ORIGINAL PAPER

Coumarin interacts with auxin polar transport to modify rootsystem architecture in Arabidopsis thaliana

Antonio Lupini • Fabrizio Araniti • Francesco Sunseri •

Maria Rosa Abenavoli

Received: 24 September 2013 / Accepted: 17 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Coumarin is a highly active allelopathic com-

pound which plays a key role in plant–plant interactions

and communications. It affects root growth and develop-

ment of many species, but its mode of action has not been

clarified yet. It has been hypothesized that auxin could

mediate coumarin-induced effects on root system. Through

morphological and pharmacological approaches together

with the use of Arabidopsis auxin mutants, a possible

interaction between coumarin and auxin in driving root

system development has been investigated in Arabidopsis

thaliana (Col-0). Coumarin strongly affected primary root

elongation and lateral root development of Arabidopsis

seedlings. In particular, 10-4 M coumarin significantly

inhibited primary root elongation increasing lateral root

number and root hairs length. Further, coumarin addition

was able to restore the negative effects of TIBA and NPA,

two auxin transport inhibitors, which caused a complete

inhibition of lateral root formation. Arabidopsis auxin

mutants differently responded to coumarin compared to

wild type (Col-0). In particular, lax3 mutant showed the

lowest (42 %) inhibition of primary root length, whereas,

eir1-4 mutant had higher inhibition (53 %) compared to

Col-0; conversely, aux1-22 mutant did not show any effect

in response to coumarin. An increase of lateral root number

was observed in pin1 mutant only. Finally, coumarin

increased the root hairs length in eir1-4, lax3, pin1 and

pin3-5 mutants, but not in aux1-22. These results suggested

a functional interaction between coumarin and auxin polar

transport in driving root development in A. thaliana.

Keywords Arabidopsis thaliana � Auxin � Coumarin �Mutants � Root morphology

Introduction

Exploration and exploitation of soil resources such as

water, mineral nutrients by plants is closely associated with

root development (Schlichting 1986) and in particular with

its architecture (root system architecture, RSA), which is

an integrative result of lateral root initiation, morphogen-

esis, emergence and growth (Dubrovsky and Forde 2012)

as well as root hair formation and elongation (Nibau et al.

2008). However, root system is extremely plastic organ,

able to adapt in response to external cues such as nutrients

(Lopez-Bucio et al. 2003), soil moisture and matrix (Hodge

2006) and allelopathic compounds (Whitehead et al. 1982;

Rice 1984). Coumarin is an active allelochemicals widely

distributed in plants kingdom which plays an important

role in plant–plant interaction and communication (Zobel

and Brown 1995). Released into the environment, couma-

rin affected plant growth and development of many spe-

cies, and especially their root system, one of the main

target of allelochemicals (Rice 1984). The negative effects

of coumarin on cell division and root polarity have been

known for a long time (Goodwin and Avers 1950; Avers

and Goodwin 1956), although Neumann (1959) demon-

strated that coumarin markedly stimulated the elongation

of excised segments of Helianthus hypocotyls, comparing

its action to that of the auxin. Coumarin influenced root

morphology and histology (Svensson 1971, 1972; Ku-

pidlowska et al. 1994; Abenavoli et al. 2001), showing a

A. Lupini � F. Araniti � F. Sunseri � M. R. Abenavoli (&)

Dipartimento Agraria, Universita Mediterranea di Reggio

Calabria, Salita Melissari, 89124 Reggio Calabria, RC, Italy

e-mail: mrabenavoli@unirc.it

123

Plant Growth Regul

DOI 10.1007/s10725-014-9893-0

selective effect on maize and Arabidopsis root types,

inhibiting the primary root elongation and stimulating lat-

eral root formation (Abenavoli et al. 2004, 2008). More

recently, Lupini et al. (2010), by morphological and elec-

trophysiological approaches, indicated the root zone within

20 mm from tip of maize primary root as the most sensitive

to coumarin, suggesting that this effect could be mediated

by auxin. However, the exact mechanism of coumarin on

root growth has not been clarified yet. A complex network

of molecular signaling probably governs coumarin mor-

pho-physiological responses, where auxin transport and/or

biosynthesis could play an important role.

Auxin is considered to be the prime candidate to control

stress-induced morphogenic response ‘‘SIMR’’, character-

ized by inhibition of root elongation and enhanced formation

of lateral roots (Potters et al. 2007). It plays a central role in

organ development and elongation, in shoot/root branching

and plastic growth responses (Zazımalova et al. 2010), in

lateral root initiation (Casimiro et al. 2003; De Smet et al.

2006), primordial (Benkova et al. 2003), and emergence

(Laskowski et al. 2006). Transported in polarized streams

(Polar Auxin Transport, PAT), via auxin transporters, auxin

action depends on its differential distribution (Dubrovsky

et al. 2011; Benkova et al. 2009). Moreover, many evidences

established that PAT is mediated by AUX1/LAX uptake PM

permeases, ATP Binding Cassette subfamily B (ABCB)

transporters, and PIN-FORMED (PIN) carrier proteins

(Petrasek and Friml 2009; Zazımalova et al. 2010). In par-

ticular, in Arabidopsis thaliana, the AUX1/LAX permeases

family encode one AUX1, and three Like AUX1 (LAX1, 2

and 3) (Parry et al. 2001), which control rapid and active

auxin influx in cells. On the other hand, eight members of the

AtPIN family (from 1 to 8) have been identified: PIN5, PIN6

and PIN8, localized to endomembranes, regulate the distri-

bution of cellular auxin homeostasis (Mravec et al. 2009),

and PIN1, PIN2, PIN3, PIN4 and PIN7, localized to plasma

membrane (PM), perform cellular auxin efflux (Mravec et al.

2008).

Recently, Li et al. (2011) demonstrated that 4-methyl-

umbelliferone, a coumarin derivative, increased the

expression of two auxin efflux facilitator genes (PIN2 and

PIN3) in Arabidopsis roots, suggesting that auxin redistri-

bution may directly or indirectly mediate 4-methy-

lumbelliferone-induced root branching.

In this respect, the aim of this work was to investigate,

through morphological and pharmacological approaches

together with the use of Arabidopsis auxin mutants, a

possible interaction between coumarin and auxin in driving

root system development, in A. thaliana. In particular,

auxin transport inhibitors, such as NPA and TIBA, and

several AUX1/LAX and PIN Arabidopsis mutants involved

in active auxin transport/redistribution were employed to

confirm this hypothesis.

Materials and methods

Plant materials and growth conditions

Seeds of A. thaliana (Col-0) and aux1-22, lax3, pin1, eir1-4,

pin3-5 mutants were soaked in distilled water for 30 min,

surface sterilized with 95 % (v/v) ethanol and 5 % (v/v)

commercial bleach for 5 min, rinsed 5 times with sterile

water and then placed to 4 �C for 2 days. Sterilized seeds

were plated (Petri dishes, 120 9 120 mm) on solidified-agar

medium [0.75 % (w/v)] plus sucrose [0.5 % (w/v)], MES

(1 g/L), pH 5.75. They were vertically placed in a growth

chamber (22 �C, 65 % RH, 16/8 h, 300 lmol photon flux

density m-2 s-1) (Lupini et al. 2013). Five uniform seed-

lings, 4-day old, were then transferred to a single plate and

grown with same medium containing different coumarin

(MW 146.15, C99 % purity, Sigma Aldrich) and/or IAA

concentrations ranged from 10-15 to 10-4 M for 7 days.

Furthermore, to determine the effective role of auxin in

driving coumarin-effect on root system, a pharmacological

approach using 1.5 9 10-5 M 2,3,5-triiodobenzoic acid

(TIBA) and 5 9 10-6 M naphthylphthalamic acid (NPA)

(Casimiro et al. 2003) was employed.

Root measurements

Roots image was captured by scanning (STD 1600, Regent

Instruments Inc., Quebec, Canada). Briefly, after 7-day of

treatment the Petri dish containing Arabidopsis seedlings

were positioned on the scanner and a image was captured at

1,200 dots per inch (dpi) of resolution. The root length was

measured using WinRhizo Pro system v. 2002a software

(Instruments Regent Inc., Quebec, Canada) and lateral

roots number was counted manually from the image (Ab-

enavoli et al. 2008). According to Dubrovsky and Forde

(2012), to determine lateral root density, the primary root

was divided in two zones: root branching zone that extends

rootward from the shoot base to the last emerged lateral

root, and lateral root formation zone that spreads rootward

from the end of the root branching zone to last lateral root

primordium. Lateral root density has been expressed as

branching density, defined as the number of lateral roots

per unit length of the root branching zone.

Root hair length (lm) and density (root hair number

mm-2 of primary root) were determined by using a ste-

reoscopic microscopy (Olympus SZX9) and Image Tool v.

3 software (UTHSC, San Antonio, USA).

Mitotic sites determination

Number of mitotic sites was evaluated as described by

Canellas et al. (2002) with some modifications. Briefly,

Arabidopsis seedlings were harvested after 7-day of

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123

treatment and their roots were washed in 50 mM phosphate

buffer (pH 7.4), then transferred in 0.5 % KOH (w/v) and

cooled at 75� C for 20 min. The roots were rinsed once

again in 50 mM phosphate buffer (pH 7.4) and then stained

for 16 h in the dark in haematoxylin solution, containing

0.025 g haematoxylin, 0.0125 g ferric ammonium sulphate

and 1.5 mL of 45 % (v/v) acetic acid. Finally, the roots

were distained in 80 % lactic acid (w/v) at 75 �C for

10–20 s, and the mitotic sites were counted by stereoscopic

microscopy (Olympus SZX9).

Statistical analysis

All experiments were laid out in a completely randomised

design with at least fifteen replications for each. The data

were analyzed by one-way ANOVA comparing within and

among treatments, and means were separated by Tukey’s

Honestly Significant Difference (HSD) test (p B 0.05)

using Systat software (Systat Software Inc, Chicago, USA).

In the mutant experiments, the data were analyzed by

Student’s unpaired t test comparing between treated and

untreated roots.

Results

To assess coumarin responses on Arabidopsis root system,

a coumarin dose response of Arabidopsis seedlings (4 day-

old) of primary root elongation and lateral root number was

performed. Coumarin slightly increased primary root (PR)

length up to 10-6 M, whereas, the highest concentration

(10-4 M) significantly inhibited it (Fig. 1). In addition, at

lowest concentrations, coumarin weakly affected lateral

roots number (LR), which was instead significantly

increased (85.3 % respect to control), at the highest one

(10-4 M) (Fig. 1). Based on these preliminary results,

10-4 M coumarin was adopted in the subsequent experi-

ments as the more effective and interesting dose driving

root modifications.

After 7 days of treatment, IAA, ranging from 10-15 to

10-4 M concentrations, with or without 10-4 M coumarin,

affected Arabidopsis root system (Fig. 2). In particular, the

exposure to 10-15 and 10-10 M IAA together with 10-4 M

coumarin caused a significant reduction of the PR length,

which disappeared at the higher IAA levels (10-6 and 10-4

M) compared to IAA alone (Fig. 2a). On the other hand,

the simultaneous presence of coumarin and IAA signifi-

cantly stimulated, especially at lower auxin concentrations

(\10-6 M), the lateral roots number (twofold respect to

IAA alone) (Fig. 2b).

Since IAA moves out the plant cells through an efflux

carrier apparatus sensitive to two synthetic inhibitors,

TIBA and NPA, their effects with or without coumarin

have been tested. Arabidopsis roots treated with TIBA or

NPA showed a significant reduction of the PR length than

Fig. 1 Primary root length (open circle) and lateral root number

(closed circle) of Arabidopsis roots exposed to coumarin for 7 days at

different concentration. The values are presented as mean ± SE

(n = 15). Different letters indicate means that differ significantly,

according to Tukey’s HSD test at p B 0.05

Fig. 2 Primary root length (a) and lateral root number (b) of

Arabidopsis roots exposed to IAA for 7 days at different concentra-

tion with (white) or without (black) 10-4 M coumarin. The values are

presented as mean ± SE (n = 15). Different letters indicate means

that differ significantly, according to Tukey’s HSD test at p B 0.05

Plant Growth Regul

123

untreated ones (36.4 and 46 %, respectively). This effect

was further increased, although not significantly, by the

addition of 10-4 M coumarin in the medium (Fig. 3a). The

application of both auxin transport inhibitors completely

abolished the lateral roots formation (Fig. 3b), which was

instead restored to the control value after coumarin supply

(Fig. 3b).

To assess the effective role of coumarin on lateral roots

formation, mitotic sites were also observed by colorimetric

method (Fig. 4). Coumarin alone increased mitotic sites

number by threefold (18.8 ± 1.8) compared to control

(5.6 ± 0.7) (Fig. 5), and was able to restore it after NPA or

TIBA treatment which, on the other hand, completely

abolished them (Fig. 5).

Moreover, to confirm the probable interaction of cou-

marin on auxin transport and/or distribution Arabidopsis

mutants were employed. The Fig. 6 shows the visual

effects of coumarin on roots of Arabidopsis mutants and

the reference Col-0 (hereafter wild-type, wt). Coumarin

caused an inhibition of the PR length by 57 % in wild-type

respect to untreated one, whereas all mutants showed a

lower inhibition (Fig. 7a). In particular, in lax3 mutant

coumarin inhibited by 42 % the PR length, whereas the 53,

47 and 43 % of inhibition were observed in eir1-4, pin3-5

and pin1-1 mutants, respectively (Fig. 7a). By contrast,

Fig. 3 Primary root length (a) and lateral root number (b) of

Arabidopsis roots exposed to 0 or 10-4 M coumarin and specific

auxin transport inhibitors for 7 days. The values are presented as

mean ± SE (n = 15). Different letters indicate means that differ

significantly, according to Tukey’s HSD test at p B 0.05

Fig. 4 Visualization of the mitotic sites in Arabidopsis root exposed to 0 (a) and 10-4 M coumarin (b)

Fig. 5 Mitotic sites number of Arabidopsis roots exposed to 0 or

10-4 M coumarin and specific auxin transport inhibitors for 7 days.

The values are presented as mean ± SE (n = 15). Different letters

indicate means that differ significantly, according to Tukey’s HSD

test at p B 0.05

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coumarin significantly increased lateral roots number in wt

by 40 % (Fig. 7b) and this root response, although lower

than wt, was also observed in pin1 (17 %), but not in the

others mutants (Fig. 7b). These last results caused conse-

quently modifications on branching density, which was

increased in Arabidopsis wild type by 141.3 %, compared

to untreated (Fig. 8). Similar to wild-type were the

responses in pin3-5 (?62.6 %) and pin1 (?41.2 %)

mutants when exposed to coumarin whereas other mutants

did not show any significant changes (Fig. 8).

Coumarin also affected root zones distribution (root

branching and lateral roots formation zone) in Arabidopsis

seedlings (Fig. 9). In wild-type, coumarin increased root

branching zones from 51 to 72 % of the total root length

(Fig. 9). Furthermore, the aux1-22 (54.1–60.9 %), pin3-5

(63.2–77.7 %) and eir1-4 (47.7–61.8 %) mutants exhibited

a slight increase on branching zone when exposed to

coumarin (Fig. 9), not difference was observed in lax3

(46.7–45.6 %) and finally, pin1 mutant showed the highest

increase of the root branching zones, from 44.6 to 80.2 %

(Fig. 9).

The effects of coumarin were also evident in both root

hairs length (RHL) and density (RHD) (Figs. 10, 11). As in

wild-type (?153 % respect to untreated seedling) the RHL

was strongly increased in pin3-5 (?263 %), pin1 (210 %)

mutants, whereas a slight effect was showed in lax3 (29 %)

and eir1-4 (16 %) mutants (Fig. 11a); contrary, aux1-22

mutant did evidence any modification in response to

coumarin (Fig. 11a). Yet, RHD (root hairs number mm-2

primary root) was also modified by coumarin supply

(Fig. 11b). In Arabidopsis wild-type coumarin significantly

increased the RHD by 44.1 % respect to untreated seed-

lings (Fig. 11b). lax3 mutant showed the highest response,

as the coumarin induced a strong increase of the RHD

(132 %) followed by pin1 (56 %) and eir1-4 (39 %)

(Fig. 11b).

Discussion

The results of present paper have provided convincing

evidence that coumarin affected root development of A.

thaliana seedlings, causing an inhibition of primary root

(PR) length and a stimulation of lateral roots (LR) devel-

opment. However, coumarin-induction of LR number was

not quantitatively correlated with coumarin-inhibition of

primary root elongation. Indeed, the increase in lateral

roots number and the shortening in primary root elonga-

tion, at the same coumarin concentration (10-4 M), was

85.3 versus 48 %, respectively, suggesting that this com-

pound positively regulated the LR development. This

typical SIMR phenotype induced by coumarin which

comprised a mixture of inhibition and activation has been

already observed in maize (Abenavoli et al. 2004) after

coumarin treatment and under several abiotic stresses

(Potters et al. 2007), such as phosphate starvation (Lopez-

Fig. 6 Representative root phenotype of Arabidopsis reference and mutants in response to the coumarin supply

Plant Growth Regul

123

Bucio et al. 2002), salt stress (Zolla et al. 2010) and

brassinosteroids treatments (Bao et al. 2004). Among sig-

nals that mediate SIMR, auxin patterning and/or redistri-

bution plays a central role (Potters et al. 2007). The

phosphorus-deficiency SIMR in Arabidopsis is linked to

changes in auxin sensitivity (Lopez-Bucio et al. 2002), the

cadmium SIMR in Arabidopsis and the aluminum SIMR

are correlated with changes in auxin distribution (Kol-

lmeier et al. 2000; Doncheva et al. 2005) and the UV-B

SIMR is associated with altered auxin metabolism (Jansen

et al. 2001). Thus, to examine the auxin role in coumarin

SIMR responses in Arabidopsis roots, IAA with or without

the allelochemical were applied in the growth medium. In

absence of coumarin, up to 10-6 M IAA did not cause

significant effects on both root traits, while the higher IAA

concentrations dramatically decreased primary root length

increasing the number of lateral roots. These effects were

further marked in treatments with a combination of 10-4 M

coumarin and lower IAA concentrations. These results

indicated that at low IAA levels, coumarin and auxin

produced largely additive effects on both inhibition of

primary root elongation and promotion of lateral root for-

mation. However, at highest IAA concentrations, no addi-

tive effects were observed with coumarin. Probably, as

proposed by Bao et al. (2004) in brassinosteroids and auxin

interaction, at low IAA levels, coumarin could increase the

endogenous auxin level, transport or sensitivity promoting

its activity, while at highest, signals activating lateral root

formation may be saturated, masking the additive effects of

Fig. 7 Primary root length (a) and lateral root number (b) of

Arabidopsis mutants exposed to 0 or 10-4 M coumarin for 7 days.

The values are presented as mean ± SE (n = 15). ***, **, * = sig-

nificantly different at p B 0.001, 0.01, 0.05, respectively, according to

Student’s unpaired t test

Fig. 8 Branching density of Arabidopsis mutants exposed to 10-4 M

coumarin for 7 days. The values are presented as mean ± SE

(n = 15). ***, **, * = significantly different at p B 0.001, 0.01,

0.05, respectively, according to Student’s unpaired t test

Fig. 9 Root zone distribution of Arabidopsis mutants exposed to 0

(-) and 10-4 M (?) coumarin for 7 days. The values are presented as

percentage of the distribution along primary root (n = 15)

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123

auxin and coumarin. Since auxin-coumarin interaction may

involve auxin polar transport (PAT), TIBA and NPA

effects on root traits, alone or in combination with cou-

marin, were tested. The results indicated that, in the pre-

sence of both inhibitors, the degree of inhibition of primary

root elongation conferred by coumarin did not vary. Con-

versely, both inhibitors alone not only blocked lateral root

formation in wild type seedlings but also reduced coumarin

promotion of lateral root formation. These results further

confirmed a potential interaction between coumarin and

auxin in the promotion of lateral root formation and pro-

viding additional evidence that SIMR induced by coumarin

may act through PAT modulation to activate lateral root

development. Recently, Chavez-Aviles et al. (2013) dem-

onstrated that SIMR induced by osmotic stress implicated

the modulation of PAT triggering LR development in

Arabidopsis seedlings.

Furthermore, coumarin not only restored the lateral

roots number to control value in presence of auxin trans-

port inhibitors but also the mitotic sites number, suggesting

that it was able to overcome the inhibitory effect of both

auxin transport inhibitors positively acting on lateral root

initiation and emergence of lateral root primordia.

Since local auxin gradient in the roots is the result of the

directional cell–cell transport coordinated by influx

(AUX1/LAX family) and efflux (PIN family) systems

(Mravec et al. 2008), coumarin could interact with the

auxin transport influencing either or both, the PIN or

AUX1 proteins. To better understand this interaction,

Arabidopsis mutants failed in influx or efflux carriers, have

been employed. Except for aux1-22, all Arabidopsis

mutants exhibited a significant inhibition of primary root

elongation in response to coumarin respect to untreated

seedlings. AUX1 is an auxin influx facilitator participating

in both acropetal and basipetal auxin transport at the root

tip (Swarup et al. 2001). The coumarin resistance of the

aux1-22 mutant therefore suggested that auxin transport

within the root tip may have a positive role in mediating

the coumarin effect on primary root inhibition. Moreover,

except to pin1 mutant and Col 0, coumarin did not cause

any increase in lateral roots number, indicating a possible

modulation of the PIN2, PIN3 and AUX1/LAX carriers in

lateral roots development induced by this compound. The

results on root branching density and root zone distribution,

processes largely known to be dependent on auxin flux (De

Smet et al. 2007; Marchant et al. 2002; Peret et al. 2009),

further confirmed that coumarin may modulate auxin dis-

tribution through influx or/and efflux proteins. Finally, root

Fig. 10 Effect of coumarin on the development of root hairs in Arabidopsis reference and mutant (bar = 150 lm)

Fig. 11 Root hair length (a) and Root hair density (b) of Arabidopsis

mutants exposed to 0 or 10-4 M coumarin for 7 days. The values are

presented as mean ± SE (n = 30). ***, **, * = significantly differ-

ent at p B 0.001, 0.01, 0.05, respectively according to Student’s

unpaired t test

Plant Growth Regul

123

hair formation, process strongly influenced by auxin bal-

ance (Evans et al. 1994; Jones et al. 2009), in wild type and

mutants in response to coumarin have been investigated.

Except to aux1-22 mutant, coumarin treatment led to an

increase in root hair elongation. Considering that this

process is a result of increased auxin concentration by

AUX1-dependent transport in epidermal cells (Evans et al.

1994; Sabatini et al. 1999; Jones et al. 2009), coumarin

might be act via the modulation of auxin influx in root once

again. Furthermore, not differences in root hair density

have been observed in aux1-22 and pin3-5 mutant in

response to coumarin.

In conclusion, the results suggested a functional inter-

action between coumarin and auxin polar transport, and

particularly with AUX1 carrier which determined rootward

auxin transport. Further studies will be needed to better

delineate coumarin mechanisms on regulating auxin

abundance, subcellular trafficking and/or transport activity

of auxin carriers. The dissection of such downstream

organizing events will be a future challenge.

Acknowledgments We would like to thank Eva Benkova (VIB

Department of Plant Systems Biology, UGent), Malcolm Bennett and

Ranjan Swarup (Plant Sciences Division, School of Biosciences,

University of Nottingham) for providing Arabidopsis mutants used in

the present work.

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