Fe-deficiency increases Cu acquisition by wheat cropped in a Cu-contaminated vineyard soil


  • V. Chaignon,

    1. Institut National de la Recherche Agronomique, UMR Sol and Environnement, Place Viala, F–34060 Montpellier Cedex 2, France
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  • D. Di Malta,

    1. Institut National de la Recherche Agronomique, UMR Sol and Environnement, Place Viala, F–34060 Montpellier Cedex 2, France
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  • P. Hinsinger

    Corresponding author
    1. Institut National de la Recherche Agronomique, UMR Sol and Environnement, Place Viala, F–34060 Montpellier Cedex 2, France
      Author for correspondence: Philippe Hinsinger Tel: +33 4 99 61 22 49 Fax: +33 4 67 63 26 14 Email: hinsinger@ensam.inra.fr
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Author for correspondence: Philippe Hinsinger Tel: +33 4 99 61 22 49 Fax: +33 4 67 63 26 14 Email: hinsinger@ensam.inra.fr


  •  This work evaluated the influence of iron deficiency and phytosiderophore exudation on the acquisition of copper by two bread wheat (Triticum aestivum) genotypes (‘Aroona’ and ‘Songlen’) grown in a calcareous vineyard soil, which had been contaminated by repeated applications of copper fungicides.
  •  A pot experiment was designed with a cropping device that enabled an easy access to shoots, roots and rhizosphere soil. Before being placed in contact with the soil, wheat seedlings had been subjected to, or not subjected to, either zinc or iron starvation, in order to stimulate the release of phytosiderophores.
  •  Under zinc starvation, the zinc-efficient genotype (‘Aroona’) acquired more soil copper. By contrast, iron starvation resulted in an enhanced release of phytosiderophores in both genotypes (before being grown on the soil), in elevated copper concentrations in shoots and roots and in a significantly increased acquisition of copper by wheat.
  •  Iron deficiency thus resulted in elevated acquisition of copper from the copper-contaminated soil, possibly through enhanced phytosiderophore release. Phytotoxic concentrations were, however, not attained in wheat shoots, as a major proportion of acquired copper accumulated in the roots.


The repeated use of Bordeaux mixture (Ca(OH)2 + CuSO4) as a fungicide to control vine downy mildew has led to a long-term accumulation of Cu in the topsoils of vineyards in Europe and throughout the world (Brun et al., 2001). From an environmental perspective, a major question is that of the bioavailability (and toxicity) of such accumulated Cu for a range of living organisms, including cultivated plants. The vine itself does not seem to be sensitive, partly because its roots colonize mostly the deeper soil layers, which are least contaminated (Delas, 1984). The acquisition of soil Cu by plants thus deserves to be investigated for those plants that are grown in association with vine (grassed vineyard) or which succeed the vine. However, plants can alter the chemical mobility and thereby the acquisition of metals in the root environment, i.e. the rhizosphere, in various ways (Marschner, 1995; McLaughlin et al., 1998; Hinsinger, 2001).

Chemical conditions in the rhizosphere can indeed be drastically different from those in bulk soil and a range of mechanisms are implied in the acquisition of trace metals by plants (Marschner, 1995; Hinsinger, 1998; McLaughlin et al., 1998; Hinsinger, 2001). These mechanisms vary widely with plant species and environmental conditions; some are induced or stimulated in response to nutrient deficiencies (Marschner, 1995; Hinsinger, 2001). Under Fe deficiency, which is by far the most documented case, net excretion of protons and enhanced reduction activity are typical root responses of dicotyledonous and monocotyledonous plants, with the exception of grasses (Marschner & Römheld, 1994). Enhanced release of carboxylic or phenolic compounds has also been reported as a response to Fe deficiency (Zhang et al., 1989; Ohwaki & Sugahara, 1997). In Fe-deficient grasses (graminaceous plants), as first reported by Takagi (1976), roots release amino acids, the so-called phytosiderophores (PS). These root exudates can chelate Fe(III) from insoluble sources (Takagi, 1976; Rengel, 1997) and then mediate its uptake (as Fe(III)-PS) into the plant (Marschner & Römheld, 1994). However, studies on graminaceous species have shown that the release of phytosiderophores is also enhanced under Zn deficiency (Zhang et al., 1989) and even Cu deficiency (Gries et al., 1998). In these cases, it is uncertain whether phytosiderophore release is a primary response to Zn or Cu deficiency or is caused by a chain of events that involve internal plant Fe deficiency, owing to impaired utilization of plant Fe (Walter et al., 1994; Rengel, 1997; Gries et al., 1998). In addition, Treeby et al. (1989) have shown that Zn, Cu and Mn concentrations in plants increased with increasing release of phytosiderophore in barley as a response to Fe deficiency. Many of the former studies have focused primarily on Fe and Zn deficiencies, which are acknowledged as a frequent nutritional constraint for plants, especially in calcareous soils.

Nevertheless, in addition to Fe and Zn deficiencies, calcareous soils can be highly contaminated by other trace metals, such as Cu in a large proportion of vineyards for example. In Southern France, in the last 20 years about one-third of the Languedoc vineyards (formerly above 450 000 ha) has been shifted to other land uses, including various crops such as bread and durum wheat as the dominant species. In such conditions, there is a need to estimate the bioavailability and risks of toxicity of soil Cu for these new crops. In Switzerland, Coullery (1996) reported that cereals and forage grasses exhibited the largest shoot Cu concentrations over a range of species grown in calcareous, Cu contaminated (formerly vineyard) soils. One may thus question whether Fe deficiency and enhanced phytosiderophore release can lead to increased acquisition of Cu to concentrations that are phytotoxic or toxic for animals.

The aim of the work was to study whether the exudation of phytosiderophores, in response to Zn or Fe deficiency, by roots of two bread wheat genotypes cropped in a Cu contaminated calcareous soil is involved in the acquisition of Cu by the plant.

Materials and methods

Plant and soil characteristics

Two bread wheat genotypes, classified as Zn-efficient (Triticum aestivum, cv. Aroona) and Zn-inefficient (Triticum aestivum, cv. Songlen) were chosen for their capacity to release phytosiderophores at different rates (Rengel & Römheld, 2000). The soil was calcareous and had been collected in February 2000 in a vineyard from a small agricultural watershed at Roujan (south France, 60 km West of Montpellier; Ribolzi et al., 2000). The soil had been sampled from 2–15 cm depth, air dried and sieved at 2 mm. The clay, sand and CaCO3 contents were 182, 188 and 273 g kg−1, respectively. The pHwater was 8.7, while the cation exchange capacity was 9.4 cmolc+ kg−1. The total Cu and Zn content (HF-ClO4 extraction; Baize, 2000) were 144 µg g−1 and 67 µg g−1, respectively. The ethylenediaminetetraacetic acid (EDTA)-extractable Cu and Zn content were 51 and 3 µg g−1, respectively. The ‘free’ Fe content, according to the Mehra–Jackson procedure (Baize, 2000), was 7.3 g kg−1.

Bioassay: hydroponic stage

The cropping device used for the present bioassay was adapted by Chaignon and Hinsinger (2002) from that designed by Guivarch et al. (1999). It enables an easy recovery of both shoots and roots. Because it also provides an access to rhizosphere soil, this bioassay can also be used to study chemical changes occurring in the rhizosphere that affect the bioavailability of trace elements to plants (Guivarch et al., 1999). The bioassay is a two-stage procedure: in the first stage, plants are grown for 3 wk in hydroponic conditions (hereafter called ‘hydroponics stage’) in pots made of two PVC pipes, the small one (32 mm external diameter) being inserted into the large one. The large pipe was closed at the bottom by a nylon mesh (30 µm pore diameter) and the small pipe by a grid (1.5 mm pore diameter). The time needed for the roots to fully cover the mesh as a dense root mat was 3 wk. Two seeds of wheat were sown on top of the grid in each pot and germinated for 1 wk with a nutrient solution containing 600 µm CaCl2 and 2 µm H3BO3. The pots were then transferred to 6-l buckets (10 pots per bucket, 12 buckets) filled with continuously aerated nutrient solution. Three different compositions were used to design three treatments. The one-third of the pots was grown in a complete nutrient solution (+Zn/+Fe or sufficient supply) of following composition: 2 mm KNO3, 2 mm Ca(NO3)2, 1 mm MgSO4, 0.5 mm KH2PO4, 0.1 mm FeNaEDTA, 10 µm H3BO3, 2 µm MnCl2, 0.05 µm NaMoO4, 1 µm ZnSO4, 0.2 µm CuCl2. Another one-third of the pots was grown in the complete nutrient solution without Zn (–Zn or deficient supply of Zn) while the final third of the pots was grown in the complete nutrient solution without Fe (–Fe or deficient supply of Fe). All nutrient solutions had an initial pH of about 5.5 and were renewed every third day to avoid large pH changes and nutrient depletion. After 2 wks, five replicates (five pots taken from different buckets) were collected and analysed separately for each treatment. The analyses of these pots provided references (starting point or control) for the second stage of the bioassay.

Bioassay: soil stage

For the second stage of the bioassay (thereafter called ‘soil stage’), pots with 3-wk-old-wheat plants were transferred for 8 d onto small soil disks (1.5 mm thickness, 32 mm diameter) that were connected to three different nutrient solution reservoirs according to the previous treatments. Before the transfer, the soil had been divided into three subsamples and incubated for 6 d with a nutrient solution that had the same composition as the three above-mentioned nutrient solutions (+Zn/+Fe, –Zn and –Fe) minus CuCl2 and FeNaEDTA, whatever the Zn or Fe supply. Hence, the composition of the nutrient solution supplied for both +Zn/+Fe and –Fe treatments were the same at this stage of the experiment. No Cu was supplied so that soil Cu was the only source of Cu for the plants. The FeNaEDTA was omitted because EDTA might have affected the speciation of Cu in the soil. For each treatment, five replicates were prepared by placing five pots with plants (from hydroponic stage) onto five soil disks. Five additional soil disks were prepared for each treatment and left uncropped. At the end of the soil stage of the bioassay (after 8 d of contact with the soil), all replicates were collected and analysed separately for each treatment. The two stages of the bioassay were conducted in a growth chamber with the following day/night conditions: 16 h, 25°C, relative humidity (r.h.) 75% and a photon flux density of about 550 µmol m−2 s−1 (in the range 400–700 nm)/8 h, 20°C, r.h. 100% and darkness.

Collection and quantification of root exudates (phytosiderophores)

For the collection of root exudates, intact plants were used before being harvested at the end of each stage of the bioassay. The roots were repeatedly rinsed for 1 min under a gentle stream of deionized water and then transferred to small Petri dishes containing 15 ml of aerated deionized water for 3 h. The procedure started 3 h after the onset of the light period in order to coincide with the peak period of exudation of PS (Takagi et al., 1984). To prevent microbial degradation of the exudates during their collection and further treatment, 0.01 g l−1 of Micropure (Roth GmbH, Karlsruhe, Germany), was added to the deionized water. At this concentration (i.e. 10% of that recommended by the manufacturer for water purification), the collection solution was supposedly bacteriostatic (Gries et al., 1998), while it was not expected to affect the measurement to any great extent (Neumann & Römheld, 2001). After a 3-h collection period, the solutions were filtered through an ashless filter paper (Whatman 40). The plants were then harvested as described below.

The amount of phytosiderophore in the root exudates was determined indirectly by the amount of Cu mobilized from a Cu-loaded resin (Chelite P, Serva, Heidelberg, Germany) as previously described by several authors (Treeby et al., 1989; Cakmak et al., 1994; Walter et al., 1994; Cakmak et al., 1996; Rengel, 1997). The Cu-Chelite P complex was prepared by stirring 5 g of Chelite P in 500 ml of 50 mm CuSO4 for 15 min This suspension was filtered and washed with deionized water until the water was free of Cu. Then, the Cu-loaded resin was equilibrated with 500 ml of 10 mm MES (2-(N-morpholino) ethane-sulfonic acid) buffer (pH 5) and used for Cu mobilization tests. A volume of 2 ml of root exudates was mixed with 2 ml of Cu-loaded resin suspended in MES buffer (pH 5) and 6 ml of deionized water for 45 min, before being filtered through an ashless filter paper (Whatman 40). The concentration of Cu in the filtrates was measured by flame atomic absorption spectrometry (AAS Varian SpectrAA-600, Mulgrave, Australia) and the amount of phytosiderophore in the root exudates was calculated as mobilized Cu equivalents per two plants per 3 h.

Plant and soil analyses

After the collection of root exudates, roots and shoots were harvested and weighed. Shoots were oven dried at 105°C and roots were frozen at –20°C. Copper bound to root cell walls was determined as described by Chaignon and Hinsinger (2002). Briefly, a subsample of 0.4 g of fresh (thawed) roots was shaken end-over-end with 20 ml of 1 mm HCl for 3 min, then 180 µl of 1 m HCl were added to yield a final concentration of 10 mm HCl. After shaking for a further 5 min, the suspensions were filtered through an ashless filter paper (Whatman 40) and Cu was assayed by flame atomic absorption spectrometry. This was achieved only for plants harvested at the end of the soil stage of the experiment as the amount of roots of hydroponically grown plants was not large enough to yield an accurate measurement of apoplasmic Cu through this procedure. Preliminary experiments had been conducted to verify that the concentrations of HCl and durations of the extraction steps did not result in a damage of plasma membrane and, hence, in an overestimate of apoplasmic Cu (Iwasaki et al., 1990). For this purpose, K efflux from wheat roots was monitored over time with K-sensitive microelectrodes in 1 mm and 10 mm HCl. Negligible efflux was found over the short periods selected for the extraction procedure, even for frozen roots, suggesting that the extracted Cu accounted only for apoplasmic Cu. The remaining portion of roots was oven dried at 105°C. The oven-dried roots and shoots were digested separately in a 1 : 1 mixture of hot, concentrated HNO3 and HClO4 (AOAC, 1975). Concentrations of Cu, Zn and Fe in the digests were determined by flame atomic absorption spectrometry (AAS).

The cropped and uncropped soils from the three treatments were collected at harvest and oven dried at 105°C. The soil pH was determined in a CaCl2-background electrolyte as follows: one g of dry soil was shaken for 2 h with 5 ml of 50 mm CaCl2. The suspension was then centrifuged at 15 000 g for 3 min and pH measured in the supernatant.


Visual symptoms and growth

Visual Fe deficiency symptoms (leaf chlorosis) were observed for both genotypes after less than 2 wks of growth under Fe starvation but appeared for ‘Songlen’ first. At the end of the soil stage of the bioassay, the shoots were lighter green than control shoots (i.e. at the end of the hydroponic stage of the bioassay). In addition, the roots were darker than control ones. Under deficient supply of Zn, no visual symptoms were observed for either of the two wheat genotypes.

Regardless of the treatment, shoot dry biomasses of both wheat genotypes increased significantly during the soil stage of the bioassay (Table 1). This trend was also observed for root dry biomasses. Whatever the wheat genotype, Zn deficiency did not result in altered growth of shoots or roots. By contrast, Fe deficiency led to a significant decrease of shoot biomass (Table 1).

Table 1.  Dry biomass of shoots and roots (mean value ± standard deviation of five replicates) expressed as g per pot (i.e. per two plants)
 Hydroponic stageSoil stage
  1. Mean values with different letters within a line are significantly different (at P < 0.05) as measured by an LSD test.

Shoot dry mass0.6 ± 0.0a0.6 ± 0.2a0.5 ± 0.2a1.9 ± 0.2c1.7 ± 0.2c1.1 ± 0.4b
Root dry mass0.1 ± 0.0a0.2 ± 0.1a0.2 ± 0.0a0.4 ± 0.2b0.5 ± 0.2b0.5 ± 0.1b
Shoot dry mass0.5 ± 0.3ab0.6 ± 0.1b0.3 ± 0.1a1.1 ± 0.1c1.2 ± 0.4c0.7 ± 0.3b
Root dry mass0.1 ± 0.1a0.2 ± 0.1a0.1 ± 0.1a0.5 ± 0.1bc0.5 ± 0.1c0.3 ± 0.1b

Root and shoot Cu concentrations

In hydroponics, Cu concentrations in roots and shoots of both ‘Aroona’ and ‘Songlen’ varied with Zn and Fe supply (Fig. 1a). At the end of the soil stage, Cu concentrations in roots and shoots of both wheat genotypes significantly increased only under Fe deficiency (Fig. 1b). For both wheat genotypes, Cu concentrations in the shoots significantly decreased relative to hydroponically grown plants, while a systematic increase was found for root Cu concentrations. In both ‘Aroona’ and ‘Songlen’, the percentage of Cu bound to root cell walls decreased from about 80% to 40% as a result of Fe starvation (Fig. 1b).

Figure 1.

Copper concentrations (µg g−1 dry matter (DM)) of shoots (grey) and roots (dark grey) of ‘Aroona’ and ‘Songlen’ wheat genotypes grown in nutrient solution (hydroponic stage) (a) and on soil (soil stage) (b), under sufficient (+Zn/+Fe), Zn deficient (–Zn) and Fe deficient (–Fe) supply. Percentages of total root Cu bound to root cell walls are indicated in white for soil-grown plants (not determined for hydroponically grown plants). For each bioassay stage procedure, and shoot and root separately, mean values with different letters are significantly different (P < 0.05) as measured by an LSD test. For any given Fe/Zn supply treatment and genotype combination, * indicates that Cu concentration at the end of the soil stage significantly differed (P < 0.05) from that measured at the end of the hydroponic stage. The error bars are standard deviations.

Plant Cu and soil Cu acquisition

Figure 2a shows that the effects of Fe starvation on the amount of Cu in hydroponically grown plants were significant compared with the control. The amount of plant Cu (biomass × Cu concentration) in both wheat genotypes was larger under Fe starvation, and significantly larger for ‘Aroona’. This was further confirmed at the soil stage of the bioassay (Fig. 2b). The amounts of plant Cu in both ‘Aroona’ and ‘Songlen’ significantly increased by 270–315% under Fe deficient supply, compared with plants under a sufficient supply of Fe and Zn (Fig. 2b). No significant difference in the amount of plant Cu was found between sufficient and deficient Zn supplies in either genotype. The shoot : root ratios of the amount of plant Cu at the end of the soil stage were lower than those of hydroponically grown plants, and more so for ‘Songlen’ than ‘Aroona’ (Table 2). Copper uptake from soil by wheat per unit of soil mass (Cu acquisition) was deduced from the amount of plant Cu by subtracting the initial amount of Cu in hydroponically grown plants and expressing the final result relative to the mass of dry soil supplied (Table 3). Copper acquisition from soil was three- to four-fold larger for wheat grown under Fe starvation, compared with plants under sufficient supplies of Fe and Zn. Under Zn-deficient supply, Cu acquisition from soil increased (approx. two-fold) only for ‘Aroona’. Wheat genotypes extracted between 4% and 19% of soil total Cu content (Table 3).

Figure 2.

Amounts of Cu in plant parts (grey, shoot-accumulated Cu; dark grey, root-accumulated Cu) expressed as µg per pot (per two plants), for the two wheat genotypes under sufficient and deficient Fe and Zn supply, measured at the end of the hydroponic stage (a) and at the end of the soil stage (b). For each bioassay stage, mean values with different letters are significantly different (P < 0.05) as measured by an LSD test. The error bars are standard deviations.

Table 2.  Shoot–root ratios of the amount of Cu in plant parts, deduced from the amount of Cu in the shoots (in µg per pot) divided by the amount of Cu in the roots (in µg per pot)
 Hydroponic stageSoil stage
Table 3.  Soil Cu acquisition by wheat (total amount of Cu acquired from the soil, mean value ± standard deviation of five replicates), expressed as µg g−1 soil and as percentage of total soil Cu content
 Soil stage
  1. Mean values with different letters within a line are significantly different (at P < 0.05) as measured by an LSD test.

µg g−1 soil6 ± 3a14 ± 6b25 ± 4c
% of total soil Cu content4 917
µg g−1 soil8 ± 3a 6 ± 4a29 ± 7b
% of total soil Cu content6 419

Root and shoot concentration and acquisition of Zn and Fe from soil

In hydroponically grown plants, Zn concentrations in both ‘Aroona’ and ‘Songlen’ decreased about twofold under Zn starvation and significantly increased in shoots and roots under Fe starvation compared with plants under sufficient supplies of Zn and Fe (Table 4). Concentrations of Zn in the ‘Songlen’ genotype were larger than in ‘Aroona’ under Fe deficiency. At the end of the soil stage, the same trend was observed when comparing the various treatments and genotypes. A significant increase in Zn concentration of both shoots and roots occurred for Fe-deficient plants. Significant amounts of soil Zn were acquired only by ‘Aroona’ (Table 4). In addition, the acquisition of Zn from the soil was larger for ‘Aroona’ grown under Fe-deficient supply than Fe-sufficient supply or Zn-deficient supply.

Table 4.  Zinc and iron concentrations (µg g−1 dry matter) of shoots and roots of wheat grown in nutrient solution (hydroponic stage of the experiment) and soil (soil stage of the experiment), and acquisition of soil Zn and Fe (total amounts of Zn and Fe acquired from the soil, µg g−1 soil). All values are expressed as mean value ± standard deviation of five replicates
 Hydroponic stageSoil stage
  1. Mean values with different letters within a line are significantly different (at P < 0.05) as measured by a LSD test.

Shoot Zn concentration  61 ± 9c  26 ± 6b  91 ± 9d  21 ± 3ab  15 ± 1a  85 ± 14d
Root Zn concentration 116 ± 17b  67 ± 7ab 256 ± 58c 107 ± 101ab  44 ± 21a  88 ± 32ab
Soil Zn acquisition     –     –     –   5 ± 5a   8 ± 5a  17 ± 5b
Shoot Zn concentration  62 ± 13b  32 ± 14a 316 ± 44d  23 ± 3a  12 ± 2a 105 ± 6c
Root Zn concentration 124 ± 15a  68 ± 11a 408 ± 271b  68 ± 33a  37 ± 13a 184 ± 112a
Soil Zn acquisition     –     –     –   1 ± 15 a   0 ± 5a   0 ± 16 a
Shoot Fe concentration 121 ± 39b 126 ± 18b  81 ± 9a  70 ± 10a  75 ± 9a  67 ± 16a
Root Fe concentration1791 ± 185ab2271 ± 1025b1056 ± 394a1609 ± 1321ab1102 ± 617a1027 ± 201a
Soil Fe acquisition     –     –     – 104 ± 24a  96 ± 18 a 122 ± 37a
Shoot Fe concentration 154 ± 18b 217 ± 87b 171 ± 35b  80 ± 18a 103 ± 25a  74 ± 20a
Root Fe concentration1710 ± 556a1790 ± 420a 976 ± 526a 933 ± 458a 970 ± 291a1530 ± 850a
Soil Fe acquisition     –     –     –  83 ± 80a  61 ± 14a 131 ± 67 a

In hydroponically grown plants, regardless of genotype, Fe concentrations observed in shoots and roots under Fe-deficient supply were lower than under sufficient Fe or deficient Zn supply. Nevertheless, differences were seldom significant (Table 4). For ‘Aroona’ and ‘Songlen’, respectively, Fe acquisition from the soil reached 96 and 61 µg g−1 soil under Zn-deficient supply, 104 µg g−1 and 80 µg g−1 soil under sufficient supply and 122 µg g−1 and 131 µg g−1 soil under Fe-deficient supply (Table 4). However, there was no significant difference between the treatments.

Phytosiderophore release and rhizosphere pH changes

In hydroponically grown plants adequately supplied with both Zn and Fe or under deficient supply of Zn, the release of PS remained low in both genotypes (Fig. 3a). By contrast, under Fe-deficient supply, the release of PS was markedly enhanced in both genotypes (Fig. 3a). This difference was not found for plants at the end of the soil stage of the bioassay. No significant pH change was found in the rhizosphere soil except for ‘Aroona’ under sufficient supply of Zn and Fe, for which there was a slight pH increase relative to control, uncropped soil (Table 5).

Figure 3.

Amounts of released phytosiderophores (PS) expressed as µmol PS per pot (per two plants) per 3 h, for the two wheat genotypes under sufficient and deficient Fe and Zn supply, measured at the end of the hydroponic stage (a) and at the end of the soil stage (b). For each bioassay stage, mean values with different letters are significantly different (P < 0.05) as measured by LSD test. The error bars are standard deviations.

Table 5.  Values of pH in uncropped (control) and cropped (rhizosphere) soil for the two wheat genotypes (mean value ± standard deviation of five replicates). For each nutrient supply (treatment), control and rhizosphere mean values are compared by an LSD test
 Soil stage
  • *

    Significant at P < 0.05; ns: not significant.

Control (uncropped) soil6.58 ± 0.056.61 ± 0.066.58 ± 0.06
Aroona rhizosphere soil6.73 ± 0.04*6.60 ± 0.12 ns6.60 ± 0.03 ns
Songlen rhizosphere soil6.60 ± 0.07 ns6.55 ± 0.04 ns6.64 ± 0.05 ns


The experiment aimed to compare wheat growth, wheat Cu, Zn and Fe concentrations, Cu acquisition from soil and phytosiderophore release as affected by Zn and Fe deficiency.

Effect of Zn deficiency

Visual Zn deficiency symptoms, such as necrotic patches on leaves and reduction in shoot length (Marschner, 1995) did not appear on either of the two bread wheat genotypes. In addition, Zn concentrations in the leaves of both genotypes remained slightly above the Zn critical deficiency level, which is about 10–20 µg g−1 dry matter (Reuter & Robinson, 1997), and more so for ‘Aroona’ than ‘Songlen’ (Table 4). The short duration of growth under Zn starvation (14 days) and the wheat species used could partly explain such results. Unlike several previous reports (Cakmak et al., 1994, 1996; Erenoglu et al., 1996; Rengel & Römheld, 2000), we did not find any significant enhancement of PS release as a response to Zn starvation (Fig. 3). This might, however, be simply the consequence of not having reached a severe Zn deficiency in plant shoots in the present work.

The concentrations of Fe and Cu in either the shoots or the roots of both genotypes were not significantly altered by Zn starvation (Table 4 and Fig. 1). However, since severe Zn deficiency was not reached, the present experiment cannot fully discount the possible implication of the Zn status of wheat plants in the acquisition of other divalent metals such as Fe and Cu, as already pointed out by earlier works in the case of Fe (Walter et al., 1994; Rengel & Römheld, 2000). In the case of ‘Aroona’, the acquisition of soil Cu significantly increased under Zn starvation (Table 3). Additional work is thus needed to check how the acquisition or translocation of Cu is affected by Zn deficiency in this wheat genotype.

Effect of Fe deficiency

Unlike Zn starvation, Fe starvation had a major effect on wheat growth (Table 1), Cu, Zn and Fe concentrations in wheat (Fig. 1 and Table 4) and significantly enhanced the release of PS (Fig. 3). It is well documented that, like other grasses, wheat responds to Fe deficiency by enhanced PS release which has been described as Strategy II of Fe acquisition (Marschner & Römheld, 1994; Marschner, 1995). There were only minor differences between the two bread wheat genotypes in the severity of Fe deficiency symptoms: ‘Aroona’ appeared less susceptible to Fe deficiency than ‘Songlen’. The growth of both wheat genotypes was considerably reduced under Fe deficiency: the dry biomass of Fe-deficient whole plants was 40% lower than that of adequately supplied plants (Table 1). However, Fe deficiency did not result in a significant change in Fe concentration in either roots or shoots of any of the two genotypes, except for hydroponically grown ‘Aroona’ shoots (Table 4). Conversely, Fe deficiency resulted in much increased concentrations of both Cu and Zn in the shoots and roots of both wheat genotypes (Fig. 1 and Table 4). This effect was significant in most cases and coincided with a much (significantly) increased release of PS in both genotypes at the end of the hydroponic stage of the experiment (Fig. 3). This suggested the possible implication of the release of PS in the acquisition of Cu (Fig. 1) and Zn (Table 4) for both hydroponically grown plants and, more interestingly, for plants grown on the calcareous soil as sole source of Cu and Zn. In the latter case, however, the relationship between Cu or Zn acquisition and PS release was not as clear when PS release at the end of the soil stage of the experiment was taken into account. The release of PS at the end of the hydroponic stage of the experiment (i.e. immediately before placing the plants in contact with the soil for the second stage of the experiment) increased two- and three-fold in response to Fe deficiency in ‘Songlen’ and ‘Aroona’, respectively (Fig. 3). In comparison, the acquisition of soil Cu increased three- and four-fold in response to Fe deficiency in ‘Songlen’ and ‘Aroona’, respectively (Table 3). It is noteworthy that the acquisition of Cu was only about 5% of total soil Cu for plants adequately supplied with Fe (and Zn), while it increased to about 20% for Fe-deficient plants (Table 3), which is considerable in the context of the fairly large total Cu content of the contaminated soil studied in the present experiment. In addition, the rate of PS release by Fe-deficient plants ranged between 0.1 µmol and 0.2 µmol per pot (for a 3-h period of measurement) prior and after the soil stage of the experiment (Fig. 3), suggesting that over the 8-d period of contact with the soil, the plants released at least c. 1–2 µmol PS per pot. This is an underestimate because it assumes that plants did not release any PS except during this peak period of the day, which does not hold true. In comparison, the amount of Cu acquired by the plants over this 8-d period was about 1 µmol Cu per pot (as deduced from Fig. 2). Meanwhile, the acquisition of Fe from soil was about 2–4 µmol Fe per pot and that of Zn was 0–0.5 µmol Zn per pot (as deduced from Table 4). Such results point to a potential role of PS release in the acquisition of not only Fe but also Cu from the calcareous, contaminated soil used in the present experiment. This is in line with the results of Treeby et al. (1989) who reported an increase in shoot Cu, but also Zn and Mn concentrations in Fe-deficient barley relative to Fe-sufficient barley. This was, however, found for an uncontaminated soil, which contained only 24 µg Cu g−1 soil, whereas the soil presently used contained 144 µg Cu g−1 soil, of which as much as about 20% was acquired by Fe-deficient wheat plants over the 8-d period of contact. As suggested by Treeby et al. (1989), PS do not only complex Fe; they can also complex other metals such as Cu, Zn and Mn, or Co and Ni, as mentioned by Mench and Fargues (1995). Murakami et al. (1989) measured the affinity of PS for Fe, various divalent metals and showed that they ranked as follows: Mn2+ < Fe2+ < Zn2+ < Ni2+ < Fe3+ < Cu2+ (Hinsinger, 2001). This suggests that the stability of the complex that PS form with divalent Cu is even greater than that of the complex formed with trivalent Fe. This provides a theoretical basis for the strong implication of PS in the acquisition of Cu by wheat, as suggested by the present results.

In the calcareous soil used in the present experiment, significant acquisition of soil Zn was only found for ‘Aroona’, which is the Zn-efficient genotype (tolerant to Zn deficiency), and Zn acquisition (Table 4) was well-positively correlated with PS release at the end of the hydroponic stage (Fig. 3a): both parameters increased about threefold in response to Fe-deficiency for the ‘Aroona’ genotype. This suggests that, as for soil Cu, there is some implication of PS in the acquisition of soil Zn by ‘Aroona’. If not related to the amounts of PS released, the greater efficiency of ‘Aroona’ in the use of soil Zn, compared with ‘Songlen’ (Table 4), might be related to a greater ability to take up PS–Zn complexes and make use of these as a source of Zn.

The present results do not provide proof of a direct causal relationship between the release of PS and the acquisition of soil Cu or Zn in wheat. They simply show that both the release of PS and the acquisition of Cu (and that of Zn in ‘Aroona’) are coincidentally enhanced under Fe deficiency. Previous works have shown that Fe deficiency can induce an increased uptake capacity (increased sink) not only of Fe, but also of other metals such as Cd, Zn and Cu (Cohen et al., 1998; Korshunova et al., 1999). This might occur as a consequence of an induction of the synthesis of metal transporters at the plasma membrane of root cells and of the poor selectivity of those transporters. In addition, although it has been used by many other authors, the method that we used for measuring the amount of PS released by wheat roots has several drawbacks. A major one is that it is not specific for PS (Gries et al., 1998): other complexing molecules among root exudates, such as organic and phenolic acids, amino acids (e.g. histidine) or proteins (e.g. nicotianamine), might also have contributed some proportion of the mobilization of Cu bound to the resin in the proposed method. Strictly speaking, it is thus more indicative of the complexing power of root exudates than of the amount of PS. A chemical analysis of the composition of root exudates would have helped clarify this point.

Cu acquisition and phytotoxicity in calcareous soils

The chemical mobility and hence the acquisition of trace metals is often considered as minimal, or at least decreased in calcareous soils, compared with acidic soils (Alloway, 1995). In the case of Cu, however, several studies have shown that, although it is less than that achieved by plants grown in very acidic soils, a fairly large acquisition of Cu can occur in calcareous soils, especially when the root compartment is taken into account (Mitchell et al., 1978; Cherrey et al., 1999; Brun et al., 2001). Our results show that a substantial proportion of total soil Cu in the present calcareous soil can be acquired by wheat plants, and more so under Fe deficiency (Table 3). They also confirm that a major proportion of plant Cu is accumulated in the root compartment (Fig. 2). As reported for diverse plant species such as rape (Chaignon & Hinsinger, 2002), maize (Brun et al., 2001), ryegrass (Cherrey et al., 1999) and ectomycorrhizal pine seedlings (van Tichelen et al., 2001), wheat plants appeared to be capable of regulating and hence restricting the translocation of Cu into their aerial parts, resulting in much larger Cu concentrations in the roots than in the shoots. Part of this phenomenon was probably achieved by binding Cu into root cell walls (Iwasaki et al., 1990; Marschner, 1995), as suggested by the large proportion of apoplasmic Cu found in roots of soil-grown plants (40–80% of total Cu contained in the roots; Fig. 1). Cell walls behave as ion exchangers because of their pectic polysaccharide and glycoprotein constituents (Allan & Jarrell, 1989) and exhibit a large affinity for metal cations such as Cu (Sattelmacher, 2001). A much smaller proportion of Cu was bound to the cell walls of Fe-deficient wheat plants in the present experiment (approx. 40% relative to 80% in adequately supplied plants). This is in line with the results of Awad and Römheld (2000) who showed that released PS can decrease the fraction of metals bound to the root apoplasm, such as Zn, Fe, Ni and Cd. Cakmak et al. (1996) had previously referred to the high capacity of PS to mobilize Zn from the apoplasm in roots of wheat grown under Fe-deficient conditions. Similarly, the present results suggest that the enhanced release of PS as a response to Fe deficiency might have contributed to the increased mobilization of Cu from the root apoplasm and, hence, to an increase in physiologically active Cu (i.e. Cu taken up into the root cells). This point would need to be addressed in detail in the future, as it can dramatically influence Cu phytotoxicity.

However, the present experiment did not provide any direct evidence of Cu phytotoxicity, even for those plants that achieved the largest acquisition of soil Cu (i.e. Fe-deficient plants). Copper concentrations in plant shoots remained much below those reported as phytotoxic levels (about 75 µg g−1 dry matter in wheat, according to Reuter & Robinson, 1997). This might partly be due to a dilution effect, as 3 wk-old plants were grown for a rather short period (8 d) on top of the contaminated soil (Chaignon & Hinsinger, 2002). Earlier and more sensitive indicators of Cu phytotoxicity (e.g. root elongation or enzymatic activities as proposed by Mocquot et al., 1996) should have been used for concluding whether Cu phytotoxicity occurred or not.

Nevertheless, our results clearly suggest that Fe deficiency in wheat can substantially increase the acquisition of Cu from a Cu-contaminated, calcareous soil, possibly through an enhanced release of PS by wheat roots. Indeed, Fe deficiency resulted in a two- to three-fold increased release of PS, which coincided with a three- to four-fold increased acquisition of soil Cu. Within only 8 d of growth, Fe-deficient wheat plants thereby acquired about 20% of the fairly high total Cu content of the contaminated soil under study (144 mg kg−1). Although Fe-deficiency was artificially induced in the present work by starving the plants before growing them in contact with the soil, it is well known that a major constraint for plant growth in calcareous soils is the restricted mobility and availability of micronutrients such as Fe and Zn (Marschner, 1995). Phytosiderophores are thus expected to be released in larger amounts by grasses grown in calcareous soils, which is considered as a fairly efficient strategy to avoid lime-induced Fe chlorosis (Marschner & Römheld, 1994; Marschner, 1995). However, to further confirm the ecological relevance of the present results, further research is needed in less artificial conditions of growth (in the longer term, conventional pot or field experiments). Under such conditions, lower levels of Fe deficiency might be experienced by plants, compared with the present experiment (no Fe supplied for 2 wks), even when grown in calcareous soils.

In conclusion, this research has demonstrated that Fe deficiency can increase the acquisition of Cu in bread wheat grown in a Cu-contaminated, calcareous soil. For both wheat genotypes under study, enhanced release of PS in Fe-deficient plants coincided with significantly elevated concentrations of Cu in both shoots and roots, with a major proportion of Cu accumulating in the roots. Despite this much-elevated acquisition of Cu, concentrations of Cu in wheat shoots remained below phytotoxic levels, possibly because of the short duration of the experiment (8 d of contact with the soil). The present experiment suffers from several disadvantages which are related to its rather artificial conditions and to the particular arrangement of soil/roots in the cropping device. However, this approach provided a unique tool for addressing the issue of Cu acquisition by soil-grown plants since it enabled easy access to the root compartment in which most of plant Cu is accumulated. It also provides easy access to rhizosphere soil in order to investigate those soil–root interactions occurring in the rhizosphere that ultimately determine the acquisition of Cu by the plants. This experiment should, nonetheless, be supplemented with conventional pot experiments and ultimately with in situ measurements in order to validate the present findings in less artificial growth conditions. Considering that the soil used in the present experiment was calcareous and contained a moderately high level of total Cu compared with reported values for other vineyard soils, this work clearly suggests that further investigations are needed to identify those soil situations where Cu can reach phytotoxic values for plants or toxic levels in the edible parts of the plants for animals or humans. This work also suggests that grasses such as wheat might be particularly vulnerable compared with other plant species, at least when growing in calcareous environments, as a consequence of their ability to release PS and thereby acquire considerable soil Cu. Further research is needed to determine the processes involved in the acquisition of Cu at the soil–root interface and at the root apoplasm–symplasm interface.


Professor Zdenko (Zed) Rengel (University of Western Australia) is gratefully acknowledged for his advice when initiating this research and for kindly supplying the seeds of the two wheat genotypes. We thank Dr Claude Plassard for her help in the preliminary experiment for assessing the apoplasmic Cu extraction (electrophysiological measurements). The comments and criticisms of Professor Erik Smolders and an anonymous referee for improving the quality of this paper are also acknowledged. Financial support of this work was provided by the French Ministry of Environment through its PNETOX (Programme National de Recherche en Ecotoxicologie) programme.