May 22, 1987 - Scalo, Rome, Italy. ABSTRACT. The action of ... Maize seeds were continuously rinsed with tap water for 12 h and germinated on filter paper ...
Plant Physiol. (1988) 87, 176-178 0032-0889/88/87/0176/03/$01 .00/0
Effect of Exogenous Putrescine, Spermidine, and Spermine on K+ Uptake and H+ Extrusion through Plasmamembrane in Maize Root Segments' Received for publication May 22, 1987 and in revised form September 25, 1987
MARINA DE AGAZIO*, MARIA CARMELA GIARDINA, AND STEFANO GREGO2 Institute of Biochemistry and Plant Ecophysiology, National Research Council, 00016 Monterotondo Scalo, Rome, Italy The purpose of this research was to study the effect of polyamines on K+ influx and H+ extrusion in apical root segments of Zea mays. K+ influx and H+ extrusion were stimulated both by a 'washing' procedure and then by the fungal phytotoxin, fusicoccin, after being inhibited by 'cutting' (10, 11). Results are reported that indicate a specific action of polyamines on K+ influx and H + extrusion, reflecting in our opinion, a physiological role of polyamines at the plasma membrane level.
ABSTRACT The action of exogenous polyamines (putrescine, spermidine, and spermine) on 'washing' and fusicoccin-stimulated K+ uptake and H+ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K+ influx and HI extrusion without interfering with K+ uptake and HI extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K+ uptake and HI extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K+.
The naturally occurring polyamines elicit a variety of physiological responses ranging from promotion of growth to regulation of senescence (1, 2, 8, 16). When exogenously applied to ageing and detached tissue, polyamines interact with membranes inducing changes which, in turn, lead to retardation of senescence. In fact, exogenous polyamines are able to preserve Chl retention in thylakoid membranes of barley chloroplasts (14), to stabilize oat leaf protoplasts against lysis (7), or to reduce membrane permeability in discs of beet root storage tissues (13). However, to date, it is not yet clear whether the polyamines associate unspecifically with negatively charged phospholipids because of their polycationic nature (15) or whether their interaction with cellular macromolecules is more specific (3, 17). Recently it was demonstrated that polyamines selectively rigidify the surface of the plasma membrane, reducing fluidity of microsomal membrane from primary leaves of bean, and that some of the physiological effects of polyamines could reflect membrane rigidification rather than a true physiological response (15). As transport processes of anions, cations, and organic substances through the plasma membrane are its main function, it is interesting to know whether polyamines, interacting with the membrane, can interfere with these processes. It was demonstrated that high external concentrations of Ca2+ ions, which, like polyamines, are able to rigidify the membranes, decrease K+ uptake in wheat and cucumber (4) and ATPase activity in barley roots (5). A relationship between the inhibition of both K+ uptake and ATPase activity and Ca2+ induced reduction in mobility of the lipid polar head groups was suggested (4, 5).
MATERIALS AND METHODS Plant Material. Maize seeds (Zea mays L., hybrid line Plenus S 516) were supplied by the Dekalb Center (Chiarano, Italy). Maize seeds were continuously rinsed with tap water for 12 h and germinated on filter paper wetted with 0.5 mm CaSO4 at 27°C in the dark for 3 d. Washing Procedure. Tipless root apical segments (1 cm) of primary maize roots were collected and washed for 2 h in 0.5 mM CaSO4 solution using 4 g fresh weight of tissue/L washing solution in a thermoregulated bath at 30°C in the dark. Polyamine Treatment. Rpot segments after the washing procedure were treated with polyamines (putrescine, spermidine, and spermine, chloride forms dissolved in 0.5 mm CaSO4 solution) for 1 h in the dark in a thermoregulated bath at 30°C with shaking. Polyamines were used at a concentration of 1 mM, unless otherwise indicated. Influx Experiments. K+ influx determinations, measured with 86RbCl as a tracer, were made using 150 mg samples of excised roots in 0.1 mm phosphate (K+ salt, pH 6) + 0.2 mM CaSO4 as previously reported (6). 86RbCl (specific activity 1.24.102 K Bq g-1) was supplied by Amersham International Limited, Amersham, Bucks., U.K. For the determinations of kinetic constants 1 mM Tris-MES buffer + 0.2 mM CaSO4 (pH 6.0) was used and the polyamines were added during K+ uptake. The calculation was done using a linear regression program which gave best fit estimates for Km and Vmax. In the experiments with fusicoccin a concentration of 10 /LM was used during K+ uptake. HI Extrusion Experiments. Samples of 150 mg of root apical segments were washed for 2 h and then transferred into 5 ml of aerated 1 mm Tris-MES buffer (pH 6.0) + 0.2 mM CaSO4 with or without putrescine, spermidine, and spermine at a final concentration of 1 mm. After addition of polyamines, the pH of the samples was adjusted to 6.0 by Tris, 10 mm. Samples were allowed to equilibrate for 30 min at 30°C and then KCI was added to a final concentration of 20 mm. After 15 min from the addition of KCI, fusicoccin was added to a final concentration of 10 AM. The H + concentration changes were measured by titrating in a Radiometer TTT80 with 0.2 mm NaOH. During titration, N2 was bubbled through the liquid. The H + extrusion was measured
l Supported by CNR, Italy. Special grant IPRA-Subproject 1, paper No. 1726. 2 Present address: Institute of Agricultural Chemistry. University of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy. 176
POLYAMINE EFFECTS ON K+ UPTAKE AND H + EXTRUSION 15 min after adding KCI and 1 h after fusicoccin treatment, using different samples. Data reported in the tables and figure refer to a single typical experiment in three replicates. Three series of independent experiments were carried out giving reproducible results.
RESULTS K + influx through the plasma membrane was measured in root apical segments from 3-d-old maize seedlings washed for 2 h in CaSO4 solution and pretreated for 1 h with different concentrations of putrescine, spermidine, and spermine in separate solutions. Washing of root apical segments was necessary to reactivate the K+ transport system strongly inhibited by 'cutting' (10). Figure 1 shows the effect of different concentrations of putrescine, spermidine, and spermine on K+ uptake. Spermidine appears to be the most effective inhibitor over the entire range of concentration. The resulting K+ uptake inhibition increased from 11% at 0.5 mm to 82% at 20 mm concentration. Spermine exhibits a smaller inhibiting effect while putrescine shows a slight but reproducible activation at 0.5 mm followed by an inhibiting effect lower than those of the other polyamines. In Table I the vallues of the kinetic constants Km and Vmax for K+ uptake in the fpresence and absence of polyamines are reported. The concenitration range of KCl was between 2 and 50 4
-C
4-'
3
.-c
CO)
0 L) %4.-
2
0)
Co
1
0
mM. The increase in the value of apparent K,, with no change of the value of Vmax in the presence of polyamines suggests that the mechanism of inhibition is competitive. Since the activation of K+ uptake induced by fusicoccin is additive to that induced by washing, the effect of the polyamines on K+ uptake stimulated by fusicoccin was examined in washed root apical segments. The results show that polyamines do not inhibit the stimulation by fusicoccin of K+ uptake (Table II). The effect of polyamines on proton extrusion induced by washing and then by fusicoccin was investigated, since this process is involved in the transport of cations through the plasma membrane (12). The experiments were carried out in the presence of high concentrations of KCl (20 mM) which is known to enhance H+ extrusion (9). Table III shows that, similar to the results of the K + uptake experiments, polyamines inhibit H + extrusion in washed root segments without interfering with the extrusion stimulated by fusicoccin. Also in this case putrescine exhibits a smaller inhibiting effect than those of spermidine and spermine.
DISCUSSION It has been recently suggested that the decrease of plasma membrane fluidity caused by polyamines is due to a nonspecific effect incurred when they act as polyvalent cations and associate with the negatively charged head groups of the bilayer phospholipids (15). In this context the inhibition, induced by polyamines, of K+ uptake and H+ extrusion through the plasma membrane reported in this paper could be explained as a consequence of the rigidification of membrane, in analogy with Ca2+, which at high external concentrations is known to reduce the mobility of lipid polar head groups (5) and to inhibit K + uptake (4) and ATPase activity (5). On the other hand, the membrane rigidification previously reported was evident at high concentration (50 mM) while at 1 mm, the concentration used in our experiments. It was found that putrescine did not show any effect and spermidine and spermine decreased membrane fluidity by only 10% (15). it is unlikely that the inhibition of K+ uptake of about 50%, found at 1 mm, can be ascribed only to a nonspecific membrane rigidification. Our results support the different hypothesis that the effect of polyamines on K+ uptake and H+ extrusion is to be ascribed to a specific interaction of polyamines with the
E
177
plasma
membrane. In fact, the
analysis
of kinetic
con-
stants indicate that polyamines behave as competitive inhibitors 0
5
10
15
20
polyamines (mM ) FIG. 1. Effect of diifferent concentrations of polyamines on K + ("mRb) uptake. (0), Control; (O), putrescine; (A), spermidine; (EJ), spermine. Data reported refer tto a single typical experiment in three replicates. Standard errors did n Lot exceed 5%.
of K+. Moreover, polyamines inhibit preferentially K+ uptake and H+ extrusion activated by washing and have no effect on that activated by fusicoccin. A nonspecific effect on plasma membrane,
rigidification,
indiscriminately
biochemical functions of membrane. In conclusion we suggest that the inhibiting effect of exogenous polyamines on K+ uptake and H+ extrusion through plasma membrane reflects a true physiological role. In fact, we would hazard the hypothesis that the endogenous polyamines are in-
Table I. Kinetic Constants for K+ (86Rb) Uptake in the Presence and Absence of Putrescine, Spermidine, and Spermine in Root Apical Segments of Z. mays Root apical segments (1 cm long) were washed for 2 h in 0.5 mm CaSO4 and transferred into 5 ml of 1 mM Tris-MES buffer + 0.2 mm CaSO4 (pH 6.0) supplied with or without putrescine, spermidine, and spermine adjusted to pH 6.0. Uptake measurements were made for 60 min. Data reported refer to a single typical experiment in three replicates. Treatment R Km (5 mM) correlation coefficient ,tmol g- I fresh wt h - ± SD mM + SD Control 0.99 6.06 ± 0.18 11.76 ± 0.29 Putrescine 0.99 8.69 ± 0.44 12.05 ± 0.48 0.99 Spermidine 22.22 ± 1.00 13.80 ± 0.62 0.96 Spermine 12.50 ± 0.44 9.09 ± 0.34
Vad
178
Plant Physiol. Vol. 87. 1988
DE AGAZIO ETAL.
Table II. Effect of Putrescine, Spermidine, and Spermine oni K Uptake Stimullated by Ftusicoccini in Washed Root Apical Segments oJ Z. inavs Root apical segments (1 cm long) were washed for 2 h in 0.5 mm CaSO, and then treated for 1 h in the presence or absence of putrescine. spermidine, and spermine. Samples were transferred into 5 ml of 0(.1 mM phosphate (K+ salt, pH 6.0) + 0.2 mm CaSO4 and uptake measurements were made for 30 min with or without 10 AM fusicoccin (FC). Data reported refer to a single typical experiment in three replications. Treatment 1 mM Control
Putrescine Spermidine Spermine
K ("'Rb) Uptake + FC
- FC
3.85 3.34 1.85 2.40
,umol g 'fresh 6.96 5.69 + 4.60 + 5.47 +
+ 0.08 + 0.14 + 0.05 + 0.05
wvt h 0.36 0.20 0.17 0.31
AFC SD 3.11 2.84 2.75 3.07
Table III. Effect of Putrescine, Spermidinie. and Spermine on H ELvtrusioni Sitmiildlated bY K titid Fusicoccini in Washed Root Apical Segments Root apical segments (1 cm long) were washed for 2 h in 0.5 mM CaSO4 and transferred into 5 ml of 1 mm Tris-MES buffer + 0.2 mm CaSO4 (pH 6.0) supplied with or without putrescine, spermidine, and spermine adjusted to pH 6.0. KCI at final concentration of 20 mm was added and after 15 min H* extrusion was titrated with 0.2 mm NaOH. In another set of samples, 15 min after KCI addition, fusicoccin (FC) was added at final concentration of 10 p.M and H+ extrusion was titrated after 1 h. Data reported refer to a sintle typical experiment in three replications.
H Extrusion
Treatment
(1 mM)
KCI (20 mM) .eq H' g ' fresh wt 15 mini
geq H g
i.e
SD
Control 0.67a + 0.04 Putrescine 0.39' + 0.02 0.Oa- 0.00 Spermidine Spermine 0.0;' + 0.00 a It represents ,ueq of H- extruded in 15 min after KCI in 1 h after FC addition.
volved in the response of the root to cutting by selectively blocking the basal K + uptake and H + extrusion. Further studies are in progress on this suggestive hypothesis and its relative implications. was a
8.
gencrous gift from Prof. A. Ballio.
LITERATURE CITED 1. ALTMAN A, U BACHRACH 1981 Involvement of polyamines in plant growth and senescence. In CM Caldarera, V Zappia, V Bachrach, eds, Advances in Polyamines Research. Vol 3. Raven Press, New York, pp 365-375 2. BAGNI N, D SERAFINi-FRACAssINI, P TORRIGIANI 1981 Polyamines and growth in higher plants. In CM Caldarera, V Zappia, V Bachrach, eds, Advances in Polyamines Research, Vol 3. Raven Press, New York, pp 377-388 3. BALLAS SK, N MOHANDAS, LJ MARTON, SB SHOHET 1983 Stabilization of erythrocyte membranes by polyamines. Proc Natl Acad Sci USA 80: 19421946 4. BENGTSSON B 1982 The effect of Ca' -stress on fluxes of Ca` and K- in wheat and cucumber. Physiol Plant 56: 415-420 5. CALDWELL CR, A HAUG 1981 Temperature dependence of the barley root plasma membrane-bound Ca' and Mg '-dependent ATPase. Physiol Plant 53: 117- 124 6. DE AGAZIO M, MC GIARDINA 1984 Loss of recovery capacity of plasmalemma K+ influx after cutting in chlorsulfuron pretreated maize roots. Plant Phvsiol 76: 940-942 7. FUHRER J, R KAUR-SAWUNEY. LM SHIH. AW GALSTON 1982 Effccts of
-
si)
t
(0.25 7.09" + 0.32 6.72" 0.24 5.91" 0.30 It represents ,ueq of H extruded 6.16
addition.
l .3-diaminopropane and spermidince on senescence of oat leaves. Plant Phvsiol 70): 1597-16t)0 KAUR-SAWHNEY R. LM SHIII. LiE FLORES. AW GALSION 1982 Rclattioni polvaminc synthesis and titer to aging ind senescenec in oait leascs. Plant Physiol 69: 4)5 -410( LADO P. Ml DE MICHELIS. R CERANA. E MARRE 1976 FuLsicoccin-induccd. K--stimulated proton secretion and acid-indUced growth ot apical root segments. Plant Sci Lett 6: 5-21) LEONARD RT. JB HANSON 1972 Induction and development of incrcised iOll absorption in corn root tissuc. Plant Phvsiol 49: 430-435 MARRL E 1977 Effects of fusicoccin and hormones on plant cell membrane activities. In E Marre. 0 Cifferi. eds. Regulation of Cell Membranc Acti\ities in Plants. Elsevier. Amsterdam. pp 185-202 MARRF E 1979 Integration of solute transport in cereals. In DL Laidman. RG Jones. eds. Rcecnt Advances in the Biochemistrv of Cereals. Acldcmic Press. New York. pp 3-25 NAIK BI, SK SRIVASTAVA 1978 Effect of polvamines on tissuc permeabilitv. Biochemistry 17: 1885-1887 PoPovIC RB. DJ KYLE. AS COHFN. S ZALIK 1979 Stabilization of thliakoid mcmbranes by spermine during stress-induced senescence of barlscy lca disc. Plant Physiol 64: 721-726 ROBERTS DR, EB DUMBROFI. JE TIhoMPSON 1986 Exogenous polsamines alter membrane fluidity in bean leaves-a basis for potential misinterprctation of their true physiological role. Planta 167: 395-4(01 SLOCUM RD. AW GALSTON 1985 Changes in polyamine hiosynthesis Issociated with postfertilization growth and deselopment in toba cco ovarv tissuics. Plant Physiol 79: 336-343 SOLAINi G. B TADOLINI 1984 Spermine binding to submitochondrial particecs and activation of adenosinc triphosphatase. Biochem J l18: 49'-499 exogenous
9.
Acknowledgmnents-Fusicoccin
gM) iesh ivt h fehw i
FC (10
1i). 11. 12.
13. 14. 15.
-
16. 17.
of
