Short-term effects of coumarin along the maize primary root axis

Abstract

The short-term effects of coumarin on three different maize primary root zones, transition zone (TZ, 3 mm) and two non-growing zones (NGZ1 and NGZ2 at 20 and 50 mm, respectively), were studied in order to investigate the effect of the allelochemical on maize root elongation rate (RER). The RER, plasma membrane (pm) H⁺-ATPase activity, quantitative pH changes and cell membrane potentials were evaluated. The results showed that coumarin caused at the TZ (1) an increased RER; (2) an enhancement of pm H⁺-ATPase activity and proton extrusion; and (3) a transient depolarization followed by a hyperpolarization of cell membrane potential. These observations were not evident in the NGZ1 and NGZ2 of the maize root. Coumarin-treatment in the NGZ1 did not change RER, but caused a membrane depolarization, while the NGZ2 was mostly insensitive to the allelochemical. These data suggested that the primary maize root was sensitive to coumarin within a 20 mm section from the root tip, but the more distal NGZ2 was not involved in coumarin-elicited physiological responses.

Introduction

Coumarin is a simple allelopathic compound found in many plants,¹ widely distributed in both natural plant communities and crops,² where it plays an important role in plant-plant interactions and biocommunication.^3,4 Like other allelochemicals, such as phenolic compounds released from living plants into the environment,⁵ coumarin can influence many physiological and biochemical processes: root growth,^6–9 nitrate uptake and metabolism,^10,11 respiration and photosynthesis¹² and germination.^13–15 The effect of coumarin is species-specific^7,15 and concentration-dependent, often stimulatory at low and inhibitory at high concentrations.^9,14 Coumarin has been widely studied for allelopathic effects on root growth, organ considered the primary target of this compound.¹⁶ Earlier investigations indicated clearly that coumarin changed root cell polarity of growth, causing an inhibition of longitudinal root cell elongation accompanied by a simultaneous stimulation of radial expansion.^6,17 These effects were also observed in alfalfa grass, where the thickness of seminal roots was enlarged abnormally because of an inhibition of the longitudinal root growth.⁸ Recently, Abenavoli et al.¹⁵ demonstrated a selective and species-specific effect of coumarin on the root growth of individual roots: 100 µM coumarin inhibited primary root length and stimulated lateral root formation in Arabidopsis. By contrast, in maize seedlings, coumarin did not inhibit growth of the primary root relative to seminal and nodal roots.⁹ However, these effects were observed after long-term coumarin exposure (48 h), while little information is available for the short-term effects of coumarin on the morphological and physiological responses of maize roots. This knowledge may be more useful for a better understanding of the allelochemicals mode of action. Furthermore, plant root axes are characterized by having different zones with diverse anatomical, morphological and physiological traits which can respond differently to nutrient, water and allelochemicals which are heterogeneously distributed in soils. Particularly, the transition zone (TZ), localized 1.7–3.4 mm from the tip, has been characterized as having special cell physiological properties which allow the root to respond to a wide range of environmental signals.^22–24 Furthermore, coumarin-induced swelling in the root apex, similar to that caused by mechanical impedance in the TZ behind the meristem, has been reported.²²

The differential short-term responses of three different primary root zones of maize, TZ, non-growing zones 1, 2 (NGZ1 and NGZ2) to localized coumarin treatment have been investigated. In particular, the ability of coumarin to locally influence the cell plasma membrane, an early event of the allelochemical action,²⁵ along the maize root axis has been studied. To address these both questions, the effect of coumarin on pm H⁺-ATPase activity, proton efflux and cell plasma-membrane electrical potential difference in each zone of the primary maize root was evaluated and the results are reported in this paper.

Results

A different response pattern of RER to 0, 25, 50 and 100 µM coumarin treatments within the TZ, NGZ1 and NGZ2 of intact root maize seedlings was clearly evident (Fig. 1). In the TZ, a significant increase of RER was induced by 50 and 100 µM coumarin (0.0221 and 0.0258 mm min⁻¹, respectively) compared to the control (0.0133 mm min⁻¹) and 25 µM coumarin treatment. By contrast, in the NGZ1 and NGZ2, RER was not significantly affected by all coumarin concentration treatments (Fig. 1).

Figure 1.

Effect of coumarin supply (30 min) in agar medium on root elongation rate of TZ (3 mm), NGZ1 (20 mm) and NGZ2 (50 mm) of primary maize root. Means with different letters are significantly different (p < 0.05, Tukey's test) with regard to root zones.

A similar response pattern within the TZ, NGZ1 and NGZ2 was observed for pm H⁺-ATPase activity when exposed to coumarin. Indeed, in the TZ, H⁺-ATPase activity markedly increased by 64% with respect to the control after 100 µM coumarin exposure, with lesser stimulation caused by 50 µM and none with a 25 µM coumarin treatments (Fig. 2). In NGZ1 and NGZ2, H⁺-ATPase activity was not significantly modified by treatment at all three coumarin concentrations (Fig. 2).

Figure 2.

H⁺-ATPase activity (nmol P_i µg⁻¹ prot. h⁻¹) of plasma membrane vesicles isolated from root of maize seedlings exposed for 30 minutes to different coumarin concentrations. Data are the mean of five replicates and bars indicate the standard error. Within each root zone, different letters indicated difference at p < 0.05 (Tukey's test).

For the TZ, the proton efflux was significantly stimulated by 50 and 100 µM coumarin treatments by 50.3 and 36.6% respectively, compared to the control, while this increase was not observed after 25 µM coumarin exposure (Fig. 3). By contrast, in the more distal root zones (NGZ1 and NGZ2), the proton efflux was significantly inhibited by 50 or 100 µM coumarin treatments by 71–79% and 68–74%, respectively, while 25 µM coumarin showed a similar behavior to the control (Fig. 3).

Figure 3.

Effect of coumarin on proton efflux from the TZ (3 mm), NGZ1 (20 mm) and NGZ2 (50 mm) of primary maize root. Means with different letters are significantly different (p < 0.05, Tukey's test) with regard to root zones.

Cell membrane potentials, before, during and after the coumarin treatment were recorded (E_m), in order to detect the initial cell responses within the various root zones of intact primary maize roots. After insertion of a microelectrode into the mature epidermal cells and the stabilization of E_m (<10 min) in a nutrient solution without the coumarin, the E_m was significantly different among the three root zones (Fig. 4). Then, the bathing medium was changed to one containing coumarin at different final concentrations and the effect on the E_m was evaluated within each of the root zones. In the TZ, all the coumarin concentrations caused an immediate and transient depolarization (less negative electrical potential), followed by a more negative hyperpolarization (Fig. 4A–C). While the depolarization response was not significantly different among the various coumarin treatments, the hyperpolarization phase varied in relation to the allelochemical concentrations (Table 1). Indeed, 50 µM coumarin caused a hyperpolarization of about 21 ± 1.8 mV, which was statistically different from 25 µM (9 ± 3.6 mV), but similar to that induced by 100 µM coumarin (14 ± 2 mV) (Table 1). In the NGZ1, the coumarin addition only elicited a depolarization with no subsequent hyperpolarization (Fig. 4D–F). At the highest coumarin concentrations (50 and 100 µM), the depolarization of the cell membrane potential (26.3 ± 4.1 and 21.7 ± 2.3 mV, respectively) was significantly different from that measured with a 25 µM coumarin treatment (10 ± 0.6 mV) (Table 1). By contrast, in the NGZ2, coumarin did not cause any significant effects on the cell plasma membrane potential (Fig. 4G–I and Table 1).

Figure 4.

Electrophysiological traces of membrane potential of the TZ (3 mm), NGZ1 (20 mm) and NGZ2 (50 mm) of primary maize root exposed to 25 µM (A, D and G, respectively), 50 µM (B, E and H, respectively) or 100 µM coumarin (C, F and I, respectively). The data displayed the curves from representative membrane potential recording of five similar experimental results for 30 minute of exposure. Arrow indicates the starting time of coumarin perfusion.

Table 1.

Depolarization (DEP) and hyperpolarization (HYP) of root membrane potential along primary maize root by different concentrations of coumarin

Coumarin (µM)			Distance from tip (mm)
	3		20		50
	DEP	HYP	DEP	HYP	DEP	HYP
25	4 ± 1a	9 ± 3.6a	10 ± 0.59a	0	0	0
50	3 ± 0.54a	21 ± 1.82b	26.33 ± 4.1b	0	0	0
100	3 ± 0.57a	14 ± 2ab	21.66 ± 2.3b	0	0	0

Mean ± SE. Different letters (along columns) for the different concentrations indicate significant differences at p < 0.05 (Tukey's test).

Discussion

Coumarin treatments in the TZ, NGZ1 and NGZ2 of maize primary roots elicited differing morpho-physiological responses. More specifically in the TZ, but not at greater distances (NGZ1 and NGZ2), coumarin caused (1) an increased RER; (2) an higher H⁺-ATPase activity; (3) an enhancement of proton extrusion; and (4) a transient depolarization followed by a sharp hyperpolarization of membrane potential. This coumarin-induced pattern in TZ was similar to that induced by auxin in oat and maize coleoptiles^26,27 and may suggest an auxin-like behavior or/and an interaction with the auxin signaling pathways for this allelochemical. A possible interference of coumarin on auxin metabolism was already reported in Petunia hybrida.²⁸ From these results, a mechanism action of coumarin in the TZ of maize primary root, based on the classic acid growth theory, could be proposed. Coumarin interacts with the plasma membrane causing an immediate transient depolarization and then subsequently, directly or indirectly, stimulated the pm H⁺-ATPase activity resulting in an increased H⁺ efflux with consequently more negative hyperpolarization of the plasma membrane. The increase of the proton release determined an acidification of the apoplast, thereby facilitating root growth rate as confirmed by the increased root elongation rate in the TZ after coumarin treatments (Fig. 1).

In contrast to the TZ, coumarin-response pattern in the NGZ1 displayed (1) an unchanged RER and pm H⁺-ATPase activity; (2) an inhibition of proton extrusion; and (3) a sharp depolarization of membrane potential. Therefore, the only common effect induced by coumarin in both root zones is to the depolarization of the membrane, possibly suggesting that, although coumarin is electrically neutral, its uptake occurred via H⁺-coupled mechanism. This cotransport mechanism, proposed by Pang et al.²⁹ for undissociated phenolic acids, could be responsible for the substantial membrane depolarization occurring after coumarin treatment. How can we explain the other contrasting responses to coumarin between the TZ and NGZ1 of maize primary root? Assuming that coumarin could exhibit an auxin-like behavior or/and interact with the auxin signalling pathways, probably a lower auxin sensitivity or concentration in the NGZ1 could be limiting the coumarin mediated responses in this root zone. Indeed, an asymmetrical auxin distribution along root axis of Arabidopsis thaliana (0 to 3 mm, 3 to 10 mm and 10 to 20 mm) with the highest concentrations in the root tip/meristem/elongation zones and lowest toward the basal region was observed.³⁰ A lower auxin content in the NGZ1 could not be adequate to reach the level required for the auxin-induced activation of the pm H⁺-ATPase, which in turn led to decreasing H⁺ efflux resulting in change in the RER. For example, an adequate auxin budget plays a central role in controlling lateral root initiation in Arabidopsis thaliana.³¹

Finally, the NGZ2 was the region of the maize primary roots less affected by coumarin treatment since no change in the root elongation rate, pm H⁺-ATPase activity and plasma membrane potential was measured, although a decrease in the H⁺ efflux was observed. As cells mature along the developing root axis, differential changes in gene expression may explain these differences.³³ The presence of lignified sclerenchymatous fibers³² and suberized endo- and exodermal cells³⁴ in the more mature root regions of maize may limit the coumarin-plasma membrane interactions.

Overall, these short-term coumarin treatment experiments confirm the plasma membrane transport activity as an early target for the allelochemicals. Further, the experiments indicated the presence of a threshold concentration dividing the stimulatory from non-effect for the allelochemical. Indeed, 25 µM coumarin did not produce any physiological responses along primary maize root while 50 and 100 µM coumarin, as observed in a previous study on root anatomy, morphology and physiology in a durum wheat cultivar showed a stimulatory effect on root elongation rate.¹⁰ Finally, the results showed that the transition zone was the region of the maize primary root most responsive to the coumarin treatments. The important “sensing zone” role of the root tip was also evoked for other environmental cues such as chilling,³⁵ water stress³⁶ and nutritional signals such as nitrate³⁷ and phosphate.³⁸

An ecological role could be attributed to this localized root tip response to coumarin. The root tip is the zone first encountering and interacting with the soil environment in which, in addition to nutrient and water resources, allelochemicals such as coumarin could be present. Indeed, plant residues, litter decomposition in the top soil and rain leaching from foliage may provide sources of coumarin.³⁸ Furthermore, root exudates also contain coumarin providing a mechanism to sense resource competition from other plants.⁴⁰ Finally, coumarin concentrations in the soil will depend on microbial activity, but the ranges used for experimental measurements seem feasible for those occurring in nature.³⁹ Hence, during exploration for nutrients and water, the root could be exposed to a locally allelochemical-enriched soil, which is sensed by a root tip zone of environmental perception and signal transduction.

In conclusion, these results suggested several important considerations: (1) the TZ of primary maize root is the most sensitive to coumarin; (2) the morpho-physiological responses of the more apical root zones to the coumarin showed an auxin-like pattern. Further studies are necessary to better understand if the change in bioelectric pattern of membranes induced by coumarin could be due to an H⁺-coupled transport of the allelochemical as suggested for monocarboxylic and benzoic acids²⁹ or to alterations in the flux of ions as reported for monoterpenes^41,42 and then a subsequent direct or indirect action in the TZ auxin perception system.

Materials and Methods

Plant material and growth condition.

Maize (Zea mays L., cv Cecilia, Pioneer, Italia) seeds, previously immersed in deionized water for 48 h, were germinated over aerated 0.5 mM CaSO₄ solution, in controlled conditions (continuous darkness; 24°C and 70% RH). After 72 h, homogeneous seedlings were transferred into hydroponic culture containing 1 l of aerated one-fourth strength Hoagland solution (NS, nutrient solution). The pH was adjusted to 6.0 with 0.1 N KOH. The seedlings were maintained in a growth chamber at 24 ± 1°C with a 14 h photoperiod, at a photon flux density of 300 µmolm⁻²s⁻¹ at plant height and 70% RH for seven days until the experimental measurements. All reagents used were of the highest analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO USA).

Root elongation rate.

Root elongation rate (RER) of three different zones (TZ, NGZ1 and NGZ2 at 3, 20 and 50 mm of distance from the root tip, respectively) of primary root of intact maize seedlings was determined as previously described by Sivaguru and Horst,⁴³ with minor modifications. Briefly, agar dissolved in 0.5 mM CaSO₄ (0.75% w/v) was layered in Petri dishes (120 × 120 mm) and divided in three segments. Either zero, 25, 50 or 100 µM coumarin was added to the cooled agar solution only into the middle agar segment (1 mm width, Fig. 5). Primary root of intact maize seedlings was then vertically placed in the agar plates so that the various root zones, TZ, NGZ1 or NGZ2 were in contact with the coumarin-treated agar segments. Then an image of individual root segment was captured after 0 and 30 min of coumarin exposure using a digital camera (Olympus C-5050). The length of each root zone was measured using the WinRHIZO pro STD 1600 software (Instruments Régent Inc., Canada) and, RER was calculated as the increase of the root length during the 30 min of coumarin treatment (Fig. 5). Care was taken to ensure that the maize roots were positioned vertically so that gravity-induced curvature did not interfere with the measurements.

Figure 5.

Experimental set up for root growth rate measurements.

H⁺-ATPase assay.

Isolation of plasma membrane vesicles. Plasma membrane vesicles were isolated from primary root zones of maize seedlings using a small-scale procedure from Giannini et al.⁴⁴ modified by Santi et al.⁴⁵ Treated and control maize root zones (1–1.5 g) were homogenized in extraction buffer (250 mM sucrose, 10% (v/v) glycerol, 10 mM glycerol-1-phosphate, 2 mM MgSO₄, 2 mM EDTA, 2 mM EGTA, 2 mM ATP, 2 mM DTT, 5.7% (w/v) choline chloride and 25 mM BTP buffered to pH 7.6 with MES and 1 mM PMSF and 20 mg/ml chimostatin freshly added before homogenization), filtered and centrifuged twice at 12,700 g for 3 and 25 min, at 4°C. The suspension was layered over a 25/38% discontinuous sucrose gradient (10 mM DL-α-glycerol-1-phosphate, 2 mM MgSO₄, 2 mM EGTA, 2 mM ATP, 1 mM PMSF, 2 mM DTT, 20 mg/ml chimostatin, 5.7% choline chloride, 5 mM BTP buffered at pH 7.4 with MES) and centrifuged at 12,700 g for 60 min at 4°C. The vesicles, banding at the 25/38% interface layers, were collected and centrifuged at 14,000 g for 45 min at 4°C. The pellets, resuspended in a medium (20% glycerol (v/v), 2 mM EGTA, 2 mM EDTA, 0.5 mM ATP, 1 mM PMSF, 2 mM DTT, 20 mg/ml chimostatin, 5.7% choline chloride, 5 mM BTP buffered at pH 7 with MES), were immediately frozen in liquid N₂ and stored at −80°C until use.

Protein assay. Total soluble protein was estimated according to the Bradford⁴⁶ using bovine serum albumin as standard.

ATPase activity. ATP-hydrolyzing activity was determined by measuring the release of inorganic phosphate, as described by Forbusch⁴⁷ at 38°C. Assays were performed at 38°C in a 0.6 mL assay medium containing 50 mM BTP-MES, pH 6.5, 5 mM MgSO₄, 5 mM ATP, 0.6 mM Na₂MoO₄, 100 mM KNO₃, 1.5 mM NaN₃, 0.01 % (w/v) Brij₅₈, with or without 100 µM vanadate (V₂O₅), an inhibitor of P-type H⁺-ATPase.⁴⁷ Sodium azide and KNO₃ were used as selective inhibitors of mitochondria and tonoplast H⁺-ATPase, respectively. The difference between these two activities was attributed to the pmH⁺-ATPase. The reaction was initiated by the addition 0.5–1.5 µg of membrane protein and was stopped after 30 min with a solution containing: 0.6 M HCl, 3% (w/v) SDS, 3% (w/v) ascorbic acid and 0.5% (w/v) ammonium molybdate at 2°C. The enrichment degree in plasma membrane of vesicles was determined in the presence of 0.1 mM V₂O₅, 1 mM NaN₃ and 150 mM KNO₃, selective inhibitors of plasma membrane, tonoplast and mitocondrial ATPase, respectively.

Proton efflux assay.

H⁺ efflux was measured from the change in pH of an unbuffered solution bathing the root (modified from Glass et al.⁴⁸). Primary root of intact maize seedlings was positioned in a chamber that was partitioned into three compartments each of which incorporated three distinct zones (TZ, NGZ1 and NGZ2) isolated by silicone grease. Each root zone compartment contained 0.5 mM CaSO₄ pH 6.0 solutions, with or without (control) 25, 50 or 100 µM coumarin added. The pH of each compartmental solution was monitored for 30 min using a pH electrode (4.5 mm diameter tip, Thermo Scientific, Auchtermuchty, Scotland, model CMAW711). The extrusion rate was expressed as µmolH⁺g⁻¹ fresh weight h⁻¹ and was calculated from the measured change in pH.

Membrane potential measurements.

All electrophysiological experiments were performed on intact, 100 mm long, primary roots. The membrane electrical potential of the outermost layer of cells was measured at TZ, NGZ1 and NGZ2 using a standard glass microelectrode technique. Single-barrelled microelectrodes were prepared using filamented borosilicate glass as described previously.^50,51 The microelectrode was backfilled with 200 mM KCl solution using a 70 mm long Microfil needle (World Precision Instruments Inc., Stevenage, UK). For the electrode impalement the primary root of intact maize seedlings, 7 day-old, was placed in a Plexiglass chamber and perfused with a solution containing 0.5 mM CaSO₄, 2 µM KNO₃, 1 mM MES-NaOH (pH 6) and 25, 50 or 100 µM coumarin. Impalements with microelectrodes were always made in mature epidermal cells and measured the voltage difference (mV), between the inside of the cell and the external bathing solution. The values from −70 to −140 were considered to define a successful cell microelectrode impalement and measurement. The initial impalement of an epidermal cell could be confirmed visually and by the accompanying jump in the voltage recorded after which it was not possible to see the precise location of the tip. Before the coumarin treatment, a time interval (10 min) of stable cell electrical membrane potential was recorded (data not shown).

Statistical analysis.

Data were firstly checked for deviations from normality and homogeneity of variances. The Em, proton efflux, pm H⁺-ATPase activity were analyzed using one-way ANOVA (coumarin concentration) with a completely randomized design with 5 replicates, while RER data were representative of 10 replicates. The Tukey's test was used for comparing the means within each root zones. Statistical analysis was run using Systat v. 8.0 software package (SPSS Inc.).