THE INHIBITORY EFFECTS OF COUMARIN

ON THE GERMINATION OF DURUM WHEAT

(Triticum turgidum ssp. durum, CV. SIMETO) SEEDS

M. R. ABENAVOLI,1,* G. CACCO,1 A. SORGONA,1 R. MARABOTTINI,2

A. R. PAOLACCI,2 M. CIAFFI,2 and M. BADIANI1

1Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale(BIOMAA), Facolta di Agraria, Universita Mediterranea di Reggio Calabria, Loc. Feo di

Vito, I-89124 Reggio Calabria RC, Italy2Dipartimento di Agrobiologia e Agrochimica, Universita degli Studi della Tuscia, Via S.C.

De Lellis, I-01100 Viterbo VT, Italy

(Received January 15, 2005; revised October 4, 2005; accepted October 5, 2005)

Published Online April 6, 2006

Abstract—The event chain leading to germination blockage in durum wheat

(Triticum turgidum ssp. durum Desf.) seeds exposed to the allelochemical

coumarin (2H-chromen-2-one) was studied. The physiological and biochem-

ical aspects thought to be critical for a successful seed germination were

measured. At concentrations above 200 2M, coumarin inhibited seed

germination in a concentration-dependent manner. Inhibition occurred early

during seed imbibition (phase I), was rapid, and irreversible. During phase I,

coumarin inhibited water uptake, electrolyte retention capacity, and O2

consumption. Later on, coumarin delayed the reactivation of peroxidases,

enhanced the activity of superoxide dismutase, decreased the activities of

selected marker enzymes for metabolic resumption, and repressed the

transcription of molecular chaperons involved in secretory pathways.

Insufficient and/or late seed rehydration caused by coumarin could have

delayed membrane stabilization or decreased respiratory O2 consumption,

both of which are conducive to an overproduction of reactive O2 species.

Being unbalanced by an adequate upsurge of antioxidant defense systems, the

resulting oxidative stress might have ultimately interfered with the germina-

tion program.

Key Words—Allelochemicals, coumarin, seed germination, antioxidants,

molecular chaperons, durum wheat, Triticum turgidum ssp. durum Desf.

0098-0331/06/0200-0489/0 # 2006 Springer Science + Business Media, Inc.

489

Journal of Chemical Ecology, Vol. 32, No. 2, February 2006 (#2006)

DOI: 10.1007/s10886-005-9011-x

* To whom correspondence should be addressed. E-mail: mrabenavoli@unirc.it

Abbreviations: !-AMY = !-amylase; "-AMY = "-amylase; APX = ascorbate peroxidase; AsA =

ascorbic acid; BiP = lumenal binding protein; CAT = catalase; DHAR = dehydroascorbate reductase;

DHAsA = dehydroascorbic acid; ER = endoplastic reticulum; G6PDH = glucose-6-phosphate

dehydrogenase; GluK = glucokinase; GPX = hydrogen-donor aspecific (guaiacol) peroxidase;

GSH = reduced glutathione; GS-SG = glutathione disulfide; MDH = malate dehydrogenase;

MDHAR = monodehydroascorbate reductase; OPPP = oxidative pentose phosphate pathway;

PCR = polymerase chain reaction; PDI = protein disulfide isomerase; PyrK = pyruvate kinase;

ROS = reactive oxygen species; RT = reverse transcription; SOD = superoxide dismutase; TRX =

thioredoxin h.

INTRODUCTION

Coumarins form a large class of allelochemicals widely distributed in both

natural plant communities and crops (Zobel and Brown, 1995). Being localized

on the leaf, seed surface, and pollen wall, and released into the environment by

living plants or by decomposing plant material, coumarins are involved in

ecological interactions in both managed and natural plant communities

(Rice, 1984; Zobel et al., 1991; Bertin et al., 2003; Bais et al., 2004).

Coumarin (2H-chromen-2-one), the simplest compound of this class,

affects root form and function (Abenavoli et al., 2001, 2004), decreases

respiration and photosynthesis (Moreland and Novitzky, 1987), and influences

nitrogen uptake and metabolism (Abenavoli et al., 2001, 2003). Coumarin is a

strong inhibitor of seed germination. This may confer an advantage to the

producing plant species by reducing competition in its immediate environment,

and/or by delaying germination of its own seeds under unfavorable conditions

(Zobel and Brown, 1995).

To explain this inhibitory action, coumarin has been hypothesized to be

either a blocker of the cell cycle (Zobel and Brown, 1995), uncoupler of

oxidative phosphorylation (Ulitzer and Poljakoff-Mayber, 1963; Khan and

Zeng, 1985), or to interfere with amino acid transport and protein synthesis

(Van Sumere et al., 1972). Aliotta et al. (1992, 1993) reported that coumarin is

an inducer of coat-imposed dormancy through the inhibition of water uptake

during seed imbibition. Such a multiplicity of proposed roles suggests that the

key mechanism(s) by which coumarin affects seed germination remains to be

identified.

In the present work, we looked at the early events leading to the blockage

of germination in durum wheat (Triticum turgidum ssp. durum Desf. cv.

Simeto) seeds exposed to coumarin. To fulfill such an aim, and keeping as a

reference scheme the triphasic germination time course (Bewley, 1997), the

effects of different coumarin concentrations were evaluated on relevant phys-

iological and biochemical processes associated with seed imbibition and

germination.

490 ABENAVOLI ET AL.

METHODS AND MATERIALS

Germination Experiments. Seeds of T. turgidum ssp. durum Desf., cv.

Simeto were surface-sterilized for 20 min into 20% (v/v) NaClO solution, and

rinsed several times with sterile distilled water. Fifteen seeds were evenly placed

into Petri dishes (9 cm diam) on moist filter paper soaked with 5 ml sterilized

aqueous coumarin solution adjusted to pH 5.8, whose final concentrations ranged

from 0 to 1000 2M. Seeds were incubated in darkness in a growth chamber at

24-C and 70% RH. Seeds showing at least a 2-mm-long extrusion of the radicle

after 36 hr of imbibition were considered to have successfully completed their

germination. Germination counts were expressed as percentages of the total

number of seeds.

In a series of recovery experiments, Simeto seeds were exposed to 0, 100, or

1000 2M coumarin for 1, 3, 6, 12, or 24 hr and then transferred under sterile

conditions onto filter paper containing 5 ml sterile distilled water until the end

of germination (36 hr). This is referred to as the ab initio experiments. In a

complementary set of experiments aimed at evaluating the time window that

coumarin inhibition is effective, the Simeto seeds were initially placed into

distilled water for 1, 3, 6, 12, or 24 hr and then exposed to 0, 100, or 1000 2M

coumarin for the time remaining to complete germination (36 hr). This is

referred to as the in itinere experiments.

Water Content. The amount of water taken up by the seeds was determined

as the percent increase in weight of imbibed seed with respect to the initial dry

weight of the seed (Aliotta et al., 1994; Baskin et al., 1998). Prior to weighing,

seeds were paper-blotted for 10 sec.

Respiration. Oxygen consumption in germinating seeds was measured with

a Clark-type electrode (Hansatech Ltd., King’s Lynn, UK). For each measure-

ment, two seeds (approx. 150 mg fw each) were placed for approximately 20 min

into a cuvette containing 1 ml air-saturated distilled water at 25 T 0.5-C.

Electrolyte Leakage. For each measurement, 2.0 g seeds (ca. 15 seeds) were

bathed in 0, 100, or 1000 2M aerated coumarin solutions (20 ml) and incubated at

25-C for 10 min. The conductivity of the solution was then measured by a

MPC227 conductimeter (Mettler-Toledo, Greinfensee, Switzerland).

Assays of Metabolites and Enzymes. After 1, 3, 6, 12, 24, or 36 hr during

imbibition, seeds were collected, immediately frozen with liquid N2, and kept at

j80-C until used. Frozen seeds were ground in liquid N2, with mortar and

pestle. The fine powder (ca. 1 g) was then homogenized with 4 vol. of ice-cold

0.1 M HEPES buffer (pH 7.50), made with 5 mM 2-mercaptoethanol, 10 mM

MgCl2, 2 mM dithiothreitol, 2 mM Na2 ethylene diamine tetraacetic acid,

0.1 mM phenylmethylsulfonyl fluoride, 1% (w/v) bovine serum albumin, and

1% (w/v) insoluble polyvinylpyrrolidone. To measure the ascorbate peroxidase

(APX) activity, 1 mM ascorbic acid (AsA) was added to the grinding buffer

491INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

(Amako et al., 1994). When assaying the glutathione (GSH) pool, 0.1 N hydro-

chloric acid was used as the extraction medium (Schwanz et al., 1996). The super-

natants, obtained after filtering the seed extracts through four layers of cheesecloth

and centrifuging at 39,000 � g for 30 min, were used as sources of analytes.

All tested metabolites and enzymes were assayed spectrophotometrically

(Lambda 5 double-beam spectrophotometer; Perkin-Elmer, Norwalk, CT, USA),

essentially as reported by Sanita di Toppi et al. (2005). Ascorbate peroxidase

activity (EC 1.11.1.11) was monitored by following the oxidation of AsA at 290

nm in the presence of H2O2 as the cosubstrate. Superoxide dismutase activity

(SOD, EC 1.15.1.1) was followed by the superoxide-dependent oxidation of

hydroxylamine to nitrite, followed by the colorimetric detection of nitrite at 540

nm; by intercepting O2j, SOD is able to inhibit the final colorimetric reaction in

a concentration-dependent manner. Catalase activity (CAT, EC 1.11.1.6) was

determined by following the consumption of H2O2 at 240 nm. Dehydroascor-

bate reductase activity (DHAR, EC 1.8.5.1) was monitored at 265 nm as it was

formed from dehydroascorbic acid (DHAsA). Monodehydroascorbate reductase

activity (MDHAR, syn. ascorbate free radical reductase, EC 1.6.5.4) was

determined by following the NADH consumed to reduce the monodehydro-

ascorbate radical generated from AsA by the action of ascorbate oxidase.

Ascorbate and DHAsA were assayed according to the method Wang et al.

(1991), in which the AsA-mediated reduction of Fe3+ to Fe2+ is followed by the

formation of a red chelate among Fe2+ and 4,7-diphenyl-1,10-phenanthrolin

(bathophenanthroline), which absorbs at 534 nm. A standard curve covering the

range 0–10 nmol AsA was used. Total and oxidized glutathione (glutathione

disulfide, GSSG) were measured by following the 5,50-dithiobis(2- nitrobenzoic

acid)-GS-SG reductase recycling procedure proposed by Griffith (1985); GSSG

was determined after removal of GSH from the plant extracts by derivatization

with 2-vinylpyridine. Changes in absorbance of the reaction mixtures were

measured at 412 nm and 25-C. GSH was determined by subtracting GSSG (as

GSH equivalents) from the total glutathione content.

Glucokinase activity (GluK, EC 2.7.1.1) was determined by monitoring the

rate of glucose-6-phosphate dehydrogenase-coupled reduction of NADP+

(Espen et al., 1995). Malate dehydrogenase activity (MDH, EC 1.1.1.37) was

determined by following the NADH oxidation by !-oxalacetate at 340 nm

(Queiroz, 1969). Glucose-6-phosphate dehydrogenase activity (G6PDH, EC

1.1.1.49) was assayed by measuring the reduction of NADP to NADPH in the

presence of glucose-6-phospate (Deutsch, 1983). Pyruvate kinase activity

(PyrK, EC 2.7.1.40) was determined by monitoring the rate of NADH oxidation

at 340 nm (Pirovano et al., 1996). Both !-amylase (!-AMY, EC 3.2.1.1) and

"-amylase ("-AMY, EC 3.2.1.2) activities were determined by monitoring the

release of maltose units (assayed against a maltose standard at 546 nm) at 25-C,

by using soluble starch as the substrate. !-AMY activity was measured at pH

492 ABENAVOLI ET AL.

7.0, and "-AMY at pH 4.8, a value that renders !-AMY inactive (Bergmeyer et

al., 1983).

Total soluble protein was estimated according to the method of Bradford

(1976) using bovine serum albumin as the standard. All of the above analyses

were performed at 4-C.

Nucleic Acid Extraction and Analysis. The procedures used for total RNA

extraction, its electrophoretic (25 2g per sample) transfer to nylon membrane,

prehybridization, hybridization and washing of the nylon filters, and Northern

blotting were the same as employed by Ciaffi et al. (2000).

cDNA probes were labeled by incorporating digoxigenin-11-dUTP via

polymerase chain reaction (PCR), following the method of Ciaffi et al. (1999). As

DNA templates, the following sequences were used: PDICDW-Fl-Rl

(AJ277379), a 1.695-kbp reverse transcription (RT)-PCR product containing

the entire coding sequence of protein disulfide isomerase (PDI) gene obtained

from ripening caryopses of T. turgidum ssp. durum cv. Langdon and cloned into

pGEM\-T (Promega Italia, Linate, Italy; Ciaffi et al., 2000); a partial 1.0-kbp

cDNA clone [in pBluescript\ (SK-)] coding for the lumenal binding protein

(BiP), isolated from Triticum aestivum L. (Grimwade et al. 1996); an RT-PCR

product isolated from T. aestivum by using a pair of primers designed based on

the thioredoxin h (TRX) gene sequence reported by Gautier et al. (1998).

Experimental Replication and Statistics. Each reported value represents the

mean of measurements carried out at least in triplicate and obtained from three

independent experiments T SD. Two-way ANOVA was performed for

germination (ab initio and in itinere experiments), water content, conductivity,

O2 uptake, and the antioxidant metabolites and enzyme activity parameters, to

test the effects of coumarin concentration and time of imbibition. The data were

checked for deviations from normality and homogeneity of variances prior to

analysis. A posteriori comparisons were carried out using the Tukey test to

check the significant differences of each parameter among coumarin concen-

trations within each time of imbibition.

One-way ANOVA and, then, a posteriori comparisons by Tukey’s test were

carried out to check the significant differences of germination parameters (dose–

response curve) among the coumarin concentrations. Statistical analysis was con-

ducted using the Systat v.8.0 software package (SPSS Inc., Evanston, Il, USA).

Nucleic acid extraction and analysis were carried out on seeds obtained

from three independent experiments.

RESULTS

Seed Germination. Two hundred 2M coumarin was the concentration

threshold beyond which coumarin inhibited seed germination (Figure 1a). A

493INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

FIG. 1. Effects of coumarin on the germination of durum wheat cv. Simeto seeds.

(a) Dose–response curve; (b) the ab initio experiment: seeds were exposed to coumarin

for the indicated times and transferred into water until completion of germination (36 hr);

(c) the in itinere experiment: imbibition started in water followed by exposure of the

seeds to coumarin for the indicated times. Each value is the mean T SD (N = 9).

(a) Different letters indicate significant difference at P < 0.05 (Tukey’s test). (b, c)

Different letters within time of imbibition groups indicate significant difference among

treatments (P < 0.05, Tukey’s test). No letters within time of imbibition groups indicate

no significant difference among the treatments (P > 0.05, Tukey’s test).

494 ABENAVOLI ET AL.

maximum of more than 60% inhibition was reached with the highest coumarin

concentration, i.e., 1000 2M. No macroscopic change in morphology was

observed in coumarin-inhibited seeds (not shown).

Based on Figure 1a, 100 and 1000 2M were adopted in the subsequent

experiments as representatives of the no-effect and of the inhibitory coumarin

concentration ranges, respectively.

Exposing seeds to different coumarin levels was interrupted at variable

periods, allowing them to recover in distilled water (36 hr; referred to as the ab

initio experiment in Methods and Materials). A 1-hr exposure to 1000 2M

coumarin was sufficient to cause a 20% inhibition of germination. Inhibition

increased almost linearly with increasing exposure to the allelochemical,

reaching 50% inhibition after 24 hr (Figure 1b).

In a complementary experiment, seeds were first placed into distilled water

for variable periods, and then exposed to coumarin until the end of germination

(36 hr; the in itinere experiment). Exposure to 1000 2M coumarin 1, 3, or 6 hr

after the beginning of imbibition, but not to 100 2M at these same times,

inhibited germination up to 90% with respect to control seeds (Figure 1c).

However, inhibition was significantly decreased when seeds were exposed to

1000 2M coumarin either 12 or 18 hr after the beginning of imbibition, and com-

pletely disappeared when the treatment was administered after 24 hr (Figure 1c).

Early Events in Germination. After 1 hr exposure, either 100 or 1000 2M

decreased the amount of water taken up by seeds by about 40 or 80%,

respectively (Figure 2a). However, seeds recovered to the control level after 3–5

hr. Thereafter, coumarin had no remarkable effect on water (Figure 2a).

A thirty min exposure to 1000 2M coumarin, but not 100 2M, caused a 20%

higher solute leakage with respect to the control, and this remained constant

until the end of the experiment (Figure 2b).

Respiratory O2 consumption showed a complex, apparently multiphasic,

pattern both in control and in treated seeds (Figure 2c). Coumarin tended to

abolish the burst of O2 consumption during phase I, in a concentration-

dependent manner. Both 100 and 1000 2M significantly potentiated the

respiratory burst occurring during phase II.

Antioxidant Systems. During germination, AsA was often almost undetect-

able, although drastic but transient increases were observed after 3 or 12 hr of

exposure to either 100 or 1000 2M, respectively (Figure 3). In contrast, the

concentration of its oxidized form, DHAsA, was higher and appeared to

drastically increase from 24 hr of imbibition onwards, but not in the presence of

1000 2M coumarin.

In the case of GSH, balance among the reduced and oxidized form (GSSG)

was almost constantly in favor of the former during germination (Figure 3).

However, at the beginning of imbibition, seeds contained almost exclusively

GSSG, which increased after 1 hr of water uptake (phase I), regardless of the

495INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

496 ABENAVOLI ET AL.

presence of coumarin. Control and 1000-2M-treated seeds showed almost

identical GSH and GSSG profiles, whereas 100 2M coumarin decreased the

GSH level and increased the GSSG level (Figure 3).

In both treated and control seeds, APX activity was detectable only after 24

hr of imbibition (phase III; Figure 3). Enzyme activity dramatically increased

thereafter, except in 1000-2M-treated seeds, where only 25% of control activity

was present after 36 hr. In contrast, both DHAR and MDHAR activities were

detectable, in both treated and control seeds, during the early stages of imbibition

(Figure 3). Coumarin (1000 2M) inhibited MDHAR activity after 1 hr of

imbibition and then again after 24 hr (about j70% compared to control), a 30%

reduction compared with control seeds and being apparent at the end of

germination.

Like APX, GPX activity was also undetectable during the first 12–24 hr of

imbibition, with its latency prolonged by the presence of 1000 2M coumarin.

Again, like APX, GPX activity increased dramatically during the 12- to 36-hr

period up to 400-fold. However, the late burst of activity was almost completely

abolished in seeds exposed to 1000 2M coumarin (Figure 3).

Contrary to the peroxidases, SOD activity peaked during the early periods

of imbibition (phase I; Figure 3). Coumarin delayed the early upsurge and

increased its amplitude, in a concentration-dependent manner. A second peak in

SOD activity occurred after 24 hr of imbibition (phase III), but not in the 1000-

2M-treated seeds. No CAT activity was detected in germinating seeds.

Marker Enzymes for Metabolic Reactivation. Enzymatic markers for

metabolic reactivation, such as !- and "-AMY (starch mobilization), GluK

and PyrK (reactivation of glycolisis), G6PDH (oxidative pentose phosphate

pathway, OPPP), and MDH (Krebs cycle) were all detectable from the start of

germination (Figure 4).

From 12 hr onwards, 1000 2M coumarin caused a 20% decrease in !-AMY

activity. This same coumarin level also prevented the gradual increase in GluK

activity that occurred in control seeds during the first 12 hr of imbibition, and

caused a 20–40% inhibition of enzyme activity through the end of the

experimental period. Coumarin at 100 2M also had an inhibitory effect during

the early stages of germination, but GluK tended to recover to the control level

during phase III. No clear difference among the treatments was observed for

FIG. 2. Water uptake during germination of Simeto seeds in the presence of varying

levels of coumarin (a). Solute leakage in germinating Simeto seeds exposed to different

levels of coumarin (b). Oxygen consumption in germinating Simeto seeds exposed to

different levels of coumarin (c). Symbols for coumarin levels as in Figure 1c. Roman

numerals and dotted lines are intended to indicate different phases during seed

germination. Each value is the mean T SD; N = 3. Statistics as in Figure 1b, c.

497INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

498 ABENAVOLI ET AL.

PyrK and MDH activities. G6PDH was initially depressed by the presence of

1000 2M coumarin, but from 3 hr of imbibition onwards there was no significant

difference with respect to the control. Conversely, this same activity was

stimulated by the presence of 100 2M coumarin during phase III.

Molecular Chaperones and Protein-Modifying Enzymes. The time courses

for BiP and PDI transcript accumulation were similar (Figure 5). In both cases,

a weak Northern hybridization signal appeared after 1 hr of imbibition (phase I)

and tended to increase in intensity thereafter. A strong BiP and PDI hy-

bridization signal appeared after 24 hr of imbibition in the presence of 0

and 100 2M coumarin, but not in 1000-2M-treated seeds. Compared to BiP and

PDI, TRX transcripts were abundant from the early stages of imbibition, and

did not increase thereafter (Figure 5). In contrast to BiP and PDI, 1000 2M

coumarin enhanced the accumulation of the TRX transcripts from 6 hr of im-

bibition onwards.

DISCUSSION

Our results confirm that coumarin inhibits seed germination (Aliotta et al.,

1992, 1994) and that a threshold concentration of about 200 2M is required for

inhibition to occur in durum wheat seeds. Notwithstanding, 100 2M coumarin

was employed here because it is more representative of the levels of cinnamic

acid derivates commonly found in soils (Macias, 1995).

Coumarin inhibited durum wheat germination in a concentration-dependent

manner. Inhibition by coumarin was rapid, apparently irreversible, and cumu-

lative, as it increased along with increasing time of exposure. Our results suggest

that coumarin irreversibly blocks or prevents one or more key germination

event(s) during phase I or early phase II. Weak or no inhibitory effects were

observed when seeds were exposed to the allelochemical during phase III.

Aliotta et al. (1994) reported that inhibition of radish seed germination

caused by 5-methoxypsoralen, which belongs to the coumarin family, was

associated with an inhibition of water uptake. Such inhibition only became

FIG. 3. Antioxidant metabolites [ascorbate (AsA) and glutathione (GSH)], and their

respective oxidized forms [dehydroascorbate (DHAsA) and glutathione disulfide (GS-

SG)], and antioxidant enzymes [ascorbate peroxidase (APX), superoxide dismutase

(SOD), and guaiacol peroxidase (GPX)] and antioxidant-regenerating enzymes [dehydro-

(DHAR) and monodehydroascorbate reductase (MDHAR)], in germinating Simeto seeds

exposed to different levels of coumarin. Symbols for coumarin levels as in Figure 1c.

Each value is the mean T SD (N = 3). Statistics as in Figure 1b, c.

499INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

evident during phase II and was protracted during phase III. In the present work,

coumarin reduced water uptake during phase I, but the decrease was recovered

during phase II. This suggests that germination arrest occurred during phase I.

Interestingly, both 100 and 1000 2M coumarin inhibited water uptake, but only

FIG. 4. Marker enzymes for metabolic reactivation during germination of Simeto seeds

exposed to different levels of coumarin. !-AMY, !-amylase; "-AMY, "-amylase,

respectively. GluK, glucokinase; PyrK, pyruvate kinase; MDH, malate dehydrogenase;

G6PDH, glucose 6-P dehydrogenase. Symbols for coumarin levels as in Figure 1c. Each

value is the mean T SD (N = 3). Statistics as in Figure 1b, c.

500 ABENAVOLI ET AL.

the latter inhibited seed germination. Only the lesser coumarin concentration

allowed reaching that minimal rehydration threshold during phase I that must be

achieved for the germination program to be successfully executed.

Water influx into the cells of dry seeds provokes a transient perturbation to

membrane structure, which causes an immediate and rapid leakage of solutes

into the surrounding medium. After a short time of rehydration, stable

configuration is restored and solute leakage stops (Bewley, 1997). Here, as a

consequence of early inhibition of water uptake, 1000 2M coumarin could delay,

or even prevent, the recovery of a stable membrane configuration. Indeed,

phenolic compounds can induce rapid depolarization of plant membranes (Glass

and Dunlop, 1974) as well as peroxidation of membrane lipids (Baziramakenga

et al., 1995), both of which lead to an increased efflux of solutes.

Interference with membrane functions, and/or delay in taking up an

adequate amount of water (Bove et al., 2001), might also explain the early

inhibition of O2 consumption induced by 1000 2M coumarin during phase I.

Indeed, Moreland and Novitzky (1987) suggested that coumarins, being able to

perturb the membrane systems that sustain electron transport, can inhibit both

respiration and photosynthesis.

FIG. 5. Typical Northern blot hybridization profile of the lumenal binding protein (BiP),

protein disulfide isomerase (PDI), and thioredoxin h (TRX) mRNA transcripts

accumulation during the germination of Simeto seeds exposed to different levels of

coumarin. Each lane contained 25 2g total RNA. rRNA, ribosomal RNA.

501INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

Water uptake, membrane perturbation, and resumption of respiration may

all promote the generation of reactive oxygen species (ROS), including

superoxide radical anion, hydroxyl radical, and H2O2, during seed germination

(De Gara et al., 1997). However, ROS scavenging may be problematic during

germination because, in dry seeds, SOD, which converts superoxide into H2O2,

is present with modest activity. Likewise, APX, which removes toxic levels of

H2O2, is almost absent during the early stages of germination (De Gara et al.,

1997; Figure 3). Therefore, a primary antioxidant strategy during the early

stages might be reduction of DHAsA to AsA by using GSH as the electron

donor, which is catalyzed by DHAR. Indeed, our results indicate that during the

early stages of germination, seeds are substantially devoid of AsA, APX, and

CAT activities, but contain DHAsA and show DHAR activity. Coumarin at

1000 2M inhibited DHAR and MDHAR activities, and decreased DHAsA

levels, but this occurred only during phase III, i.e., after the arrest of germi-

nation had already occurred. Late effects of coumarin were also apparent as far

as the glutathione pool and redox ratio is concerned.

Delayed resumption of APX activity could leave the 1000-2M-treated seeds

without an adequate ROS scavenging capacity during germination, things being

made worse by the absence of a measurable CAT activity. De Gara et al. (1991)

reported that a decrease in APX activity may be related to a loss of seed

germination capacity in Dasypyrum villosum (L.) Borb. Again, however, the

coumarin-dependent inhibition of APX reactivation, albeit drastic, became

apparent in a late stage of the germination process, so that it probably cannot be

regarded as the primary target of coumarin action.

In contrast, the drastic and transient increase in SOD activity induced by

1000 2M coumarin was much closer in time to phase I, during which coumarin-

induced arrest of germination was supposed to occur. This transitory stimulation

of SOD activity might indirectly indicate a need to face an early burst of

superoxide production. However, neither superoxide nor H2O2, the product of

SOD catalysis, were measured in this study.

Instead, 1000 2M coumarin prevented the activation of the other

peroxidase, GPX, well in advance of phase III. This suggests a rather direct

link, on a time basis, with the unknown early event targeted by coumarin. IAA

at supraoptimal concentrations may inhibit germination of apple seeds and thus

participate in the maintenance of dormancy (Nikolaeva et al., 1987). We might

speculate that the 1000-2M-treated seeds, being deficient in GPX, were also

lacking in IAA oxidase activity (Foyer et al., 1997). The resulting excess of

IAA could then contribute to the blocking of germination. If, instead, a major

role of GPX during seed germination is protection from oxidative damage

(Stacy et al., 1996), then lack of GPX activation might worsen the consequences

of APX inhibition (see above), thus further weakening the overall ROS

scavenging capacity of the 1000-2M-treated seeds. According to Bewley

502 ABENAVOLI ET AL.

(1997), within a few hours from the beginning of imbibition, glycolisis, OPPP,

and the Krebs cycle are activated and seeds achieve their full metabolic status.

Since a limited number of marker enzymes was considered here, and cou-

marin’s effects on their in vivo activity could not be evaluated, considering the

results obtained with 1000 2M coumarin, no obvious failure in the reactivation

of the main ATP- and NAD(P)H-producing pathways is apparent.

In germinating cereal seeds, !-AMY activity is rapidly induced through

de novo synthesis and secretion of the enzyme by the aleurone layer. In contrast,

"-AMY is constitutive, deposited during seed development in a zymogenic

form, which is converted into the active enzyme during germination (Kruger,

1979). Khan (1969) reported that 340 2M coumarin completely inhibited !-

AMY synthesis in barley seeds. Accordingly, here, 1000 2M coumarin inhibited

!-AMY activity, but such effect only became evident in an advanced stage of

germination.

One function of the endoplastic reticulum (ER) is to synthesize a large

group of vacuolar or secreted proteins, such as proteases, hydrolases, and

amylases. Among the intrinsic components of the ER, central roles in the

correct folding, and oligomerization, and formation of disulfide bridges of na-

scent secretory proteins are assigned, respectively, to chaperonine-like enzymes,

such as BiP, and to protein-modifying enzymes, such as PDI. The former is

likely to bind transiently to all nascent protein chains and to associate per-

manently to malfolded proteins; the latter is a member of the thioredoxin su-

perfamily, responsible for the introduction and isomerization of disulfide bonds

that are often present in secreted- and cell surface proteins (Ciaffi et al., 2000).

Coumarin (1000 2M) had opposite effects on the transcript abundances of ER-

resident- (BiP and PDI) and of cytosolic molecular chaperons (TRX h in the

present study). Livesley et al. (1992) reported that the germination rate of bread

wheat (T. aestivum L.) seed populations was correlated with the PDI activity of

microsome-enriched fractions extracted from the aleurone layer. Our results

indirectly support these findings and suggest that one coumarin target could be

the transcriptional activation of genes involved in the assemblage and

stabilization of secretory proteins, which could decrease !-AMY synthesis and

secretion (Figure 4), eventually affecting storage mobilization and metabolic

reactivation during seed germination. On the other hand, early increase in the

TRX transcript abundance suggests that exposure to 1000 2M coumarin causes

oxidative perturbation in the cytosol, requiring the activation of compartment-

specific mechanisms of redox homoeostasis.

In summary, beyond a concentration threshold of about 200 2M, coumarin

was able to rapidly and significantly inhibit the germination of durum wheat

seed during the early stages of imbibition. This inhibitory effect produced a

series of changes that were either transitory (e.g., the inhibition of seed water

uptake), too late to be directly traceable to the early arrest during the ger-

503INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

mination process, or whose extent was much lower than proportional to the

severity of the inhibition observed. As a result, we cannot precisely identify the

primary structure or function targeted by coumarin. Neither can we assess

whether one or more critical events were affected.

We are prompted to speculate that, in Simeto seeds exposed to 1000 2M

coumarin, a less-than-sufficient rehydration in the very early stage of imbibition

may prevent or delay the attainment of a stable configuration of membrane

systems. This could lead to a prolonged loss of osmotically active substances

and substrates, and delay resumption of respiration. A reduced utilization of O2

as the terminal electron acceptor may lead to an increase in mitochondrial pO2,

and result in an electron overflow. Both of these conditions are conceivably

conducive to increased ROS generation. This may explain the observed drastic

increase in SOD activity during phases I–II, with compensatory purposes, which

could in turn result in a burst of H2O2. H2O2 overproduction could escape the

control operated by the endogenous scavenging systems, since both GPX,

during phase II, and APX, later on, failed to reactivate in the 1000-2M-treated

seeds. This may eventually cause oxidative stress, which explain the enhanced

transcription, already occurring during phase II, of genes involved in redox

homoeostasis, such as TRX. Since redox regulation is of critical importance for

most, if not all, processes in the aerobic cell, including signal perception and

transduction, cell-to-cell communication, and the cell cycle itself (Foyer et al.,

1997), an altered redox balance caused by exposure to coumarin may ultimately

hamper, or even block, the execution of the germination program.

Acknowledgment—The Universita Mediterranea di Reggio Calabria is acknowledged for

financial support (Intramural Research Grants 2000 and 2001 to MRA and MB).

REFERENCES

ABENAVOLI, M. R., DE SANTIS, C., SIDARI, M., SORGONA, A., BADIANI, M., and CACCO, G. 2001.

Influence of coumarin on the net nitrate uptake in durum wheat. New Phytol. 150:619–627.

ABENAVOLI, M. R., SORGONA, A., SIDARI, M., BADIANI, M., and FUGGI, A. 2003. Coumarin inhibits

the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture. J. Plant

Physiol. 160:227–237.

ABENAVOLI, M. R., SORGONA, A., ALBANO, S., and CACCO, G. 2004. Coumarin differentially affects

the morphology of different root types of maize seedlings. J. Chem. Ecol. 30:1871–1883.

ALIOTTA, G., FUGGI, A., and STRUMIA, S. 1992. Coat-imposed dormancy by coumarin in radish

seeds: the influence of light. G. Bot. Ital. 126:631–637.

ALIOTTA, G., CAFIERO, G., FIORENTINO, A., and STRUMIA, S. 1993. Inhibition of radish germination

and root growth by coumarin and phenylpropanoids. J. Chem. Ecol. 19:175–183.

ALIOTTA, G., CAFIERO, G., DE FEO, V., and SACCHI, R. 1994. Potential allelochemicals from Ruta

graveolens L. and their action on radish seeds. J. Chem. Ecol. 20:2761–2775.

AMAKO, K., CHEN, G.-X., and ASADA, K. 1994. Separate assays specific for ascorbate peroxidase

504 ABENAVOLI ET AL.

and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate

peroxidase in plants. Plant Cell Physiol. 35:497–504.

BAIS, H. P., PARK, S.-W., WEIR, T. L., CALLAWAY, R. M., and VIVANCO, J. M. 2004. How plants

communicate using underground information superhighway. Trends Plant Sci. 9:26–32.

BASKIN, J. M., NAN, X., and BASKIN, C. C. 1998. A comparative study of seed dormancy and

germination in an annual and a perennial species of Senna (Fabaceae). Seed Sci. Res. 8:501–512.

BAZIRAMAKENGA, R., LEROUX, G. D., and SIMARD, R. R. 1995. Effects of benzoic and cinnamic

acids on membrane permeability of soybean roots. J. Chem. Ecol. 21:1271–1285.

BERGMEYER, H. U., GRASSL, M., and WALTER, H.-E. 1983. !- and "-amylases, pp. 151–153, in H.

U. Bergmeyer, J. Bergmeyer, and H. Grassl (eds.). Methods of Enzymatic Analysis, 3rd edn.,

vol. 3. Verlag Chemie, Weinheim, Germany.

BERTIN, C., YANG, X., and WESTON, L. 2003. The role of root exudates and allelochemicals in the

rhizosphere. Plant Soil 256:67–83.

BEWLEY, J. D. 1997. Seed germination and dormancy. Plant Cell 9:1055–1066.

BOVE, J., JULLIEN, M., and GRAPPIN, P. 2001. Functional genomics in the study of seed germina-

tion. Genome Biol. 3:reviews 1002.1–1002.5<http://www.genomebiology.com/2001/3/1/1002>.

BRADFORD, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities

of protein utilising the principle of protein–dye binding. Anal. Biochem. 72:248–254.

CIAFFI, M., DOMINICI, L., TANZARELLA, O. A., and PORCEDDU, E. 1999. Chromosomal assignment

of gene sequences coding for protein disulphide isomerase (PDI) in wheat. Theor. Appl. Genet.

98:405–410.

CIAFFI, M., PAOLACCI, A. R., DOMINICI, L., TANZARELLA, O. A., and PORCEDDU, E. 2000.

Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in

durum wheat (Triticum turgidum ssp. durum). Gene 265:147–156.

DE GARA, L., PACIOLLA, C., LISO, R., STEFANI, A., and ARRIGONI, O. 1991. Correlation between

ascorbate peroxidase activity and some anomalies of seedlings from aged caryopses of

Dasypyrum villosum (L.) Borb. J. Plant Physiol. 137:697–700.

DE GARA, L., DE PINTO, M. C., and ARRIGONI, O. 1997. Ascorbate synthesis and ascorbate

peroxidase activity during the early stage of wheat germination. Physiol. Plant. 100:894–900.

DEUTSCH, J. 1983. Glucose-6-phosphate dehydrogenase, pp. 190–197, in H. U. Bergmeyer,

J. Bergmeyer, and H. Grassl (eds.). Methods of Enzymatic Analysis, 3rd edn., vol. 3. Verlag

Chemie, Weinheim, Germany.

ESPEN, L., MORGUTTI, S., ALISI, C., PIROVANO, L., RAGG, E., and COCUCCI, S. M. 1995.

Germination and pH of intracellular compartments in seeds of Phacelia tanacetifolia. Physiol.

Plant. 93:577–583.

FOYER, C. H., LOPEZ-DELGADO, H., DAT, J. F., and SCOTT, I. M. 1997. Hydrogen peroxide- and

glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol.

Plant. 100:241–254.

GAUTIER, M.-F., LULLIEN-PELLERIN, V., DE LAMOTTE-GUERY, F., GUIRAO, A., and JOUDRIER, P.

1998. Characterisation of wheat thioredoxin h cDNA and production of an active Triticum

aestivum protein in Escherichia coli. Eur. J. Biochem. 252:314–324.

GLASS, A. D. M. and DUNLOP, J. 1974. Influence of phenolic acids on ion uptake. IV. Depolarization

of membrane potentials. Plant Physiol. 54:855–858.

GRIFFITH, O. W. 1985. Glutathione and glutathione disulphide, pp. 277–289, in H. U. Bergmeyer,

J. Bergmeyer, and H. Grassl (eds.). Methods of Enzymatic Analysis, 3rd edn., vol. 3. Verlag

Chemie, Weinheim, Germany.

GRIMWADE, B., TATHAM, A. S., FREEDMAN, R. B., SHEWRY, P. R., and NAPIER, J. A. 1996.

Comparison of the expression patterns of genes coding for wheat gluten proteins and proteins

involved in the secretory pathway in developing caryopses of wheat. Plant Mol. Biol. 30:1067–

1073.

505INHIBITORY EFFECTS OF COUMARIN ON GERMINATION OF DURUM WHEAT

KHAN, A. A. 1969. Interaction of growth-retarding compounds and gibberellin on indoleacetic acid

oxidase and peroxidase of cucumber seedlings. Plant Physiol. 38:731–737.

KHAN, A. A. and ZENG, G.-W. 1985. Dual action of respiratory inhibitors. Plant Physiol. 77:817–

823.

KRUGER, J. E. 1979. Modification of wheat "-amylase by proteolytic enzymes. Cereal Chem.

56:298–302.

LIVESLEY, M. A., BULLEID, N. J., and BRAY, C. M. 1992. Protein disulfide isomerase in germinating

wheat (Triticum aestivum) seed and during loss of viability. Seed Sci. Res. 2:97–103.

MACIAS, F. 1995. Allelopathy in the search for natural herbicides, pp. 311–329, in L. M. Inderjit,

K. M. M. Dakshini, and F. A. Einhellig (eds.). Allelopathy. Organisms, Processes and

Applications. American Chemical Society, New York.

MORELAND, E. D. and NOVITZKY, W. P. 1987. Effects of acids, coumarins and flavonoids on

isolated chloroplast and mitochondria, pp. 247–261, in G. R. Waller (ed.). Allelochemicals:

Role in Agriculture and Forestry. American Chemical Society, Washington, DC.

NIKOLAEVA, M. G., PODZOVA, L. M., POLIAKOVA, E. N., DALETSKAIA, T. V., RASUMOVA,

M. V., and FIRSOV, G. A. 1987. Possibility of rapid germination of seeds of woody plants

with deep dormancy. Botanicheskii zhurnal 72:480–489.

PIROVANO, L., MORGUTTI, S., ESPEN, L., and COCUCCI, S. M. 1996. Differences in trascription

products and in translation and enzymatic activities during the early stages of imbibition of

Phacelia tanacetifolia seeds with germination inhibited by light. Physiol. Plant. 96:714–721.

QUEIROZ, O. 1969. Photoperiodisme et activite enzymatique (PEP carbolixase malique) dans le

feuille de Kalanchoe blossfeldiana. Phytochemistry 8:1655–1663.

RICE, E. L. 1984. Allelopathy. Academic Press, Orlando.

SANITA’ DI TOPPI, L., MARABOTTINI, R., VATTUONE, Z., MUSETTI, R., FAVALI, M. A., SORGONA,

A., and BADIANI, M. 2005. Cell wall immobilization and antioxidant status of Xanhtoria

parietina thalli exposed to cadmium. Funct. Plant Biol. 32:611–618.

SCHWANZ, P., KIMBALL, B. A., IDSO, S. B., HENDRIX, D. L., and POLLE, A. 1996. Antioxidants in

sun and shade leaves of sour orange trees (Citrus aurantium) after long-term acclimation to

elevated CO2. J. Exp. Bot. 305:1941–1950.

STACY, R. A. P., MUNTHE, E., STEINUM, T., SHARMA, B., and AALEN, R. B. 1996. A peroxiredoxin

antioxidant is encoded by a dormancy-related gene, PER1, expressed during late development

in the aleurone and embryo of barley grains. Plant Mol. Biol. 31:1205–1216.

ULITZER, S. and POLJAKOFF-MAYBER, A. 1963. Oxidative phosphorylation in germinating lettuce

seeds. J. Exp. Bot. 14:95–100.

VAN SUMERE, C. F., COTTENIE, J., DE GREEF, J., and KINT, J. 1972. Biochemical studies in relation

to the possible germination regulatory role of naturally occurring coumarin and phenolics. Rec.

Adv. Phytochem. 4:165–221.

WANG, S. Y., JIAO, H. J., and FAUST, M. 1991. Changes in ascorbate, glutathione, and related

enzyme activities during thidiazuron-induced bud break of apple. Physiol. Plant. 82:231–236.

ZOBEL, A. M. and BROWN, S. A. 1995. Coumarins in the interactions between the plant and its

environment. Allelopathy J. 2:9–20.

ZOBEL, A. M., WANG, J., MARCH, R. E., and BROWN, S. A. 1991. Identification of eight coumarins

occurring with psoralen, xanthoxin, and bergapten on leaf surfaces. J. Chem. Ecol. 17:1859–

1870.

506 ABENAVOLI ET AL.