8 204 i 14

nd nd ‘17 2 19 395 f 29

Effect of 18 h treatment with or without 1 mM spermidine and 1 mM acetyl-spermidine. polyamine content in root apical segments of maize seedlings. Data are expressed as nmol/g(FW). Put, putrescine: Spd. spermidine; Ac-spd, acetyl-spermidine: Ac-spm. acetyl-spermine: detectable. Data are means k S.D. from a representative experiment run in triplicate.

the latter treatment. The appearance of ac-spd corresponds to a decrease of put in ac-spm-treated roots with respect to control ( - 30%) and in ac-spm + spd with respect to spd ( - 20%). Roots absorb ac-spm and its uptake is enhanced by the presence of spd. Moreover, ABA1 content, a degradation product of spd oxidation, is reduced by ac-spm added to both control ( - 84%) or spd ( - 42%) treated tissues, confirming that the in vitro inhibiting effect of ac-spm on the oxidation of spd [20] is exerted also in vivo. Besides the reduction of the ABA1 content, the inhibition of PA0 is demonstrated by measuring its activity in ac-spm-treated roots as compared to control roots. The activity of the enzyme, extracted from roots of maize seedlings treated for 18 h with 1 mM ac-spm, is reduced by 90% (0.033 U/g fresh weight) in comparison with the enzyme extracted from untreated tissues (0.312 U/g fresh weight). These data suggest a correlation between the appearance of ac-spd and the concomitant inhibition of PAO, as reported in animal cells. Two questions arise: can exogenous spd be a substrate for the acetylation processes, as suggested by the higher level of ac-spd if spd is added to ac-spm; and is ac-spd an intermediate in the spd-to-put interconversion pathway, as suggested by the decrease of put concomitant with the accumulation of ac-spd? The experiments performed with ‘“C-spd gave a negative answer to both questions (Table 2). In fact, ‘“C-ac-spd has never been found among the PA separated from root extract of seedlings grown in 1 mM ‘jC-spd f 1

alone or in combination,

ABAI,

on endogenous

y-aminobutyraldehyde;

nd.

not

mM ac-spm suggesting that the acetylation occurs in a subcellular compartment not accessible to exogenous spd. Moreover ac-spd seems not to be involved in the interconversion pathway as evidentiated by the ineffectiveness of 14C-spd to label ac-spd while ca 30% of total radioactivity of dansylated fraction was found in put, both in presence or absence of ac-spm. In conclusion the interconversion spd-to-put pathway in plants still remains unknown. Evidence has been provided that ac-spd is present in higher plants as well but under physiological conditions it is practically undetectable, probably because of its rapid turnover, in analogy to animal cells where ac-spd is degraded by PA0 to put, The presence of ac-spd has been revealed by incubating maize root tissues with ac-spm, a non competitive inhibitor of PA0 in plant cells, sugTable 2 Distribution of 14C in polyamines absorb ‘JC-spermidine

from maize roots allowed

Treatment

Dans

Put

Spd

Ac-Spd

Spd Spd + Ac-Spm

396k26 378+X

144510 113+8

49 i_ 5 110+9

nd nd

to

Fate of 14C radioactivity in apical maize root segments (1 cm long) after 18 h treatment with I mM spermidine, labeled with Y-spermidine, in presence or absence of 1 mM acetyl spermine, at different steps of polyamine separation. Data are expressed as dpm x IO-‘/g(FW). Abrevidtions as in Table I. Dans. dansylated. Data are means +_S.D. from a representative experiment run in triplicate.

M. de Aguzio et (11. Plunt gesting that also in higher plants ac-spd is degraded by PAO. The lack of radioactive ac-spd in roots treated with 1 mM 14C-spd and 1 mM ac-spm, suggests that the acetylation of spd occurs in a cell compartment which cannot be reached by exogenous spd but it involves only spd internal to the compartment. The possibility that exogenous ac-spm can be converted to ac-spd by PA0 should be excluded because the cleavage points on ac-spm by maize PA0 produce aminoaldehydes, DAP and H,O, but not ac-spd [21].

Acknowledgements The authors wish to thank expert technical assistance.

R. Buffone

for his

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14’)

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