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• The effects are reported here of coumarin, an allelopathic compound, on root anatomy and growth, nitrate uptake and translocation to the shoot, as well as respiration in Triticum durum (cv. Simeto) seedlings.
• Wheat seedlings were grown in nitrogen-free hydroponic culture; after 6 d, coumarin (at concentrations of 0, 25 µM, 0.1, 1, 2.5 and 5 mM) and/or nitrate (50 µM) were added to the nutrient solution.
• Coumarin, in the range 25 µM–1 mM, decreased the relative growth rate of roots and increased the area of the root vessels. Within this concentration range, coumarin alone did not significantly affect net nitrate uptake. In seedlings exposed simultaneously to 100 µM coumarin and to 50 µM nitrate, the net nitrate uptake was significantly stimulated. In the presence of nitrate, even the lowest coumarin concentration tested significantly stimulated nitrate translocation from the root to the shoot.
• The effects of low coumarin concentrations on root vessel size could explain this observation, though specific interactions between coumarin and systems regulating nitrate uptake and transport within the root cell cannot be excluded.
Coumarins are lactones of o-hydroxycinnamic acid, and are allelopathic compounds that originate in the phenylpropanoid pathway. They are synthesized by almost all higher plants (Murray et al., 1982), where they are found on the surfaces of leaves, seeds and fruits (Zobel & Brown, 1995). Following shedding of these organs, coumarins are likely to diffuse into the soil and affect growth and performance of plants (Brown, 1981; Rice, 1984).
Here, we describe the influence of coumarin on induction and feedback regulation of nitrate uptake in seedlings of durum wheat (Triticum durum). Histological modifications and respiratory O2 consumption, which is strongly correlated with anion uptake in plants (Marschner, 1996), are also monitored.
Materials and Methods
Plant material and growth conditions
Seeds of Triticum durum Desf., cv Simeto, were surface-sterilized for 20 min in 20% (v/v) sodium hypochlorite solution and then rinsed repeatedly in distilled water. The seeds were germinated at 24°C for 36 h and then transferred into a growing unit containing 11 l of aerated one-fourth-strength Hoagland solution (Hoagland & Arnon, 1950) without nitrogen. The pH was adjusted to 6.0 with 0.1 M potassium hydroxide (KOH). The growing units were transferred to a growth chamber at 24°C with a 14-h photoperiod, a photon fluence rate of 300 µmol m−2 s−1 at plant height and 70% rh. During the 5 d growth period, the hydroponic medium was renewed on day 2 and day 4 and the pH was maintained at 6.0 with 0.1 M KOH.
After 5 d of growth the seedlings were transferred to a nutrient solution containing coumarin at a final concentration of 0, 25 µM, 0.1, 1, 2.5 or 5 mM. For each coumarin treatment, half of the plants were given 50 µM KNO3 (final concentration) in the nutrient solution, and were referred to as ‘induced plants’. The remaining plants (‘uninduced plants’) were left without added KNO3 in the nutrient solution.
All reagents used were of the highest analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Root growth and histological analysis
For each coumarin concentration, 30 seedlings were removed from the growing units before (t1) and after (t2) 24 h of treatment. For each seedling, all the seminal roots were used for calculating the relative growth rates for root length (RGRrl) according to Hunt (1982) as modified for root length:
[RL, root length (cm).]
One root tip (0.1 cm long) was collected from each seedling, fixed in ethanol : chloroform : acetic acid (6 : 3 : 1 by volume), dehydrated in a graded series of ethanol followed by xylitol and embedded in paraffin wax (Carnoy & Ricci, 1973). Sections 10 µm-thick, obtained with a microtome from the apical meristems, were stained with light green and ruthenium red and observed through a light Zeiss Axioskop photomicroscope (Carl Zeiss, Jena, Germany). For each of the 30 sections, all the root vessels (6–8 for each section) were measured and the resulting mean area was calculated.
Net nitrate uptake
In the present experiment, net nitrate uptake (that is the difference between nitrate influx and efflux) was measured using the method of Sidari et al. (1998). For each treatment, 60 seedlings were collected after 0, 2, 4, 8 and 24 h and their roots were rinsed carefully with nutrient solution. Then, the seedlings were transferred into a solution containing 0.1 mM KNO3, which was continuously aerated by bubbling with air. Samples were taken from the solution at 5 min intervals over a 30 min total period, and their nitrate concentration was measured at 210 nm (Goldsmith et al., 1973; Albuzio et al., 1986) with a UV-vis spectrophotometer (Shimadzu Model 2100, Shimadzu Corporation, Kyoto, Japan). Reaction blanks were obtained by incubating wheat seedlings in KNO3-free solutions. The net rate of nitrate uptake was calculated from the linear phase of the nitrate consumption curve. Each nitrate uptake experiment was conducted at least in quadruplicate.
Nitrate concentrations in plant tissues
Roots and leaves from wheat seedlings (1 g f. wt each) were homogenized on ice (Ultra-Turrax T25, Yanke and Kunkel, Staufen, D) in 6 ml of distilled water. The homogenates were centrifuged for 20 min at 30 000 ×g. Nitrate was measured in the clear supernatant by the colourimetric assay of Cataldo et al. (1975) and appropriate reaction blanks were run in parallel. All measurements were conducted in quadruplicate.
Roots excised from 8 to 10 seedlings from each treatment were cut into small pieces and then placed in a cuvette containing air-saturated buffered nutrient solution at 25 ± 0.5°C, pH 6.0. Total root respiration rates were calculated from measured rates of O2 depletion, determined polarographically with a Clark-type electrode (Hansatech Ltd, King’s Lynn, UK). Root respiration was determined within 5–15 min of excision from the shoot. In preliminary experiments, this time lag was found to have a negligible effect on the rate of O2 uptake. All measurements were conducted in quadruplicate.
Calculations and statistics
To distinguish between treatment effects on induction and feedback regulation of net nitrate uptake, the following mathematical model was developed.
Induction and the subsequent feedback inhibition can be viewed as a reaction sequence consisting of two first-order reactions:
A, the initial nitrate uptake rate, rises to B, an induced uptake rate, before decreasing, after feedback inhibition takes place, to C, a final uptake rate, which is different from A. These two first-order reactions can be represented as follows:
(A0, the net uptake rate at t = 0.) Since B incorporates constitutive and induced uptake rates (positive terms) as well as the effect of feedback inhibition (negative term), it follows that:
Rearranging and writing in integrated form yields:
(I(t), the nitrate uptake rate, in µmol NO3− (g−1 root f. wt) h−1; t, the time in h and K1 and K2 are the induction and the inhibition rate constants, respectively, expressed in h−1.) A and Iconst represent the induced and the constitutive uptake rates, respectively. The t1/2-values denote the time required to attain half the full induction and half the decay of net nitrate uptake rates, and were calculated from the ratios ln2 : K1 and ln2 : K2, respectively.
The model parameters were estimated by the method of least squares (TableCurve 2D vs 4.0 software, Jandel Scientific Ekrath, Germany) using the Levenberg-Marquardt optimization algorithm. Nonlinear regressions were repeated at least four times using different initial parameter estimates for each set of data. All runs gave the same parameter estimates.
The data obtained for RGRrl, net nitrate uptake, root respiration and nitrate content were submitted to ANOVA and the significant differences between means were compared by the Tukey’s test at P = 0.05.
One-way and two-way ANOVA and curve fitting on experimental data were performed by means of the Sigma Stat vs 3.0 and the Sigma Plot vs 3.0 (Jandel Scientific, Ekrath) software packages, respectively.
Exposure of wheat roots to 1 mM coumarin caused a progressive necrosis of the apical zone and primary structure (Fig. 1a,b), although the intact portion of the primary tissue was still able to form lateral roots (Fig. 1c). At 0.1 mM, coumarin caused swelling above the apex (Fig. 2a,b). Exposure of the roots to 2.5 or 5 mM coumarin exacerbated these toxic effects (data not shown) and, at the highest concentration, even led to the formation of cracks at the surface of the elongation zone and in the external layers of the cortex (Fig. 2c). Exposure to 5 mM coumarin also increased the number of cell layers in the root cap compared both with unexposed roots and with roots receiving lower concentrations of coumarin (Fig. 2c). Since they appeared to damage root structure, and consequently its function, coumarin concentrations above 1 mM were not studied further.
Table 1 shows that, after exposure for 24 h, increasing coumarin concentrations progressively and significantly decreased the relative growth rate for root length (RGRrl). At 0.1 mM coumarin, the decrease of RGRrl tended to be stronger (−49% in respect to control) than at either 25 µM or 1 mM (Table 1). After the same exposure period, 0.1 and 1 mM coumarin significantly enhanced the diameter of the root vessels (Table 1), causing, on average, an increase of 119 and 133%, respectively.
Table 1. Root parameters measured in durum wheat seedlings after 4 h exposure to different concentrations of coumarin
Concentration of coumarin
RGRrl (cm cm−1 d−1)
Root vessel area (µm2)
Numbers in parentheses denote the standard error of the mean. n = 30. Results of ANOVA for each analysis are given with significance levels indicated: *0.05 > P < 0.01; ***P < 0.001. Means followed by different letters are significantly different (P < 0.05; Tukey’s test).
In the absence of nitrate induction (uninduced plants) coumarin was unable significantly to affect the net rate of nitrate uptake (Table 2). In the presence of 50 µM KNO3 (induced plants) a complete induction of net nitrate uptake was attained after 8 h of contact (Fig. 3). In these induced plants, 25 µM, 0.1 and 1 mM coumarin increased net nitrate uptake by 29%, 48% and 40%, respectively, compared with control plants receiving no coumarin (Fig. 3).
Table 2. Net rates of nitrate uptake rates (µmol g−1 f. wt h−1) in roots of durum wheat exposed to different concentrations of coumarin in the hydroponic medium (uninduced plants)
Concentration of coumarin
Incubation period (h)
Values given are means (SE) from four replicates. Results of two-way ANOVA are given. NS = not significant at P < 0.05.
Coumarin × time
Table 3 shows the kinetic parameters for net rate of nitrate uptake in induced wheat seedlings exposed to varying concentrations of coumarin, calculated by means of eqn 1 described in the Materials and Methods section. Compared with the behaviour of control plants, 0.1 mM coumarin significantly increased the nitrate induction rate constant, K1. At 0.1 mM, coumarin also significantly decreased the time required to attain half of the full nitrate induction of nitrate uptake, t1/2 K1 (Table 3). After 24 h, all the coumarin concentrations tested significantly enhanced estimated nitrate accumulation in the tissues (Table 3).
Table 3. Kinetic parameters related to net rate of nitrate uptake in wheat seedlings exposed to increasing concentrations of coumarin in the presence of 50 µM NO3− (induced plants)
Concentration of coumarin
A [µmol (g−1 root f. wt) h−1]
Iconst[µmol (g−1 root f. wt) h−1]
t1/2 K1 (h)
t1/2 K2 (h)
Accumulated nitrate[µmol (g−1 root f. wt) d−1]
Numbers in parentheses denote the standard error of the mean (n = 6). Results of ANOVA for each analysis are given with significance levels indicated: *0.05 > P < 0.01; **0.01 > P < 0.001; ***P < 0.001; NS, not significant. Means followed by different letters are significantly different (P < 0.05; Tukey’s test). R2 values refer to curve fitting to experimental data.
Figure 4 shows the concentration of nitrate in roots and leaves of induced wheat seedlings after 24 h of exposure to coumarin. In control plants the concentration of nitrate in roots was almost twice that in leaves. Exposure to even 25 µM coumarin drastically altered this distribution in favour of the leaves (Fig. 4). On the contrary, coumarin alone (uninduced plants) affected neither the concentration of nitrate nor its interorgan distribution (data not shown).
In roots from uninduced plants, respiratory consumption of O2 was rapidly depressed by coumarin, in a concentration-dependent manner and up to a maximum of 20% compared with that of control roots (Fig. 5a). This depressive effect on root respiration persisted after 8 and 24 h from the beginning of the treatment (Fig. 5b,c, respectively). However, in the presence of nitrate (induced plants), the rapid coumarin-dependent drop in root respiration rates (Fig. 5a) disappeared after 8 or 24 h of exposure (Fig. 5b,c). Indeed, O2 consumption rates in induced roots exposed for 8 h to 25 µM coumarin even exceeded the control value (Fig. 5b).
The results of this study indicate that coumarin is a substance with multiple physiological targets and that its effects are generally stimulatory at low concentrations and inhibitory at high concentrations (Jansson & Svensson, 1980; Brown, 1981). In accordance with Svensson (1971), we confirm that coumarin is able significantly to increase the transectional area of root vessels (Table 1). Root swelling observed above the apex, which followed treatment with 0.1 mM coumarin (Fig. 2b), could result from a coumarin-dependent increase in the number of cell radial divisions in the subapical root zone (Avers & Goodwin, 1956; Svensson, 1971). Inhibition of root growth (Table 1) could have been caused by a coumarin-induced interference with auxin metabolism and subcellular distribution (Goren & Tomer, 1971).
Roots exhibit at least two mechanisms for nitrate uptake (Hole et al., 1990; Clarkson, 1998). Uninduced plants show a low constitutive rate of nitrate uptake (often referred to as LATS, low affinity transport system). Upon exposure to nitrate (that is in induced plants) the uptake rates increase (HATS, high affinity transport system). Nitrate therefore stimulates its own uptake (Redinbaugh & Campbell, 1991; Clarkson, 1996). Subsequent feedback regulation, which causes nitrate uptake to decline, is brought about by accumulation in plant tissues of nitrate and/or its assimilation products, such as ammonium and amino acids (Hole et al., 1990; Clarkson, 1998). In the present work, net nitrate uptake in wheat seedlings was completely induced after 8 h contact with 50 µM nitrate (Fig. 3). This is in agreement with the results obtained by MacKown & McClure (1988).
These results indicate that coumarin had different effects on uninduced and induced nitrate transport systems, the latter being, in general, much more responsive to this compound. The stimulatory effects of coumarin on nitrate uptake, reported here for the first time, are at variance with the effects reported for other allelopathic compounds, such as ferulic acid, which supposedly inhibited the initial rate of nitrate uptake and prevented any subsequent development of an increased rate in Zea mays (Bergmark et al., 1992). It should be noted that the stimulatory effect of coumarin on nitrate uptake in wheat depended on its concentration and may be associated with parallel effects on root anatomy and morphology. In fact, whilst coumarin, at all the concentrations tested, was able to increase net nitrate uptake upon full induction (Fig. 3), only 0.1 mM coumarin significantly increased the rate of induction, K1 (Table 3). This concentration of coumarin might have been the most effective because it caused subapical swelling of the tip (Fig. 2b), thereby increasing the root surface available for nitrate absorption. This, in turn, may have simultaneously enhanced nitrate availability for the induction process. According to Siebrecht et al. (1995), nitrate taken up by the root apex may stimulate the induction of nitrate uptake in older root regions. On the other hand, the fact that 1 mM coumarin caused necrosis of the root apex (Fig. 1b) may explain why this coumarin concentration was unable to increase K1 (Table 3).
On the basis of the reported data, several mechanisms may explain the differential action of coumarin on induced and uninduced transport systems. Since the induced uptake system structurally (Filleur & Daniel-Vedele, 1999) and genetically (Clarkson, 1998) differs from the uninduced system, it may be that coumarin is able to modify the former into a more efficient uptake system. This might possibly occur through interactions between coumarin and systems, presumably in the membrane, that operate and regulate nitrate uptake. In a similar way, the occurrence of a reversible perturbation of cell membrane integrity was proposed by Glass & Dunlop (1974) to explain the effects of phenolic acids on ion uptake in barley roots.
A greater diameter of the xylem vessels in coumarin-treated plants (Table 1) would have increased the efficiency of translocation of nitrate from root to shoot (Fig. 4), thus relieving or retarding the onset of retroinhibitory effects following nitrate accumulation in root cells.
Nitrate uptake is costly in terms of energy supply (Salsac et al., 1987). The main source of energy in nonphotosynthesizing tissues, such as the root, is respiratory oxidation of carbohydrates. Therefore, factors that affect respiration may also influence ion accumulation (Marschner, 1996). In the present work, increasing coumarin concentration increasingly inhibited root respiration, but only in uninduced plants (Fig. 5). In the presence of nitrate, coumarin did not inhibit root respiration (Fig. 5). In terms of energy supply therefore plants would be able to sustain their increased rate of nitrate uptake.
To the best of our knowledge, this is the first report that coumarin stimulates net nitrate uptake. Another remarkable and previously unrecognized feature of coumarin is its powerful action in promoting nitrate translocation from the root to the shoot, even when present at concentrations lower than 0.1 mM. On the basis of the available data, we tentatively suggest that the effects of coumarin on root anatomy and morphology explain most of the above observations.
On the one hand, the effects observed in this study confirm the allelopathic nature of coumarin at high concentrations. On the other hand, however, it is suggested that, at micromolar concentrations, coumarin may behave as a powerful effector of changes in root structure and function and, as such, deserves further investigation.