Summary. Root elongation of Norway spruce (Picea abies (L.) Karst.) seedlings was inhibited in the presence of. 5 pM Cd but was unaffected by a Cd ...
Tree Physiology 9,349-357 0 1991 Heron Publishing-Victoria,
Canada
Cadmium uptake in Norway spruce (Picea abies (L.) Karst.) seedlings D. L. GODBOLD Forstbotanisches Institut, Universitiit Giittingen, Biisgenweg 2,3400 Gettingen,
Germany
Received September 10, 1990 Summary Root elongation of Norway spruce (Picea abies (L.) Karst.) seedlings was inhibited in the presence of 5 pM Cd but was unaffected by a Cd concentration at 0.05 pM. Nutrient solutions labeled with ‘09Cd were used to investigate the influence of pH, cations and the metabolic inhibitor 2,4-dinitrophenol (DNP) on the uptake of Cd by rcots of intact spruce seedlings. Extracellular Cd was removed by washing the roots, and the relative amounts of Cd in the root apoplast and symplast estimated. In the presence of DNP, Cd uptake was reduced at 0.05 yM (non-toxic) but not at 5 pM Cd (toxic). At 0.05 pM Cd, the uptake of Cd into both the apoplast and symplast was dependent on the pH of the nutrient solution. Lower pH decreased Cd accumulation. Aluminum supplied at 100 or 500 pM lowered the Cd concentrations of both the apoplast and symplast. An increase in Ca or Mg supply reduced the Cd concentration of the apoplast but not of the symplast. In the presence of 5 pM Mn, the concentration of Cd in the symplast decreased by 44% compared to the control (1 f.tM Mn). High concentrations of Zn or Hg did not affect the Cd concentration of the roots.
Introduction In forest ecosystems in western Germany, flux balances have indicated that Cd is either accumulating (Truby and Zijttl 1984) or, in strongly acidified sites, being mobilized and lost (Schultz 1987). In acidified soils, Cd is mobilized mainly as Cd2+ (Kiinig et al. 1985). Several investigations have shown that the uptake of Cd by plants is controlled by the availability of Cd2+ (Greger and Lindberg 1986, Cabrera et al. 1988), thus, in forests where Cd is being mobilized, much of the Cd is in a form available to plants. Studies on Cd and forest trees have been mostly concerned with the phytotoxic effects of Cd. In several tree species exposure to Cd reduced growth of roots and shoots (Mitchell and Fretz 1977, Kelly et al. 1979). In all species investigated, the accumulation of Cd increased with an increase in Cd supply. Cutler and Rains (1974) suggested that Cd uptake in barley is not under metabolic control but is primarily by diffusion. Cataldo et al. (1983) showed that, at Cd concentrations below 0.5 PM, Cd uptake in Phaseolus vulgaris was under metabolic control, whereas at concentrations above 0.5 FM, which Cataldo et al. (1983) suggested may be phytotoxic to Phaseolus vulgaris, uptake was mainly passive. In solution culture, pH and competition from other cations influence Cd uptake. John (1976) and Hatch et al. (1988) found that low pH decreased Cd uptake in several plant species. Cadmium accumulation was depressed in Lolium perenne by Ca, Mn and Zn (Jarvis et al. 1976), in Avena sativa and Lactuca sativa by Ca (John 1976),
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350
and in Phaseolus vulgaris by Ca (Hardiman and Jacoby 1984) and by Cu, Fe, Mn and Zn (Cataldo et al. 1983). In Zea mays, Tyler and McBride (1982) found no effect of solution pH or Ca on Cd uptake. In one of the few studies using tree species, Burton et al. (1986) showed that Cd accumulation in Picea sitchensis was decreased by Cu but not by Ni. In the work presented here the uptake of Cd by seedlings of Picea abies (L.) Karst. was investigated to determine whether Cd uptake is under metabolic control. The effects of pH and cations on Cd uptake were also examined. Materials
and methods
Culture of plants Seeds of Picea abies (L.) Karst. were surface sterilized in 1% w/v Ca(OCl)z, and germinated for 3 weeks on 1% (w/v) water agar, pH 4.6. The seedlings were transferred to aseptic nutrient solutions containing (FM): Ca(NO& 450, MgS02 3 15, KHzPO4 250, N&N03 225, FeC12 5, MnSO4 1, HsBOs 5, NazMo04 O.l,ZnSO4 0.1, CuSO, 0.1, CoS04 0.02, pH 4.5 and grown for a further 7 days. The solutions were constantly aerated with sterile air. Growth conditions provided a day/night temperature of 23/21 “C, 35 f 5% relative humidity, 100 pmol mm2 s-i photon flux density (Osram L18W/25 lamps), and a 16-h photoperiod. Root elongation Aseptically grown seedlings were transferred to nutrient solutions for 2 days, then transferred to nutrient solutions containing 0.05 or 5 pM Cd as CdClz for a further 6 days. Rates of root elongation were determined as previously described (Godbold and Htittermann 1985). Cadmium uptake Seedlings were transferred to nutrient solutions containing either 0.05 or 5 pM CdClz labeled with 3.7 MBq lo9CdC12 1-l for 5 h. Each vessel contained 500 cm3 of nutrient solution and eight seedlings. Three vessels were used per treatment. To estimate the effect of 2,4-dinitrophenol (DNP) on Cd uptake, seedlings were pretreated for 30 min with nutrient solutions containing 0.1 mM DNP, then transferred to the lo9Cd solutions also containing 0.1 mM DNP. To estimate the influence of pH on Cd uptake, seedlings were equilibrated for 30 min in nutrient solutions adjusted to pH 2.5, 3.5,4.5, 5.5 or 6.5, then transferred to solutions containing 0.05 pM lo9Cd adjusted to the same pH. Seedlings were exposed to nutrient solutions containing 100 and 500 pM Al, 225, 450 and 900 PM Ca, 30,160 and 3 15 pM Mg, 5 pM Zn or Mn, or 0.05 pM Hg and the effect on Cd uptake was determined. Removal of extracellular Cd Following the uptake period, roots were blotted and transferred to unlabeled nutrient
CADMIUM
UmAKE
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351
solutions (4 “C) with a 20-fold higher Cd concentration than the uptake solution. Eight seedlings (8-10 mg root dry weight) were transferred to 100 cm3 of solution. One-cm3 aliquots of the solution were taken over a 90-min period. Determination
of ‘09Cd
Roots were dried to constant weight, and the ‘09Cd activity in the roots and desorption solutions determined in a gamma counter (Philips PW 4800).
Results and discussion Measurement of root elongation is a sensitive method for estimating the toxicity of metals to plants (Godbold et al. 1984). After exposure for 2 days to 5 pM Cd, root elongation was inhibited by 18% (Table 1). An increase in the duration of exposure to Cd further increased the degree of inhibition of root elongation. Exposure to 0.05 PM Cd did not significantly inhibit root elongation over the duration of the treatment period. After exposing roots to nutrient solutions containing lo9Cd, an attempt was made to remove extracellular Cd from the roots by washing them in unlabeled nutrient solutions. Over the 90-min washing period up to 45% of the total Cd taken up by the roots was removed in the washing solutions (Figure 1). By drawing semilogarithmic plots of the efflux profiles (Figure l), fast and slowly exchanging phases could be separated. The slope of the slowly exchanging phase was plotted using a non-linear regression method (Rygiewicz et al. 1984). In the two examples shown, the r2 values for the slowly exchanging phase were -0.952 and -0.943 for the 0.05 FM and 5 pM Cd treatments, respectively. For all subsequent data, r2 values were never less than -0.92. In an investigation of methods for removal of extracellular Cd from roots, Rauser (1987) estimated that 90% of the Cd was lost from the cell wall compartment during a lo-min wash in 5 mM CaC12 (cf. Kochian and Lucas 1982, Santa Maria and Cogliatti 1988). Using longer washing times, Rauser (1987) separated Cd efflux into fast, medium and slow exchange components, which are generally assumed to correspond to the cell wall, cytoplasm and vacuole (Walker and Pitman 1976). In this study, the exchange phases of Cd efflux from roots were used to calculate the Cd concentrations of the apoplast and symplast. The intercept of the regression line of the slowly exchanging phase at time zero was used to calculate the Cd concentration
Table 1. Root elongation (k SE) of containing 0.05 or 5 pM Cd. Day
1 6
Root elongation
Picea dies seedlings
(mm
after
1 and 6 days growth
in nutrient
solutions
day-‘)
Control
0.05 PM Cd
Control
5.0 pM Cd
2.2*0.1 2.1+ 0.2
2.1 f0.2 2.5 k 0.2
3.8 k 0.2 3.1 kO.2
3.1 kO.2 1.8 HI.2
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352
0,OSpM
Cd
5,uM Cd
CT3 b 5
80
2 8
60
60
100
40
‘Y=
30
minutes
0,05,&l
90
minutes
Cd
L
5,ufl Cd
7.9
1.7
30
60
1.5
60
30
60
90
minutes
minutes
Figure 1. Semi-logarithmic plots of ‘“‘Cd elution from roots of intact Picea dies seedlings expressed as a percentage of Cd uptake. The seedlings were exposed to 0.05 or 5 pM ‘“9Cd for 5 h. The washing solutions contained a 20-fold higher Cd concentration than the uptake solutions.
of the symplast. Using this protocol an attempt was made to correct for the loss of Cd from the symplast during washing or for the incomplete removal of extracellular Cd. The concentrations of Cd in the apoplast and symplast estimated by efflux plots and by a simple 15min wash procedure are compared in Table 2. The estimates of the Cd concentration of the symplast after a 15-min wash were higher than the values obtained from the efflux plots. However the values estimated by the two techniques did not differ significantly. Although there are difficulties in interpreting data from efflux analysis, particularly if a limited number of data points are used, efflux plots
Table 2. The concentrations of Cd (nmol Cd g&-l k SE) in the apoplast and symplast dies seedlings determined by means of efflux profiles or a 15.min wash (see Figure were exposed to 0.05 or 5 pM “nCd for 5 h. Method
Efflux Wash
0.05 uM
Cd
of roots of Picea 1). The seedlings
5uMCd
Apoplast
Symplast
Apoplast
Symplast
6.5 5 0.5 5.4kO.3
9.3 * 0.7 10.4 f 0.6
770 f 170 653 f 106
851 * 190 968+ 157
CADMIUM
UPTAKE
IN SPRUCE
353
provided a better estimate of the concentrations of Cd in the apoplast and symplast than the simple wash procedure. The uptake of Cd was determined in the presence of the metabolic inhibitor 2,4-dinitrophenol (DNP) at concentrations of Cd that inhibit root elongation (5 pM) or do not affect root elongation (0.05 PM). Cadmium uptake into both the apoplast and symplast was dependent on Cd supply (Table 3). At 0.05 PM Cd, DNP reduced Cd uptake into the apoplast and symplast by 40 and 66%, respectively, whereas at 5 PM Cd, a concentration that inhibits root elongation, DNP did not significantly reduce Cd uptake into the apoplast or symplast. In an investigation of Cd uptake into barley roots at 90 PM Cd, Cutler and Rains (1974) concluded that Cd uptake was not under metabolic control but was primarily by diffusion. Cataldo et al. (1983) concluded that active processes were involved in Cd uptake at Cd concentrations below 0.5 PM Cd, whereas at higher concentrations passive processes (adsorption and diffusion) dominated. The results presented here support the conclusion of Cataldo et al. (1983) and emphasize the importance of carrying out uptake studies with heavy metals at non-phytotoxic concentrations. The lower uptake of Cd into the apoplast in the presence of DNP, may be associated with the elimination of binding sites that are dependent on an uninhibited metabolism (Ighe and Pettersson 1974). The pH and the concentration of cations in a forest soil solution can vary greatly down a soil profile (Raben 1988). The pH and the concentration of cations in the nutrient solution were found to influence Cd concentrations in both the apoplast and symplast. The concentrations of Cd in the apoplast and symplast were strongly pH dependent (Table 4). A decrease in pH resulted in a decrease in Cd concentration in both root components. However, a greater percentage of the total Cd taken up was in the apoplast at pH 2.5 (71%) than at pH 6.5 (38%). In Dactylis glomerata, Lactuca sativa, Lolium perenne, and Rorippa nasturtium-aquaticum, there is a similar pH dependence of total Cd uptake in roots (Hatch et al. 1988). This effect has also been observed with other metal cations such as Zn (Chaudrhy and Loneragan 1972), Mn (Robson and Loneragan 1970) and Al (Godbold, unpublished observations). Aluminum reduced Cd uptake into both the apoplast and symplast (Figure 2). Compared with Al, an increase in the supply of ‘Ca and Mg only decreased the Cd uptake into the apoplast. In the presence of increased concentrations of Mn, Zn, or
Table 3. The concentrations of Cd (* SE) in the apoplast and symplast of roots of Picea a&es seedlings exposed to 0.05 or 5 pM lWCd for 5 h in the presence or absence of 0.1 mM DNP. The asterisk denotes a significant difference with DNP treatment. Treatment
Cadmium
concentration
Apoplast 0.05 PM Cd 0.05 PM Cd + DNP 5lMCd 5 PM Cd+ DNP
6.5 3.9 770 585
(nmol
g&l) Symplast
+ * * +
0.5 0.4* 170 50
9.3 3.2 851 823
* k * +
0.7 0.4* 190 70
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354 Table 4. Influence of pH on the Cd concentrations (+ SE) of the apoplast shies seedlings exposed to 0.05 nM ‘09Cd for 5 h. Cadmium
PH
concentration
(nmol
and symplast
g&l)
Apoplast 2.5 3.5 4.5 5.5 6.5
0.76 3.4 8.6 17.8 31.5
0
0
apoplast
q
symplast
100 Al (MM)
500
225
of roots of Picea
Symplast f 0.01 *0.1 zkO.2 f 1.6 f3.3
0.3 I k 0.0 1 3.6 +O.l 8.2 f0.2 14.8 IL 1.4 51.0 k5.2
450 Ca
900 CUM)
Figure 2. Influence of Al, Ca or Mg on the concentrations Picea a&s seedlings exposed to 0.05 l.tM lo9Cd for significantly different (P = 0.05). The vertical bar = SE.
30
160 Mg
315
(PM)
of Cd in the apoplast and symplast of roots of 5 h. Columns marked with an asterisk are
Hg, only Mn influenced Cd uptake (Figure 3). In the presence of 5 yM Mn, the concentration of Cd in the symplast decreased by 44% compared to the control (1 pM Mn). The decrease in Cd concentrations in the apoplast at low pH was probably a result of a smaller number of dissociated carboxylate groups in the cell walls, and hence potential binding sites. Similarly, the decrease in the concentration of Cd in the apoplast at increased Al and Ca supply, or low Mg supply, may be due to competition of the elements with Cd for binding sites in the cell walls. Aluminum has a high affinity for binding sites in the cell wall, and has been shown to displace Ca (Bengtsson et al. 1988) or Ca and Mg (Godbold et al. 1988) in the apoplast. In roots
CADMIUM
UPTAKE
control
IN SPRUCE
0
apoplast
Ed
symplast
Mn
Zn
355
Hg
Figure 3. Influence of Mn, Zn or Hg on the concentrations Picea shies seedlings exposed to 0.05 FM lWCd for significantly different (P = 0.05). The vertical bar = SE.
of Cd in the apoplast and symplast of roots of 5 h. Columns marked with an asterisk are
of Zea mays, Khan et al. (1984) concluded Cd was bound to pectic residues in the cell wall. Favali et al. (1982) found that Ca has an almost identical distribution pattern to Cd within cell walls. Calcium only reduced the Cd concentration of the apoplast, whereas Al and low pH decreased Cd uptake into both apoplast and symplast. It has been suggested that Al inhibits Mg uptake by lowering Mg loading in the apoplast, thus reducing the concentration of Mg at the uptake sites at the plasmalemma (Godbold et al. 1988). A similar mechanism could lower Cd uptake into the symplast, however Al and H ions might also directly influence Cd uptake at the plasmalemma. Suhayda and Haug (1986) showed that Al inhibits membrane bound ATPase in roots of Zea mays. Unlike the other cations examined, Mn reduced Cd uptake into the symplast, without affecting the Cd concentration of the apoplast. In Phaseolus vulgaris, Cataldo et al. (1983) suggested that Cd and Mn have common uptake sites because Mn competitively inhibited Cd uptake. Cataldo et al. (1983) also showed a competitive inhibition of Cd uptake by Zn. Jarvis et al. (1976) demonstrated a decrease in Cd absorption by Lolium perenne in the presence of Mn and Zn. In the Picea abies seedlings Zn did not affect Cd uptake. At Cd concentrations commonly found in forest soils (Lamersdorf 1989), Cd uptake in Picea abies is a metabolically controlled process. At several sites in northern Germany higher Cd concentrations were determined in vital than in subvital spruce roots (Lamersdorf 1989). Cadmium uptake was reduced by both low pH and some cations. On acid forest
356
GODBOLD
sites in northern Europe the soil solutions often contain concentrations of Al similar to those used in this work (Raben 1988). Thus it is to be expected that, in some soils, Al will decrease Cd uptake by roots of forest trees. In soils, the plant availability of Cd increases as pH decreases (Brtimmer et al. 1986). However, the uptake of Cd into roots in nutrient solutions decreased with decreasing pH. In soil, a similar effect may tend to counteract that of pH on Cd availability and uptake by Picea abies. Hatch et al. (1988) attributed a low uptake of Cd with decreasing pH by soil-grown plants to the suppression of Cd uptake by hydrogen ions. Under conditions where the rhizosphere pH exceeds that of the bulk soil (Haussling et al. 1985), the roots may act as a sink for Cd. In conclusion, Cd uptake by seedlings of Picea abies from solutions containing non-toxic Cd concentrations involves metabolic processes, whereas uptake from solutions containing toxic Cd concentrations is mainly passive. Cadmium uptake is dependent on the pH of the surrounding medium and the supply of some cations. Aluminum inhibits Cd uptake at concentrations found in forest soils. References Bengtsson, B., H. Asp, P. Jensen and D. Berggren. 1988. Influence of aluminium on phosphate and calcium uptake in beech (Fagus sylvatica) grown in nutrient solution and soil solution. Physiol. Plant. 14:299-305. Burton, K.W., E. Morgan and A. Roig. 1986. Interactive effects of cadmium, copper and nickel on the growth of Sitka spruce and studies of metal uptake from nutrient solutions. New Phytol. 103:549-557. Cabrera, D., S.D. Young and D.L. Rowell. 1988. The toxicity of cadmium to barley plants as affected by complex formation with humic acid. Plant Soil 105: 195-204. Cataldo, D.A., T.R. Galand and R.E. Wildung. 1983. Cadmium uptake kinetics in intact soybean plants. Plant Physiol. 73:844-848. Chaudrhy, EM. and J.F. Loneragan. 1972. Zinc absorption by wheat seedlings: II. Inhibition by hydrogen ions and by micronutrient cations. Soil Sci. Sot. Amer. Proc. 36:327-331. Cutler, J.M. and D.W. Rains. 1974. Characteristics of cadmium uptake by plant tissue. Plant Physiol. 546-11. Favali, M.A., M. Ferrario and N. Barbieri. 1982. Subcellular distribution of potassium antimonate precipitates in plant tissues. I. Zea mays leaves. Cytobios 35: 19-28. Godbold, D.L. and A. Hiittermann. 1985. Effect of zinc, cadmium and mercury on root elongation of Picea dies (Karst.) seedlings, and the significance of these metals to forest die-back. Environ. Pollut. 38:375-381. Godbold, D.L., E. Fritz and A. HiittermM. 1988. Aluminum toxicity and forest decline. Proc. Natl. Acad. Sci. 85:3888-3892. Godbold, D.L., W.J. Horst, J.C. Collins, D.A. Thurman and H. Marschner. 1984. Accumulation of zinc and organic acids in roots of zinc tolerant and non-tolerant ecotypes of Deschumpsia caespitosa. J. Plant Physiol. 116:59-69. Greger, M. and S. Lindberg. 1986. Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). I. Cd*+ uptake and sugar accumulation. Physiol. Plant. 66:69-74. Hardiman, R.T. and B. Jacoby. 1984. Absorption and translocation of Cd in bush beans (Phasedus vulgaris). Physiol. Plant. 61:670-674. Hatch, D.J., L.H.P. Jones and R.G. Burau. 1988. The effect of pH on the uptake of cadmium by four plant species grown in flowing solution culture. Plant Soil. 105:121-126. Haussling, M., E. Leisen, H. Marschner and V. Roheld. 1985. An improved method for non-destructive measurement of the pH at the root-soil interface (rhizosphere). J. Plant Physiol. 117:37 l-375. Ighe, U. and S. Petterson. 1974. Metabolism-linked binding of rubidium in the free space of wheat roots and it relation to active uptake. Physiol. Plant. 30:24-29.
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