Putrescine-Induced Woundingand Its Effects Membrane ... · mLofthe ether phase were collected, evaporated under N2 gas at 35°C, and redissolved in 100 1iL methanol. Standards for

Plant Physiol. (1989) 90, 988-9950032-0889/89/90/0988/08/$01 .00/0

Received for publication November 9, 1988and in revised form February 20, 1989

Putrescine-Induced Wounding and Its Effects on MembraneIntegrity and Ion Transport Processes in Roots of Intact

Corn Seedlings

Joseph M. DiTomaso, Jon E. Shaff, and Leon V. Kochian*Department of Agronomy, Cornell University, Ithaca, New York 14853 (J.M.D.), and U.S. Plant, Soil and Nutrition

Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Cornell University,Ithaca, New York 14853 (J.E.S., L.V.K.)

ABSTRACT

Interactions between putrescine and membrane function wereexamined with the use of a recently developed microelectrodesystem that enables us simultaneously to quantify membranepotentials and net K+ fluxes associated with individual cells atthe root surface of an intact corn (Zea mays L.) seedling. Incontrast to the results of others, our analyses indicate that ex-ogenous putrescine (0.5 millimolar), in the absence of calcium,does not maintain membrane stability. In addition, putrescinecaused a wound response characterized by a gradual depolari-zation of the membrane potential and a considerable net effluxof K+ from the root. In the presence of calcium, both short term(20 minutes) and long term (24 hours) exposure to a high concen-tration of exogenous putrescine (5 millimolar) also caused areduction in the resting membrane potential and a significant K+efflux. However, preincubating corn roots in a solution containingthe antioxidant ascorbate ameliorated the wounding effects ofputrescine and slightly increased potassium uptake. A similarpreincubation in the absence of calcium did not protect mem-branes against putrescine-induced damage. The amelioratingeffect of ascorbate on putrescine-induced membrane damagesuggests that the wounding response of high putrescine levelsin corn roots involves the catabolism of the polyamine by a cellwall diamine oxidase, with the concomitant production of hydro-gen peroxide and free radicals resulting in peroxidative damageof the plasmalemma.

Diamines, particularly putrescine, can accumulate in plantsas a response to increased growth or to a variety of stressconditions, including potassium deficiency, water, salt, or acidstress (for a review, see ref. 23), and herbicide treatment (5).However, it has been shown that under these conditions theconcentration of tri- and tetraamines (such as spermidine andspermine) generally remains unchanged. This stress-mediatedincrease in diamines has been suggested to be an adaptiveadvantage to plants, and a number of protective functions forpolyamines have been proposed. For example, in studiesinvolving protoplasts and excised cereal leaves, exogenouspolyamine applications were effective in preventing senes-

cence by reducing Chl loss (4) and inhibiting the rise inRNAase and protease activities (1 1). Additionally, it has beensuggested (31) that due to its polycationic nature, putrescinecould be involved in the ionic regulation of the plant cell

symplasm. In response to acid stress, for example, it has beenhypothesized (25) that increases in cytoplasmic putrescinelevels could help to maintain cytoplasmic pH at a constantvalue.

It has also been suggested (13) that under stress conditions,polyamines may partially replace calcium in maintainingmembrane integrity by binding to phospholipid componentsof the membrane. The involvement of polyamines in wound-induced membrane permeability has been investigated byseveral researchers. As a measurement ofmembrane integrity,Naik and Srivastava (13) spectrophotometrically monitoredthe leakage ofthe red pigment, betacyanin, from red beet rootdiscs. They reported that exogenously applied spermidine andspermine decreased betacyanin efflux from the discs of beet(Beta vulgaris L.) roots treated with ethanol. Polyamines werealso effective in reducing betacyanin efflux in beet root tissueswounded with ethylene, ammonium sulfate (15), RNAase, orhigh temperature (1, 26).However, some confusion and controversy exists concern-

ing the role of polyamines in stabilizing membranes. In theabove cited work, Srivastava and Smith (26) reported thatexogenously applied putrescine, spermidine, and sperminedecreased betacyanin efflux in temperature stressed beet rootdiscs, while total ion efflux was shown to increase. To explainthis discrepancy, they speculated that polyamines may disruptthe plasmalemma but stabilize the tonoplast. Since betacyaninis thought to be retained in the vacuole, its efflux would bereduced. However, Smith and Croker (24) recently demon-strated that this apparent reduction in betacyanin leakage isactually due to a chemical reaction between betacyanin andpolyamines in the external solution, which causes a decolori-zation of the red pigment. These results cast doubt on anyhypothesis concerning polyamine stabilization of membranesbased on the measurement of betacyanin leakage from planttissues.Although a stress-induced increase in putrescine levels may

have protective advantages, an excessive accumulation couldalso be a cause of injury. High levels of exogenous diaminesresulted in symptoms of toxicity in broad bean ( Vicia fabaL.) (18) and in barley (Hordeum vulgare L.) (27), whileincreased endogenous putrescine may cause a reduction incell division in osmotically stressed cereal protoplasts (30) andnecrosis in salt stressed broad bean (18). Ferrante et al. (7)found that cultures of the protozoan Plasmodiumfalciparum

988

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PUTRESCINE EFFECTS ON MEMBRANE INTEGRITY AND ION TRANSPORT

degenerated when exposed to spermine and polyamine oxi-dase in combination. However, if the polyamine or the poly-amine oxidase were added alone, no symptoms were observed.They concluded that polyamine oxidation leads to cellulardisruption through the production of toxic aldehydes, hydro-gen peroxide, and free radicals. These substances have longbeen recognized to cause tissue injury through membranedestruction (20).

In plants, diamine oxidase activity has been identified in anumber of species of the Leguminosae (for a review, see ref.23) and in rice (Oryza sativa L.) (3) and oat (Avena sativa L.)(8). The catabolism of putrescine by diamine oxidase occursalmost exclusively within the cell walls (8) and results in theproduction of pyrroline, hydrogen peroxide, ammonia (28),and possibly free radicals (34). Thus, the toxic effect associatedwith high levels of exogenous putrescine may be the result ofmembrane damage caused by diamine oxidase-mediated pu-trescine catabolism.

In the present work, we investigated the role of putrescinein maintaining and/or altering membrane integrity. The in-teraction of exogenous putrescine and calcium, in terms oftheir effects on Em' and net potassium fluxes in roots of intactcorn seedlings, was studied. It has been well documented thatcorn roots are a particularly sensitive tissue, and that variousperturbations such as excision, cold shock, etc., can elicit awound response (10). This response is characterized by areduction in K+ influx and a stimulation of efflux and by adepolarization of Em. Additionally, we have recently devel-oped a microelectrode system that enables us to simultane-ously quantify membrane potentials and net ionic fluxesassociated with individual cells at the root surface of an intactcorn seedling (14). Thus, we have an experimental systemthat is well suited for studying the apparently broad spectrumof interactions between putrescine and membrane function.Results of these experiments indicate that putrescine does notreplace calcium in maintaining membrane stability. On thecontrary, the diamine caused a depolarization of the mem-brane potential and increased potassium leakage. Additionalexperiments indicate that hydrogen peroxide and free radicalproduction may be the cause of membrane damage and thusbe responsible for the phytotoxic effect of high exogenousputrescine levels.

MATERIALS AND METHODS

Plant Material

Zea mays L. seeds (3377 Pioneer) were surface-sterilized in0.5% NaOCl, and then were germinated for 2 d in the darkon filter paper saturated with 0.2 mM CaSO4. Subsequently,eight germinated seedlings were selected for uniform growth,transferred to polyethylene cups with polyethylene mesh bot-toms (two seedlings per cup), and then covered with blackpolyethylene beads. Four cups (eight seedlings) were thenplaced into precut holes in the covers of black polyethylenecontainers containing 2.4 L of aerated 0.2 mM CaSO4 solutionand were grown for 3 d. The seedlings were grown at 22°Cunder low-light conditions. The primary root of the intact 5-

'Abbreviation: Em, membrane potential.

d-old seedlings was used for all K+ flux and electrophysiologyexperiments.

Extraction and Quantification of Polyamines

Corn root sections were excised 0 to 0.5, 0.5 to 1.5, 1.5 to2.5, and 2.5 to 3.5 mm from the tip. Each section was weighedand ground in a glass homogenizer with 5% cold perchloricacid at a ratio ofabout 25 to 100 mg root tissue/mL perchloricacid. Homogenized tissue samples were extracted in perchlo-ric acid for 1 h in an ice bath. Samples were centrifuged at24,000g for 20 min at 4°C, and the supernatant phase, con-taining the 'free' polyamine fraction, was either immediatelybenzolyated or stored at -20°C in small cryogenic plasticvials. One mL of 2 N NaOH was mixed with either 500 ,uL ofthe free polyamine fraction extracted from root tissue or 50,L of each 1 mm polyamine standard in 0.01 N HCI. Afteraddition of 10 ,L benzoyl chloride, mixtures were vortexedfor 10 s and incubated at room temperature for 20 min. TwomL of saturated NaCl and 10 mL cold anhydrous diethylether were added to each sample. The tubes were capped,gently mixed, and centrifuged at 1 5OOg for 5 min at 4°C. FivemL of the ether phase were collected, evaporated under N2gas at 35°C, and redissolved in 100 1iL methanol. Standardsfor putrescine, cadaverine, spermidine, and spermine weretreated similarly to tissue extracts.The redissolved samples were injected into a fixed 20 ,L

loop for loading onto a 4.6 mm by 250 mm, 5 ,um particlesize reverse-phase (C18) column. Samples were eluted fromthe column by a Perkin-Elmer Series 410 pump at roomtemperature with a flow rate of 1 mL/min. Polyamine peakswere detected by a Perkin-Elmer LC-95 absorbance detectorat 254 nm. The isocratic solvent system consisted of 49%acetonitrile (v/v) in deionized water. Results were recordedon a Nelson Analytical 900 Series Intelligent Interface andintegrated with Nelson Analytical PC Integrator software froman IBM XT. Results are presented as nmol/g fresh weight.

Measurement of Net K+ Fluxes

Liquid membrane-type neutral carrier-based K+-selectivemicroelectrodes (tip diameter = 0.5 4m) were constructed aspreviously detailed (12) using Fluka K+-selective cocktail(catalog No. 60031, Fluka Chemical Co.). We have recentlydeveloped a technique that enables us to quantify net ionicfluxes associated with individual root epidermal cells, basedon the measurement of radial ion activity gradients in theunstirred layer at the root surface with ion-selective micro-electrodes. These steady state gradients are the result of iontransport at the root surface (influx or efflux) and the diffusionof ions either toward or away from the root (see ref. 14 for adetailed description of the experimental procedures). Briefly,the intact seedling was housed in a Plexiglas chamber attachedto the stage of an Olympus compound microscope mountedon its back on the surface ofa vibration-damped table (KineticSystems Inc.). The K+-selective microelectrode was mountedin a pressure-relieved holder on the preamplifier of a highinput resistance dual electrometer (model FD 223, WP In-struments, Inc.). The preamplifier was then mounted onto aNarashige hydraulically driven micromanipulator (model

989



Plant Physiol. Vol. 90, 1989

MO-204, Narashige USA) that was attached to the microscopestage such that the microelectrode could be lowered verticallyinto the solution and reach chosen radial distances from thehorizontally oriented root (usually 50 and 100 ,um from theroot surface).The root and vertically positioned K+-selective microelec-

trode were viewed under moderate magnification (x60-150)with the Olympus microscope. In order to measure net K+fluxes, the appropriate experimental solution was flowedthrough the chamber until the previous solution was dis-placed, and then flow was ceased. The Plexiglas chamber wasconstructed to minimize mixing of the solution surroundingthe root due to mechanical vibration and convection; we havefound that steady state ion activity gradients are establishedat the root surface approximately 5 min after flow is stopped.Subsequently, the K+ activity in the unstirred layer was meas-ured at 50 and 100 ,um from the root surface and the net K+flux at the root surface was determined from the followingequation derived from diffusion analysis of the spatial sym-metry of the K+ activity gradient:

2 WDK(CI - C2)JK - ln(Ru/R2)

where JK is the net flux of K+ (in ,umol cm-' s-'), DK is theself-diffusion coefficient for K+ (in cm2 s-'), C, and C2 arethe K+ activities at the two positions, and RI and R2 are therespective distances from the positions where the K+ activitywas measured to the center of the root. The appropriateconversion factors were used to obtain a net flux in terms of,umol g-' h-'. The net K+ fluxes determined in this study weremeasured approximately 2 cm back from the root apex.

Electrophysiological Studies

The microelectrode system was constructed such that mem-brane potentials and K+ fluxes could be measured simulta-neously. Membrane potentials were measured using a WPImodel KS-750 amplifier and microelectrodes (tip diameter =0.5 grm) made from single-barrelled borosilicate glass tubingand filled with 3 M KCI (adjusted to pH 2 to reduce tippotentials). The reference electrodes for both membrane po-tential and K+ flux measurements were also 3 M KCl-filledmicropipettes and were placed in the solution bathing theseed in order to minimize contamination of the solutionbathing the root with K+ diffusing from the reference elec-trodes. Cells of the root epidermis and cortex were impaledusing a separate hydraulically driven Narashige micromanip-ulator mounted at a second position on the microscope stage.

RESULTS

Polyamine Levels in Roots of Corn Seedlings

Endogenous free polyamine concentrations were deter-mined in the terminal 0.5 cm and the three subsequent 1-cmsegments back from the root apex (Table I). Putrescine con-tent varied little from the root apex to 3.5 cm back from thetip, averaging 289 nmol/g fresh weight. However, the concen-trations of spermidine and spermine were significantly higherin the tip section containing the meristem, and decreased

Table I. Polyamine Content in Four Corn Root SectionsSeedlings were grown hydroponically for 5 d in aerated 0.2 mm

CaSO4 solution. The primary root of six plants were used per repli-cate. Measurements are based on four replicates ± SE.

Polyamine ConcentrationCom Root Section

Putrescine Spermidine Spermine

cm from tip nmol/g fresh wt

0-0.5 283 ± 50 733 ± 137 53 ± 140.5-1.5 255±12 175±28 9±0.91.5-2.5 294±31 95±20 6±1.32.5-3.5 322 ± 23 83 ± 12 7 ± 1.3

dramatically in the other regions. Similar free polyaminelevels in corn roots were reported by Dumortier et al. (6),although they measured a gradual increase in putrescinecontent from the tip to the base of the root.

Effects of 0.5 mm Exogenous Putrescine on Em and K+Fluxes in the Presence and Absence of Ca2+

In our initial experiments, we wished to determine whethera low concentration (approximately equal to putrescine levelsin unstressed or mildly stressed plants) of exogenous putres-cine could substitute for calcium in maintaining membraneintegrity. Hence, both Em and net K+ fluxes were monitoredas measures of membrane integrity, in response to an experi-mental protocol which would remove Ca2" from the root cellwall and plasmalemma surface. First, the Em, K+-induceddepolarization of Em, and net K+ fluxes were determinedunder control (+Ca2+) conditions that would be indicative ofan unperturbed root cell plasmalemma. Representative valuesfor these parameters are seen in the initial phase of the timecourse presented in Figure 1. An initial resting Em of -164mV was measured in a bathing solution of 0.2 mM CaCl2,which is a typical value for a low salt-grown corn root. Theaddition of 50 ,M K+ (as KCI) to this bathing solution eliciteda depolarization of 46 mV, and the subsequent net K+ influxwas 2.15 ,umol g-' h-' (inset of Fig. 1). From previous work,we have found these to be fairly typical values for a normal,healthy corn root (14). In contrast, a depolarization of Em, areduction in the magnitude of the K+-induced depolarizationof Em, and an inhibition of K+ influx or a stimulation ofefflux have been shown to be characteristic of 'wounded' orperturbed roots and are associated with a decrease in mem-brane integrity (10).The addition of a low concentration of putrescine (0.5 mM)

in the presence of Ca2` elicited a transient 40 mV depolari-zation of the membrane, but within 6 min the membranepotential was only 8 mV less negative than its initial value(Fig. 1). The magnitude of the K+-induced depolarization (48mV) and the net K+ flux (2.34 ,umol g-' h-1) indicate that lowlevels of exogenous putrescine do not cause immediate mem-brane injury to corn roots. Subsequently, Ca2+ was removedfrom the cell wall and plasmalemma surface by flowing aCa2+-free solution containing 1.0 mM EGTA through the rootchamber for 15 min. Although the value of the Em wasrelatively unaffected by Ca2' removal, a destabilization of theplasmalemma was observed, as indicated by rapid and dra-

990 DiTOMASO ET AL.




B 2.34 + 0.32ECoCI2 IC 0.28 0.04

-130 I11I1305~lj\ (2\ EGTAEX

-1 ~-170

E K ClCaCp Kt EGTAputrescine + COC12

0 9 28 34 41 50 57 66 76Time (min)

Figure 1. Representative time course of the response of the corticalcell membrane potential and net K+ flux of intact low salt-grown cornroots (initial control conditions: 0.2 mm CaCl2 + 5 mm Mes-Tris buffer[pH 6]) to: first, treatment with low concentrations (0.5 mM) ofputrescine (as putrescine dihydrochloride) in the presence of 0.2 mmCaCl2, and second, the removal of calcium from the root cell plas-malemma by exposure to a solution containing 1 mm EGTA. WhenK+-induced depolarizations and net K+ fluxes were measured follow-ing a particular treatment, the root was exposed to a solution con-

taining 50 ,M KCI in addition to the particular treatment (calcium,putrescine, or EGTA). The net K+ fluxes were measured at the timepoints labeled A, B, and C on the figure and the flux values are

presented in the inset. A positive flux value indicates K+ influx. Allexperimental solutions used for the results presented here and insubsequent figures contained 5 mm Mes-Tris buffer (pH 6) in additionto the other reagents. The data presented here and in the followingfigures are representative results of experiments repeated three timeswith similar results; all K+ flux values are the means of three replicatemeasurements.

matic transient shifts in the resting potential. This destabili-zation was apparently intensified when the bathing solutionwas continuously flowed through the chamber. The removalofcalcium by EGTA chelation resulted in a marked reductionin both K+-induced depolarization and net K+ flux (Fig. 1,inset). This supports the view that calcium not only maintainsmembrane stability but plays an important role in ion trans-port processes.

In Figure 2 (continuation of Fig. 1), 0.5 mM putrescine wasadded to the bathing solution following extended EGTAtreatment. After an initial rapid 45 mV depolarization, themembrane potential continued to gradually depolarize to -86mV. The addition of external K+ had no further effect on themembrane potential, and flux measurements indicate a con-

siderable net K+ efflux from the root (-2.73 jumol g-1 h-',inset in Fig. 2). These results are characteristic of a woundresponse in corn roots. This wound response following appli-cation of low levels of exogenous putrescine in the absence ofextracellular calcium was only partially recoverable, despitethree washes in 0.2 mm CaCl2 solution. After 50 min in therecovery solution, the membrane potential had nearly re-turned to its initial value presented in Figure 1, while the netflux of K+ had only recovered by 39% (Fig. 2, inset).

Effects of 5 mm Exogenous Putrescine on Em and K+Fluxes

From our initial experiments, the application of 0.5 mMputrescine appeared to injure corn root membranes in the

E

-80

._0 0

r- -too

c

a -1200.

-140c

' -160E

0 2 6 24 47 63 73Time (min)

8t 89 102 109

Figure 2. Time course of the response of the cortical cell membranepotential and net K+ flux in intact low salt-grown corn roots toexposure to 0.5 mM putrescine in the absence of calcium (+1 mmEGTA), and to the subsequent return to the initial control conditionsof Figure 1 (0.2 mm CaCI2 + 5 mm Mes-Tns [pH 6]). The time coursepresented here is a continuation of the experiment depicted in Figure1. The net K+ fluxes were measured at time points D, E, F, and G onthe figure and the flux values are presented in the inset. A positiveflux value denotes a net K+ influx, while a negative value denotes an

efflux.

absence of calcium but had no effect in the presence ofcalcium. We next conducted experiments in which intactroots were exposed to a 10-fold increase in exogenous putres-cine (5 mM) in the presence of 0.2 mm CaCl2. This putrescineconcentration is thought to approximate the level found undersevere stress conditions (21, 33).

Initial measurements ofEm, the K+-induced depolarization,and net K+ flux were representative of a normal, healthy root.The introduction of 5.0 mm putrescine in the presence ofcalcium caused a rapid and dramatic depolarization of themembrane potential (Fig. 3). The subsequent addition of K4into the bathing medium after 5 min of putrescine treatmentcaused an initial 42 mV depolarization, followed by a more

gradual depolarization, to -71 mV. It appeared that a signif-icant portion of this depolarization is not associated with K+,but is a continued effect of putrescine exposure. This issupported by the lack of a net K+ influx measured in thepresence of 5 mm putrescine (inset, Fig. 3). Removal ofputrescine from the bathing solution resulted in only a partialrepolarization ofthe membrane potential and a small recoveryin net K4 influx. An additional CaCl2 recovery treatmentalmost completely repolarized the membrane potential to itspretreatment value, but the subsequent net K+ influx was

only 40% of the control value.These results suggest that high concentrations of putrescine

may cause membrane injury. To determine whether putres-cine-induced membrane damage is a transient effect, we

exposed 4-d-old corn seedlings grown hydroponically in 0.2mM CaSO4 to an additional 24 h treatment in CaSO4 solutionwith either 0.5 or 5.0 mm putrescine. The corn seedlingsgrown in 0.5 mM putrescine exhibited normal values for rootmembrane potential, K+-induced depolarization, and net K+influx (Table II). However, at the higher concentration of

Treatment Net K+ Flux

/zmolt h' +SE

D -2.73 + 0.1 IE -0.0t ± 0.37F -0.47 ± 0.04

EGTA + K+EGTA + putrescine G O.B3+0.t3

putrescine { COCO2

-E COC12 CoC1j2-100 ~-102© -117

-EGT K+ + CoOt2 I4® -6©

K + CoCIz -162

K + CoCt2

991




5U;2 K CoCa2 COtC2E K -1p05e1

~-I 20 - 12 -124 ~ ~ -412929-141

-160 151K + CoC12

E200 K +CaCz KC dCtC2 + putrescine K -177

-200 K CO1

K" CdaC12 CaC12 + putrescine' "#I. ' I#' I

0 2 15 27 39 46 56 63 67 76 80Time (min)

Figure 3. Time course of the response of the cortical cell membranepotential and net K+ flux in intact low salt-grown corn roots toexposure to high concentrations (5 mM) of putrescine in the presenceof 0.2 mm CaCl2, and to the subsequent removal of putrescine fromthe experimental solution. The net K+ fluxes were measured at timepoints A, B, C, and D on the figure and the flux values are presentedin the inset.

Table II. Effect of Two Concentrations of Exogenous Putrescine onMembrane Potential, K+-lnduced Depolarization, and K+ Fluxes inIntact Roots of Corn Seedlings

Seedlings were grown for 4 d in aerated 0.2 mm CaSO4 solutionand an additional 24 h in the same solution with 0.5 or 5.0 mmputrescine dihydrochloride. Measurements were made on a root ofan intact seedling in 50 jAM KCI and/or 0.2 mm CaCI2 solution bufferedat pH 6 in 5 mm Mes-Tris.

Putrescine Initial K+-inducedTreatment Em Depolarzation K+ FluxmM mv mv pmolI/g *h ± SE0.5 -158 -67 1.97 ± 0.295.0 -30 0 -2.00 ± 0.19

exogenous putrescine, the resting membrane potential wasdramatically reduced, and reached a maximum of only -30mV. The addition of K+ resulted in a large net K+ efflux(-2.00 ,mol g-' h-'). The phytotoxic effect of 5.0 mM pu-trescine became clearly visible after 48 h of exposure, as theroots appeared flaccid and translucent.

Ameliorating Effects of Ascorbate

In light of the proposed catabolism of putrescine by a cellwall-specific diamine oxidase (8), we considered the possibilitythat putrescine caused membrane damage as a result of anexcessive production of hydrogen peroxide and subsequentformation of free radicals in the cell wall solution adjacent tothe root cell plasmalemma. Thus, we hypothesized that ageneral free radical scavenger, such as ascorbic acid, mightprotect membranes against putrescine damage.To test this hypothesis, we pretreated intact corn roots with

0.5 mM ascorbate in addition to 0.2 mm CaCl2. The subse-quent addition of 5.0 mM putrescine in the presence of

ascorbate resulted in an initial 48 mV depolarization (Fig. 4).Twelve min of exposure caused no additional depolarizationof the membrane potential. Potassium uptake was measuredin the presence of putrescine and ascorbate and was found tobe slightly higher than the control value measured in theabsence of putrescine (inset, Fig. 4). Removal ofboth K+ andputrescine from the bathing solutions resulted in a repolari-zation of the membrane potential to its initial value and asubsequent reintroduction ofK+ elicited a depolarization andnet K+ influx typical of normal, healthy corn roots. Theseresults indicate that a pretreatment with ascorbate protectedcorn root membranes against wounding caused by high con-centrations of exogenous putrescine.

Influence of Ascorbate on Putrescine-Induced MembraneInjury in the Absence of Ca2+

Experiments were performed to determine if ascorbatesimilarly protected corn root membranes against damagecaused by low levels of putrescine in the absence of calcium(Fig. 5; Table III). The initial K+-induced depolarization (-51mV) and net K+ influx (1.35 ,umol g-' h-') values in 0.5 mMascorbate, 0.2 mm CaCl2, and 50 ,uM KCI were similar to thosepresented in Figure 4. Removal of K+ from the mediumresulted in a repolarization of the membrane potential to itsinitial value of- 165 mV. This membrane potential representsthe starting conditions of Figure 5. The addition of 1 mmEGTA concomitant with the removal of CaCl2 from thebathing medium elicited only a slight change in the membranepotential after 17 min of EGTA exposure. However, net K+flux measurements following the introduction ofKCI indicatea 44% reduction (0.76 Mmol g-' h-'; inset, Fig. 5; Table III)from that of the initial condition. It should be noted that theinhibitory effect ofcalcium removal on net K+ flux was greater(87% inhibition) when corn roots were not pretreated withascorbate (Fig. 1).

TetetNet K+ Flux

CoC12 + asc Tre moltm g' h'±SEA 1.61 ±0.14

-60 CaC 2 a -78 B 1.96 +0.18

I ~~~~~~~c1.86 ±0.08

-90 -102 -96 CaCl24asc-120- ~-121 -2-1122 1e K5KcJclXaursc4n g

E

K+cal+ 4C1

-IO _ "sc cadl2+asc+ K +caC12 oscputrescine -189l,p

0 1 29 37 39 46 58 61 64 85 89 99 106Time (min)

Figure 4. Time course of the influence of 0.5 mM L-ascorbic acid(abbreviated as asc in the figure) on the response of the cortical cellmembrane potential and net K+ flux in intact low salt-grown cornroots to exposure to 5 mM putrescine in the presence of 0.2 mmCaCO2, and to the subsequent removal of putrescine from the exper-imental solution. The net K+ fluxes were measured at time points A,B, and C on the figure and the flux values are presented in the inset.

992 DiTOMASO ET AL.




Net K* Flux concurrent with the removal of EGTA, and finally the re-Treatment f±mol gr' tf + SE moval of ascorbate did not cause a significant repolarizationA 0.76 ± 0.1 9 of the membrane potential nor a recovery in net K+ influx

asc + EGTA B -2.85 ±0.27 (Fig. 5, inset).K++ asc + EGTA + ut C - 1.41± 0.23i+osc+EGTA4-put0 -0.71 ± 0.29 DISCUSSION

I ~~~~~CoCt2 + OSC

asc + EGTA 1 -79 Putrescine: A Calcium Substitute?-6

-to,<-86> tOO aKs+cCt2+ -108 Polyamines have been frequently reported to have a stabi-asc + - ® CaCt2 lizing influence on membranes during periods of stress andEGTA -137 |-49 | | CCt2+asc have even been suggested to act, in part, as a calcium substi--165 -152 ¶ asc+ EGTA tute (13). However, there are several points that should be-X70 asc + EGTA + put made concerning much of the experimental evidence that hasK+aKsc+EGTAK +asc+EGTA been presented in the literature in support of this role for

80 tO 20 34#''#'K' ' ' ' polyamines. First, it has been well documented that increased0 17 24 34 58 68 so 100 120 134 levels of putrescine, a diamine, are associated with a wide

Time (min) range of plant stresses, while the same stress conditions are5. Time course of the influence of 0.5 mM L-ascorbic acid rarely coupled to a concurrent increase in triamines andated as asc in the figure) on the response of the cortical cell tetraamines such as spermidine or spermine (for a review, see.ne potential and net K+ flux in intact low salt-grown com ref. 23). Yet experimental evidence that has been used inexposure to 0.5 mM putrescine in the absence of calcium support of a membrane-protective role for polyamines isEGTA), and to the subsequent removal of first, putrescine, nearly always associated with the exogenous application ofond, EGTA (+0.2 mm CaCl2). The net K4 fluxes were meas- spermidine and spermine, but rarely with putrescine. Second,time points A, B, C, and D on the figure and the flux values most of t results demonstrating the stabilizingented in the inset. motstfte expenlmenta eut eosrtn h tblzn

effects of polyamines have been conducted with wall-lessexperimental systems such as protoplasts and organelles (2,

responses of the membrane potential and net K+ flux 17, 29). As we will suggest in the next section, researchmM putrescine clearly show that ascorbate does not concerning polyamine interactions with plant cell membranescorn root membranes against putrescine-induced in- could yield strikingly different results depending on the naturethe absence of extracellular calcium. During the course of the experimental system (intact plant tissue versus plantmin exposure to putrescine, the membrane potential systems lacking cell walls).ly depolarized to -86 mV (Fig. 5). Furthermore, K+ In the research presented here, we exposed intact corn roots[ction did not elicit further membrane depolarization to 0.5 mm exogenous putrescine in the presence and absenceused a net K+ efflux of -2.85 gmol g-' h-'. Subse- ofcalcium. This level ofexogenous putrescine should simulater, putrescine was removed from the chamber and the a moderate stress condition, particularly if symplasmicallysolution was changed three times to determine derived polyamines can easily cross the plasmalemma, as has

rthe wounding effects of putrescine could be reversed. been suggested in the literature (9, 16). In the presence ofmoval of putrescine, the reintroduction of calcium calcium, 0.5 mm putrescine had a slight stimulatory effect on

Table ll. Effect of Putrescine on K+-lnduced Depolarization and Fluxes in the Presence of AscorbicAcid and in the Absence of Calcium

Seedlings were grown hydroponically for 5 d in aerated 0.2 mm CaSO4 solution. All solutions used inchamber contained 5 mM Mes-Tris buffer at pH 6. Solutions with calcium or potassium contained 0.2mM CaCI2 or 50 gM KCI, respectively. In treatment 11, all solutions contained 0.5 mM ascorbate.Experiments were conducted three times with similar results.

Treatment K+-lnduced Depolarization K+ Flux

% of control

I. Ca2+ (control)+0.5 mM PUT 96 109+5 mM PUT 65 Efflux-5 mM PUT (recovery) 60 25

II. Ca2' + 0.5 mm ascorbate (control)+5 mM PUT 92 122-PUT 88 116+1 mM EGTA - Ca2+ 65 56+1 mM EGTA + 0.5 mm PUT - Ca2+ 0 Effiux+1 mM EGTA - PUT - Ca2+ (recovery) 0 Effiux-EGTA + Ca2+ (recovery) 0 Efflux

E-80

-It

> -140

CE-17C

1-

DI

Figure e(abbreviEmembrairoots to(+1 mMand seciured at 1are pres

The r

to 0.5protectjury inof a 24gradualintroduand catquentlybathingwhethemThe rei

993




the net flux of K+ (Table III). However, when calcium ionswere chelated from the root tissue with EGTA, exposure to0.5 mm external putrescine resulted in a depolarization of theEm, reduction in the K+-induced depolarization of Em, and adramatic alteration in K+ uptake from influx to a substantialK+ efflux (Fig. 2). This evidence strongly suggests that putres-cine does not replace calcium in maintaining membranestability and, in fact, induces a wounding response similar tothat observed following other forms of stress.

Putrescine Phytotoxicity: A Possible Mechanism forMembrane Damage

Plant tissues exposed to severe stress have been reported toaccumulate extremely high concentrations of putrescine. Forexample, Smith (21) found putrescine concentrations of 3380nmol/g fresh weight in K+-deficient corn leaves. In addition,Young and Galston (33) measured putrescine levels greaterthan 7000 nmol/g fresh weight in shoots of K+-deficient oatseedlings, and in our own studies we have found putrescinelevels to be as high as 4000 nmol/g fresh weight (13-foldincrease) in roots of herbicide-treated pea seedlings (our un-published data). Because it has been demonstrated that pu-trescine readily moves across cell membranes (16), an exoge-nous application of 5 mm putrescine would appear to be areasonable level to accumulate in the apoplasm under severestress conditions.The validity of these exogenous applications of polyamines

have been questioned by some researchers. Roberts et al. ( 19)suggested that the effects of exogenously fed putrescine maynot represent the true physiological effects of endogenousaccumulation. Also, Smith (22) noted that high concentra-tions of exogenously applied putrescine might cause phyto-toxicity by contacting the external surface of the plasma-lemma normally inaccessible to subcellularly localized endog-enous putrescine. However, these arguments would beapplicable only if the plant cell plasmalemma is relativelyimpermeable to polyamines. Although Young and Galston(32) provide evidence supporting this hypothesis, in morerecent studies, Pistocchi et al. (16) observed that a significantplasmalemma influx and efflux of putrescine could occur incultured carrot (Daucus carota L.) cells. Additionally, studiesby Friedman et al. (9) indicate that putrescine levels increasedin the xylem exudate of salt stressed sunflower (Helianthusannuus L.) plants. Although this area awaits further research,it seems reasonable to speculate that a significant increase inthe level of endogenous putrescine could cause an efflux ofputrescine across the plasmalemma into the cell wall solution,resulting in a significant increase in apoplasmic putrescineconcentrations. Consequently, it is our contention that exog-enously applied putrescine could cause similar plasma mem-brane effects to those ultimately arising from a high initialconcentration of endogenous putrescine.The results of short (Fig. 3; Table III) and long term (Table

II) exposure of intact corn roots to 5 mM putrescine provideevidence for wounding at the plasmalemma. This was seen asa depolarization in the Em and a reduction in the K+-induceddepolarization of Em, as well as a stimulation of K+ efflux.These results are in contrast to several previous studies dem-onstrating a stabilizing effect of polyamines, particularly sper-

midine and spermine, on membranes. However, a large pro-portion of these studies in plants were conducted with exper-imental systems lacking cell walls, including protoplasts (2),chloroplasts (17), and phospholipid vesicles (29). Since di-and polyamine oxidase are found exclusively in the cell wallfraction of plants, these wall-less experimental systems wouldeliminate the possible interaction between the plasma mem-brane and products of polyamine metabolism. In contrast,much of the early evidence, conducted with intact planttissues, implicates exogenous putrescine application to symp-toms of phytotoxicity (1, 18, 27). From these and our ownstudies we hypothesize that putrescine-mediated membranewounding results from the production of hydrogen peroxideand free radicals through the activity of diamine oxidase inthe cell wall. Although diamine oxidase has yet to be describedfrom corn, its activity was demonstrated in other grass species,including rice (3) and oat (8). If a similar pathway exists incorn roots, it would be expected that preincubating withascorbate, a nonspecific free radical scavenger, prior to intro-ducing a high concentration of putrescine should protect cornroot membranes against damage.The results of our electrophysiological and flux studies

support this hypothesis. The presence of 0.5 mM ascorbate inthe bathing medium ameliorated the wounding effects of 5.0mM putrescine (Fig. 4). Putrescine did not elicit an inhibitoryeffect on K+-induced depolarization of the Em and, in fact,caused a slight stimulation in the net influx ofK+ (Table III).The characteristic putrescine-induced depolarization of theEm appears to be the only significant change caused by thediamine in the presence of ascorbate. The distinct similaritiesof the kinetics of the putrescine-induced depolarization ofEmto those caused by K+ suggests that the polycation is rapidlytransported across the plasma membrane of corn roots cells.

In view of the ameliorating effects of an antioxidant onputrescine-mediated membrane damage, it might follow thata pretreatment with ascorbate would protect corn root mem-branes against the phytotoxic effect oflow levels ofexogenousputrescine in the absence of calcium. Our results, however,indicate that ascorbate does not protect corn root membranesin the absence of calcium (Fig. 5; Table III). In addition,neither Em nor K+ uptake recovered following the removal ofputrescine, ascorbate, and EGTA, or after the addition ofcalcium back to the medium. It is difficult to explain themechanism of putrescine-mediated wounding under theseconditions, but it can be concluded that the role(s) thatcalcium plays in stabilizing and protecting biological mem-branes is probably more complex than simply a physicalmaintenance of membrane integrity.

CONCLUSION

Previous hypotheses have assigned calcium-like propertiesto polyamines that provide membrane protection in responseto various plant stresses. However, we have found that inintact corn roots, putrescine does not substitute for calciumin maintaining membrane integrity. Indeed, it appears thatthe application of low levels of exogenous putrescine in theabsence of calcium causes membrane damage, based on themeasurements of root cell electrical properties and K+ trans-port as indicators of membrane integrity. Furthermore, simi-

994 DiTOMASO ET AL.




lar wound responses were elicited when roots were exposedto higher levels (5 mM) of putrescine in the presence ofcalcium. We propose that the mechanism of putrescine-in-duced wounding involves the catabolism of the polyamine bya cell wall diamine oxidase, with the concomitant productionof hydrogen peroxide and free radicals resulting in peroxida-tive damage of the plasmalemma. Because much of the pre-vious work attributing a membrane stabilizing role to polya-mines in plants was based on wall-less experimental systems,the deleterious aspects of cell wall polyamine catabolism tothe plant cell plasma membrane would not have been ob-served and the potential for polyamine-induced membranedamage would have been missed.

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9. Friedman R, Levin N, Altman A (1986) Presence and identifica-tion of polyamines in xylem and phloem exudates of plants.Plant Physiol 82: 1154-1157

10. Hanson JB, Rincon M, Rogers SA (1986) Controls on calciuminflux in corn root cells. In AJ Trewavas, ed, Molecular andCellular Aspects of Calcium in Plant Development. PlenumPress, New York, pp 253-260

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15. Parups EV (1984) Effects ofethylene, polyamines and membranestabilizing compounds on plant cell membrane permeability.Phyton 44: 9-16

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18. Richards FJ, Coleman RG (1952) Occurrence of putrescine inpotassium-deficient barley. Nature 170: 460

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29. Tadolini B, Cabrini L, Landi L, Varani E, Pasquali P (1984)Polyamine binding to phospholipid vesicles and inhibition oflipid peroxidation. Biochem Biophys Res Commun 122: 550-555

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32. Young ND, Galston AW (1983) Are polyamines transported inetiolated peas? Plant Physiol 73: 912-914

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Putrescine-Induced Woundingand Its Effects Membrane ... · mLofthe ether phase were collected, evaporated under N2 gas at 35°C, and redissolved in 100 1iL methanol. Standards for