1. Art and Diseño

Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Effect of Cu toxicity on growth of Cowpea (Vigna unguiculata)
Peter M. Kopittke1, Neal W. Menzies
School of Land and Food Sciences, The University of Queensland, St. Lucia, Qld, Australia, 4072. Ph: +61 7
3365 2079, Fax: +61 7 3365 1177, Email: [email protected]
1
Corresponding author
Abstract
Accurate determination of the rhizotoxicity of Cu in dilute nutrient solutions is hindered by the
difficulty of maintaining constant, pre-determined concentrations of Cu (micromolar) in solution.
The critical Cu2+ activity associated with a reduction in the growth of solution-grown cowpea
(Vigna unguiculata (L.) Walp. cv Caloona) was determined in a system in which Cu was
maintained constant through the use of a cation exchange resin. The growth of roots and shoots was
found to be reduced at solution Cu2+ activities ≥ 1.7 µM (corresponding to 90 % maximum growth).
Although root growth was most likely reduced due to a direct Cu2+ toxicity, it is considered that the
shoot growth reduction is attributable to a decrease in tissue concentrations of K, Ca, Mg, and Fe
and the formation of interveinal chlorosis. At high Cu2+ activities, roots were brown in color, short
and thick, had bent root tips with cracking of the epidermis and outer cortex, and had local
swellings behind the roots tips due to a reduction in cell elongation. Root hair growth was reduced
at concentrations lower than that which caused a significant reduction in overall root fresh weight.
Abbreviations: CDTA – cyclohexane-1,2-diaminetetra-acetic acid; DI – deionised; EC – electrical
conductivity; I – ionic strength; ICPAES – inductively coupled plasma atomic emission
spectrometry; ICPMS – inductively coupled plasma mass spectrometry
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Introduction
Copper is an essential plant micronutrient, required for the protein components of several enzymes
(Marschner 1995). However, when present in excess quantities, Cu is also highly toxic to plant
growth potentially causing damage resulting in complete inhibition of growth. This accumulation of
toxic Cu concentrations has been observed on both naturally ‘polluted’ soils (for example, Cu ore
outcrops) (Eleftheriou and Karataglis 1989) and on anthropogenically polluted soils (such as from
mining, smelting, waste disposal, and the use of fertilizers and fungicides).
Although the toxicity of Cu has been the subject of much research, few studies report measuring
solution Cu concentrations during the experimental period, but rather relate plant growth to the
amount of Cu initially added. This may potentially lead to erroneous results, particularly in longerterm experiments where plant uptake, adsorption onto the root apoplasm, and precipitation from
solution result in a reduction in the Cu concentration. Studying the toxicity of Cu to native
Australian tree species over a 70 d growth period, Reichman (2001) reported that even with regular
replacement additions to maintain solution Cu, concentrations varied by up to 90 % (Reichman
2001).
Ion exchange resins have been used in solution culture experiments for nearly 60 years (Arnon and
Grossenbacker 1947), and have been used to buffer solution concentrations of several ions
including H+ (pH), Ca, Mg, K, Mn, Zn, and Ni with varying success (Checkai et al. 1987; Checkai
and Norvell 1992; Hageman et al. 1961; Harper and Nicholas 1976). Resins offer the advantage of
being able to buffer nutrient concentrations at low levels without the need for organic chelates. In
general, however, the use of ion exchange resins has not been widespread, particularly in regards to
heavy metal toxicity. The use of ion exchange resins to buffer defined concentrations of heavy
metals requires accurate pH control due to the variable charge nature of the resins. Although using
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
resins to buffer Cu, Sheldon and Menzies (2005) found solution Cu concentrations to vary by up to
250 % due to changes in solution pH.
The objective of the current work was to quantify the critical free Cu2+ activity associated with a
reduction in growth of solution-grown cowpea. Automatic titration units were used to control pH,
and solution Cu concentrations were maintained constant through the use of ion exchange resins.
Growth of both the roots and shoots were related to Cu2+ activities as calculated from measured Cu
concentrations. Optical microscopy was used to examine the effect of high solution Cu on root
morphology.
Materials and Methods
Resin preparation
Approximately 400 g of Amberlite IRC-748 (Aldrich) ion exchange resin was placed into a column
and leached with methanol until the effluent was clear. Five volumes of deionised (DI) water were
then passed through the resin. Using 20 volumes of approximately 1.0 M HCl, the resin was
converted from the Na-form to the H-form, and was then leached with 20 volumes of DI water. The
resin was placed in two 5 L beakers, with Cu- and Ca-forms prepared by the addition of 2 L of the
appropriate chloride salt solution (2 mM CuCl2 or CaCl2) to the beaker. The pH of the solution was
then raised to pH 4.50 (± 0.03) using 0.1 M KOH for the Cu-form, and saturated Ca(OH)2
(approximately 0.02 M) for the Ca-form. The equilibrating chloride salt solution was replaced daily
(and further pH titrant added) until the replacement equilibration solution (without pH titrant added)
was pH 4.50 (± 0.03). The Cu saturated resin was then washed with 2 mM CuCl2 to remove/desorb
any K in the entrained solution or on the resin exchange as a result of KOH addition.
A Buchner funnel was used to remove excess moisture from the resin forms, and the moisture
content determined after oven drying a sub-sample. The Cu and Ca resins were mixed at eight ratios
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(1.3, 4.0, 5.0, 20, 45, 65, 75, and 85 % Cu-form resin) so as to yield a total resin mass of 10.5 g (0.5
g resin L-1 of the final nutrient solution). Following mixing, 20 mL of 2 mM CaCl2 added to each
resin mixture and the resins allowed to equilibrate for 14 d. By this time, exchange of Cu and Ca
between resins had resulted in the resin beads having a uniform color (Cu-saturated resin is blue,
Ca-saturated resin is white).
Due to the high affinity of the resin for both Mn and Zn (manufacturer’s guidelines), any Mn and
Zn supplied to a nutrient solution for plant growth would be rapidly taken out of solution and onto
the Cu/Ca resin mixture. Indeed, preliminary experiments indicated that unless Zn and Mn were
added as resins the plants would develop Zn and Mn deficiency. Therefore, in order to maintain
sufficient concentrations of these micronutrients in solution, Mn- and Zn-saturated resins were
prepared (as described above).
Solution culture
Experiments were carried out in controlled glasshouse conditions, with high pressure sodium lamps
supplementing natural sunlight, providing 16 h of light per day. Temperature was maintained at 30
C during the light period and 25 C during the dark.
Twenty four polypropylene containers (22 L; 265 mm diameter by 400 mm deep) were arranged in
a completely randomized design with a total of eight treatments (1.3, 4.0, 5.0 20, 45, 65, 75, and 85
% Cu-form resin) and three replicates. An air-driven water pump was placed in each container, and
the resin in a PVC container with 150 µm mesh ends (see diagram in Sheldon and Menzies (2005)).
The pump was used to force the solution through the resin, and also to aerate the solution. After an
initial 24 h aeration, each container was connected to a separate pH titration unit (TPS, miniCHEMpH) and peristaltic pump (Masterflex 5 RPM with Masterflex Tygon tubing, L/S 17) which was
used to raise and maintain the pH at 4.5 by the addition of 0.025 M Ca(OH)2. The resin mixtures
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were added to each pot, 0.5 g of each of the Mn and Zn resins incorporated, and the solutions
allowed to equilibrate for 14 d. After equilibration, the nutrient solution was found to contain
approximately 0.6 µM Zn and 0.8 µM Mn.
Cowpea seeds (Vigna unguiculata (L.) Walp. cv Caloona) were imbibed in aerated 200 μM CaSO4
solution for 2 h. Seeds were rolled in paper towel, and germinated for 36 h at 30 ºC with the ends of
the paper towel immersed in tap water. Four seedlings with radicle lengths of 10 ± 2 mm were
transferred to each container.
Nutrients were supplied through the use of a dilute basal nutrient solution (added upon initiation of
the experiment), and the incremental addition of a delivery nutrient solution at quantities calculated
to meet plant demand. Each container was filled with a basal nutrient solution, with the Cu supplied
from the ion exchange resins. GEOCHEM v2.0 (Parker et al. 1995) was used to check the
complexation of Cu by various chelates used to maintain Fe in solution. Across the Cu
concentrations of interest (and at pH 4.50) CDTA (cyclohexane-1,2-diaminetetra-acetic acid)
complexed the least Cu out of those chelates examined; approximately 0.35-1.3 % of the total
solution Cu. In contrast, EDDHA complexed 1.3-4.0 % of the total solution Cu, EDTA complexed
2.8-7.8
%,
NTA
(nitrilotriacetate)
complexed
65-91
%,
and
HEDTA
(hydroxyethylethylenediaminetriacetic acid) complexed 75-95 %. Therefore, to minimize Cu
complexation, Fe was supplied as FeCDTA (Table 1). To prepare the 10 mM FeCDTA stock
solution, 3.64 g of CDTA (Sigma) was placed in a 1 L volumetric flask and approximately 500 mL
of DI water added, with 7.2 mL of 5 M NaOH used to raise pH. 4.04 g Fe(NO 3)3.9H2O was
dissolved in 300 mL DI water, and the solution slowly added to the CDTA, with the final solution
volume raised to 1 L. Although each container was connected to a pH titration unit, the NO3-/NH4+
ratio (85:15) was predetermined so as to minimize pH variance.
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In addition to this basal nutrient solution, nutrients were supplied to the plants through the addition
of a calculated quantity of delivery nutrient solution (Table 1). Plant growth was calculated from the
exponential equation:
W t = W0 e (RGR×t )
where Wt is the plant dry mass (g) at time t (d), W 0 is plant mass at t = 0, and RGR is the relative
growth rate (g g-1 d-1) (Stadt et al. 1992). Using a RGR of 0.20 g g-1 d-1, the daily nutrient additions
(At) required in a given time interval were calculated for N (assuming a plant N concentration (C)
of 0.05 g N (g dry mass)-1), with the other nutrients added in the delivery solution in proportion to N
(Table 1):
A t = C × W0 e (RGR×t ) × (e RGR - 1)
where W0 is plant mass at t = 0 (calculated to be 0.15 g pot-1) (Taylor et al. 1998). The delivery
nutrient solution was added at the calculated rate three times during the first 7 d, then daily
afterwards. Additional DI water was supplied to the containers throughout the growth period to
maintain solution volume. Plants were allowed to grow for a total of 14 d from their introduction to
the nutrient solution until termination of the experiment.
Electrical conductivity (EC) was measured twice weekly, with the pH electrode in each container
also calibrated. Nutrient solution samples were taken on days 0, 7, and 14, filtered (0.22 µm
Millipore GSWP), acidified to pH < 2.0 using 20 µL of concentrated HCl, and refrigerated (3.5 ºC)
before analysis by inductively coupled plasma atomic emission spectrometry (ICPAES) and
inductively coupled plasma mass spectrometry (ICPMS). Upon completion of the experiment,
selected roots were taken from each treatment, stained using 0.5 % crystal violet, and examined
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using light microscopy. The fresh mass of the remaining roots and shoots was determined, the roots
thoroughly rinsed in DI water (5 mins), and the elemental concentrations of both the roots and
shoots determined using ICPAES after drying for 7 d at 65 ºC and acid digestion as described by
Martinie and Schilt (1976).
For relative shoot and root mass, a grouped regression analysis (fitting logistic curves) was
performed using GenStat 7 (GenStat 2003). Regression analysis was also used to examine the
relationship between nutrient solution Cu2+ activity (calculated from the measured Cu
concentration) and the root (linear regression model) and shoot (Mitscherlich (exponential)
regression model) Cu concentrations as determined upon completion of the trial (GenStat 2003). All
calculations of ionic strength (I) and ion activities were performed using PhreeqcI 2.11 with the
Minteq database (Parkhurst 2005). Chelated Cu accounted for < 1.3 % of the total solution Cu, with
Cu2+ accounting for > 99.9 % of the remaining soluble Cu forms.
Results
Root and shoot growth was found to decrease with increasing solution Cu2+ activity (p < 0.001)
(Figure 1). In addition, the pattern of response to increasing solution Cu2+ activity was the same for
both the shoots and the roots, with separate parameters not required to describe individual root and
shoot curves (p = 0.599) (Figure 1). Thus, the critical Cu2+ activity (corresponding to 90 %
maximum yield) of 1.7 µM was the same for both the roots and shoots (Figure 1).
Solution concentrations of Cu (buffered by the cation exchange resin) remained relatively constant
throughout the 14 d experimental period, with measured values varying by no more than 15 %
within each treatment (data not presented). Concentrations of most nutrients remained relatively
constant over time, although the average Zn concentration of 0.48 µM (supplied by the Zn-form
resin) was lower than expected. It was noted that solution Ca concentrations tended to increase over
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time due to the addition of Ca(OH)2 for pH regulation (Figure 2). This was particularly true in the
low Cu solutions where growth (and hence pH change) was greatest. It was also noted that the
concentrations of a few elements (such as K) tended to decrease slightly - particularly in the low Cu
solutions in which growth was more rapid (Figure 2). As a result of this nutrient uptake and
depletion, solution EC decreased slightly over the experimental period (Figure 2). Although it was
expected that this EC decrease would be greater in the low Cu treatments (due to more rapid
growth), similar trends were observed in all solutions (Figure 2). It is considered that the more rapid
uptake of nutrients in the low Cu solutions was offset by this higher rate of Ca(OH)2 addition for
pH regulation (Figure 2). Due to the optimization of the NO3-/NH4+ ratio, the quantity of titrant
added for maintenance of pH was comparatively small, with measurements showing that in a pot in
which no titrant was added, pH reduced from 4.5 to approximately 4.2 (data not presented).
Although nutrient supply was generally good, approximately 3 days after planting (DAP)
interveinal chlorosis was observed in the shoots of plants in the two highest Cu treatments. In
addition, during the last 2 d of growth symptoms of Mn toxicity (brown necrotic spots on the leaves
(Iwasaki et al. 2002)) were observed in the low Cu solutions. This observation of Mn toxicity was
confirmed with the analysis of the shoot tissue also indicating that Mn was toxic in these treatments
(Table 2). Shoot and root concentrations of K, Ca, Mg, Fe and Mn tended to decrease with
increasing Cu, whilst S, P and B concentrations remained steady or increased slightly (Table 2). It
was also noted that shoot concentrations of Zn and Fe in the low Cu solutions were higher than
expected (Table 2). Concentrations of Cu in both the root and shoot increased with increasing
solution Cu activity, although concentrations were approximately 10-fold greater in the roots than in
the shoots (Table 2 and Figure 3). The increase in shoot Cu with increasing solution concentrations
was almost linear, whilst root Cu increased exponentially with increasing solution Cu (Figure 3).
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Other than the obvious stunting (Figure 4a), examination of the root system revealed the formation
of toxicity symptoms in the three highest Cu treatments (1.9, 2.4 and 2.7 µM Cu2+) with the severity
of the symptoms increasing with increasing Cu2+. Roots from these high Cu treatments tended to
have slightly bent root tips, particularly at 2.7 µM Cu2+ where severe lesions due to the cracking of
the epidermis and outer cortex were also evident (Figure 4b). In addition, examination of roots at
2.7 µM Cu2+ revealed the formation of local swellings immediately behind the tip of many of the
lateral roots (Figure 4c and Figure 4d). The cells in these swellings tended to be circular in shape
rather than elongated as in the remainder of the root. It was also noted that root hair formation
tended to be inhibited in solutions with a Cu2+ activity ≥ 0.89 µM.
Discussion
Root and shoot growth of cowpea was reduced in dilute nutrient solutions (I = 3.2 mM) containing
a Cu2+ activity ≥ 1.7 µM (corresponding to 90 % maximum growth) (Figure 1). Although cation
exchange resins have previously been used to buffer Cu concentrations (Sheldon and Menzies
2005), inadequate pH control in this previous study resulted in large fluctuations in solution Cu
concentrations (Cu up to 250 % greater than the desired concentration in some treatments) due to
changes in the resins variable charge cation exchange capacity. The use of the pH stat system in the
current study not only ensured constant pH, but also relatively constant solution Cu concentrations.
However, in all previous studies other than Sheldon and Menzies (2005), solution Cu
concentrations were not buffered but typically added as an inorganic salt such as CuSO4.5H2O.
Further, in many studies solution Cu concentrations were not even measured (either during, or upon
completion of the experiment), but the concentration simply assumed to be that at which it was
added. Failure to account for Cu uptake from solution and its adsorption onto the root apoplasm
may result in the overestimation of the solution concentration, and hence underestimation of Cu
toxicity. Although solution Cu concentrations were not measured, most of these previous studies
utilised periodic renewal of the nutrient solutions to attempt to maintain constant Cu levels. The
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time between renewal, however, varied - ranging from 2 d (Liao et al. 2000), 7 d (Zhu and Alva
1993), to 21 d (Hill et al. 2000), or not at all (Taylor and Foy 1985). Further, the efficiency of
renewal on maintaining the desired Cu concentrations was not measured in any of these
aforementioned studies. Studying several native Australian tree species, Reichman (2001) found
that even with the addition of Cu every 7 d, solution Cu concentrations varied by up to 90 %,
particularly in the low Cu treatments. It is therefore possible that many of the previously reported
Cu tolerances are indeed overestimations of the true value, particularly in longer-term experiments
where Cu removal by plants would be the greatest.
Relative fresh weights of both the roots and shoots decreased in similar pattern as solution Cu
increased, with separate parameters not required to describe individual root and shoot curves (p =
0.599) (Figure 1). Similar results have been reported by Zhu and Alva (1993), Lin et al. (2003) and
Ali et al. (2002), who also found shoot growth to decrease significantly at the same Cu
concentration that caused a significant decrease in root growth. However, whilst shoot Cu
concentrations increased with increasing solution Cu, tissue concentrations of Cu were
approximately 10-fold greater in the roots than the shoots (Table 2 and Figure 3). Further, it was
noted that the decrease in root and shoot mass in solutions with a Cu2+ activity ≥ 1.7 µM (Figure 1)
corresponded with a steep increase in root Cu concentrations (Figure 3). Therefore, it is considered
that this shoot growth reduction is not due to a direct toxicity of Cu in the shoots, but rather to
nutrient deficiencies resulting from a reduced nutrient uptake by the damaged roots (Zhu and Alva
1993). Although the nutrient solutions contained sufficient nutrients, when compared to the control,
plants growing in high Cu solutions were found to have reduced shoot concentrations of K, Ca, Mg,
Fe and Mn, with concentrations often below the approximate critical concentration for deficiency
(Table 2). Indeed, interveinal chlorosis (consistent with Fe deficiency) was observed in the two
highest Cu treatments approximately 3 DAP. Decreases in shoot concentrations of Ca, Mg and Fe
and is typical of Cu toxicity (Ali et al. 2002; Lidon and Henriques 1993; Ouzounidou et al. 1995;
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Panou-Filotheou and Bosabalidis 2004) and is most likely due to a nonspecific effect of Cu on ion
absorption and translocation due to an impairment of root function (for example, membrane
leakiness, reduced ion uptake, or a reduction in radial transport or xylem loading). Induced Fe
deficiency has often been reported to be associated with Cu toxicity (Ouzounidou 1994; Taylor and
Foy 1985), and is also typical of toxicities of other metals such as Al, Co, Tl and Ni (Clark et al.
1981; Kaplan et al. 1990). It was also noted that shoot tissue concentrations of Zn and Fe in the low
Cu solutions tended to be higher than expected, although not thought to be toxic (Table 2). For
future studies, the use of preliminary investigations to optimize of solution nutrient concentrations
would be beneficial.
The approximate plant tissue Cu concentrations corresponding to 90 % maximum yield (20 µg/g
shoot, and 66 µg/g root (Figure 3)) were found to be similar to the critical concentrations of 67 µg/g
leaves and 41 µg/g root reported by Kalyanaraman and Sivagurunathan (1993) for the closely
related species Vigna mungo. In contrast, concentrations reported for other species differ
substantially; 26 µg/g shoot and 290 µg/g root in Chloris gayana (Sheldon and Menzies 2005), 340
µg/g root for Zea mays (Ouzounidou et al. 1995), 21 µg/g shoot and 170 µg/g root for Zea mays and
55 µg/g shoot and 3100 µg/g root for Phragmites australis (Ali et al. 2002), and 15 µg/g shoot and
2300 µg/g root for Triticum aestivum (Taylor and Foy 1985). This variation in critical contents is
not unexpected however, as it is considered that there is only a poor relationship between the
critical root tissue Cu concentration and growth. Examining excised root tips (the region in which
Cu would be most likely adversely effect root growth), Parker et al. (1998) reported that growth
inhibition was only weakly correlated with symplastic, apoplastic, and total Cu contents.
In addition to the shoot symptoms observed (growth reduction and interveinal chlorosis), roots
grown in the three highest Cu treatments (1.9, 2.4 and 2.7 µM Cu2+) also displayed typical Cu
toxicity symptoms with the severity of the symptoms increasing with increasing Cu2+. Roots from
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all three of the highest treatments tended to have slightly bent root tips, particularly at 2.7 µM Cu2+
where roots also displayed a deterioration and cracking of the epidermis (Panou-Filotheou and
Bosabalidis 2004) (Figure 4). It has been proposed that the epidermal cracking observed under
metal toxicity is due to the differences in elongation rate between the inner and outer cells
(Wagatsuma et al. 1987). It was also noted that roots grown at 2.7 µM Cu2+ tended to be brown in
color, short, thick, and highly branched (‘barb-wire roots’) (Kabata-Pendias and Pendias 2001; Zhu
and Alva 1993). Roots at the highest Cu also often displayed localized swellings behind the tips of
many of the lateral roots which were noted to contain plate-like cells rather than the typical
elongated cells (Figure 4). This formation of plate-like cells is attributed to a restriction in cell
elongation associated with Cu toxicity (Panou-Filotheou and Bosabalidis 2004; Savage et al. 1981).
Although the symptoms described above were observed in solutions containing ≥ 1.9 µM Cu2+, it
was noted that root hairs were more sensitive, with reductions in root hair formation occurring at ≥
0.89 µM Cu2+.
These root symptoms observed for Cu toxicity are similar to those described by others for Al
toxicity (Blamey et al. 2004; Kopittke et al. 2004; Yamamoto et al. 2001). Also, as seen for Al
toxicity, it would be expected that the Cu-induced reduction of root hair formation would result in
inhibition of nodulation by N2 fixing bacteria in leguminous species (Brady et al. 1993).
Although Mn toxicity was observed in the lower Cu treatments 2 d before completion of the
experiment, this is not considered to have substantially affected the results; the growth reduction in
these treatments would in fact reduce any differences between the low and high Cu treatments.
Further, Mn toxicity has been reported not to affect root hair growth (Wood et al. 1984), as was
evidenced in the current study by the good root hair growth observed in the low Cu solutions. This
Mn toxicity is due to the displacement of Mn from the resin by other cations with greater affinities
for the resin’s binding sites (such as Cu2+, Ca2+, and Zn2+) (manufacturer’s guidelines). Cowpea has
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previously been reported to be particularly sensitive to Mn, with toxicities often developing at
solution concentrations considered adequate for other species (Smith et al. 1983). Tissue analysis
confirmed the presence of Mn toxicity, with the shoot Mn concentrations (approximately 300 µM in
the low Cu solutions (Table 2)) greater than the 200-250 µg/g previously reported to be toxic
(Ashwath 1990; Wissemeier and Horst 1990). The Mn toxicity (and associated growth reduction)
was observed only in the low Cu treatments; growth in the higher Cu treatments was inhibited more
by Cu toxicity than Mn toxicity.
Conclusions
Unlike most previous studies, solution Cu was buffered (using a cation exchange resin) and its
concentrations measured throughout the duration of the experiment. The growth of roots and shoots
was found to be reduced at solution Cu2+ activities ≥ 1.7 µM (corresponding to 90 % maximum
growth). This observed decrease in shoot growth is not considered to be the result of a direct Cu
toxicity to the shoots, but rather a reduction in growth due to nutrient deficiencies caused by
damage to the roots; concentrations of K, Ca, Mg, and Fe decreased as solution Cu increased. At
high Cu2+ activities, roots were brown in color, short and thick, with bent root tips and cracking of
the epidermis and outer cortex. Local swellings, due to a reduction in cell elongation, were present
behind many of the root tips. Root hair growth was reduced at activities lower than that which
caused a significant reduction in root growth.
Acknowledgments
The authors recognize the comments, suggestions and ideas of Anna Sheldon and Michael Donn.
Michael Geyer is also gratefully acknowledged for his assistance with the sample preparation and
analysis.
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Table 1. Composition of the basal nutrient solution supplied at the beginning of the
experiment, and the delivery nutrient solution added throughout the duration of the
experiment in quantities calculated to meet plant demand.
Basal
Delivery
(µM)
(mM)
NO3-N
680
680
NH4+-N
120
120
K
302
400
Ca
650
150
Cl
140
234
Mg
50
75
S
502
20
P
2
20
Fe
10
5
B
3
1
Mo
0.02
0.02
Cu†
0
0
Mn†
0
0
Zn†
0
0
†
These nutrients were supplied by the Cu-, Mn-, and Zn-forms of the cation exchange resin
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Table 2. Effect of increasing Cu2+ activity (selected treatments only) on the tissue nutrient concentrations measured in the shoots and roots of
cowpea (Vigna unguiculata (L.) Walp. cv Caloona) after 14 d of growth in a dilute nutrient solution.
Roots
Shoots
Shoots
Solution Cu2+ activity
K
(µM)
------------------------ mg/g ------------------------
Ca
S
P
Mg
Cu
Fe
38.5
3.44
6.82
1.55
1.56
9.34
0.89
46.5
3.28
8.88
2.19
1.35
2.7
19.2
3.43
5.74
2.32
1.08
0.29
25.0
19.5
3.78
1.78
3.21
5.35
246
0.89
26.1
16.4
3.85
1.86
3.03
9.78
201
2.7
11.3
9.61
4.76
2.08
1.20
Critical concentration†
25-35
15-20
2.5-3.5
1.5-3.0
3.0-3.5
28.2
315
27.3
153
79.4
Approximate critical concentration for toxicity of Mn for shoots of cowpea (Reuter and Edwards 1997)
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Mn
31.4
30.7
118
182
23.9
33.7
191
145
25.1
11.6
80.9
110
71.6
50.5
70-75
20-35
Approximate critical concentration for deficiency of various nutrients for shoots of cowpea (Reuter and Edwards 1997; Smith et al. 1984)
‡
B
------------------------ µg/g ------------------------
0.29
†
Zn
38.4
314
38.2
331
39.8
33.0
300-350‡
Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Relative fresh mass (%)
125
Shoots
Roots
100
75
50
p < 0.001
2
R = 0.982
25
y
13
88
1 e
( 6.0 ( x 2.0 ))
0
0
1
Solution Cu
2
2+
3
activity (µM)
Figure 1. Effect of increasing Cu2+ activity in a dilute nutrient solution on the relative fresh
mass of roots and shoots of cowpea (Vigna unguiculata (L.) Walp. cv Caloona) after 14 d of
growth. A grouped regression (logistic) determined there was no significant difference in the
response of the shoots or roots to increasing solution Cu2+ activity (p = 0.599)
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Solution K concentration (µM)
360
320
280
240
0 DAP
14 DAP
Solution Ca concentration (µM)
200
680
660
640
0 DAP
14 DAP
620
0
1
2
2+
Solution Cu activity (µM)
3
EC (dS/m)
0.325
0.300
0.275
0.29 µM Cu2+
1.1 µM Cu2+
2+
2.7 µM Cu
0.250
0
5
10
15
Time (d)
Figure 2. Effect of solution Cu2+ activity on K concentration (top) and Ca concentration
(middle) measured 0 days after planting (DAP) and 14 DAP, and change in solution electrical
conductivity (EC) over time for selected Cu2+ activities (bottom).
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
400
40
x
y = 7.30 + 3.14 (5.60 )
p < 0.001
2
R = 0.943
30
300
Root Cu (µg/g)
Shoot Cu (µg/g)
y = 2.07 + 10.7x
20
200
10
100
0
0
0
1
Solution Cu
2
2+
p < 0.001
2
R = 0.982
3
activity (µM)
0
1
Solution Cu
2
2+
3
activity (µM)
Figure 3. Effect of increasing solution Cu2+ activity in a dilute nutrient solution on the tissue
Cu concentrations measured in the shoot (left) and root (right) of cowpea (Vigna unguiculata
(L.) Walp. cv Caloona). Dotted line corresponds to the solution Cu2+ activity (1.7 µM) found
to correspond to the 90 % relative mass for the shoot and root. A regression (solid line) of the
form: Shoot Cu = A + B (Solution Cu), was fitted to the shoot data, and of the form: Root Cu
= A + B (D)Solution Cu, to the root data.
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Kopittke, P.M., and N.W. Menzies. 2006. Plant Soil 279:287-296.
Figure 4. Roots of cowpea (Vigna unguiculata (L.) Walp. cv Caloona) after 14 d growth in a
dilute nutrient solution containing a Cu2+ activity of 2.7 µM ((a) photograph of the whole root
system (bar indicates approximately 10 mm), and optical micrographs of (b) primary root tip,
and (c) and (d) root tips of lateral roots (bar indicates 1 mm)), or a Cu2+ activity of 0.29 µM
(control) ((e) primary root tip).
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