Effects of copper concentration on mineral nutrient uptake and copper accumulation in protein of copper-tolerant and nontolerant Lotus purshianus L.

ECOTOXICOLOGY

AND ENVIRONMENTAL

SAFETY

29,2

14-228

(I 994)

Effects of Copper Concentration on Mineral Nutrient Uptake and Copper Accumulation in Protein of Copper-Tolerant and Nontolerant Lotus purshianus L. SHEN-LIN LIN Departtnettr

y~EtwironmentaI

Horticrrhttre.

Received

LIN Wu

AND

University

September

qf Cal$ornia.

Davis.

Caljlbrttia

956 16

IS, 1993

One copper-tolerant and one copper-sensitive inbred line of Lorfrs pws/tiamr.s L. derived from a copper mine waste site in Northern California and one inbred line of the same species derived from a pasture next to the mine waste were examined for the effects of excessive copper concentrations on mineral nutrient uptake and accumulation of copper in protein fractions. Plants were grown from seeds for a period of 24 days in a modified Hoagland nutrient solution culture supplemented with 3.6, and IO &I copper as copper sulfate. The basal nutrient solution without copper amendment was used as the control treatment. The uptake of Cu found in the roots was 100 times or more than that in the leaves. The root tissue copper concentrations reached a plateau under 6 PM copper treatment. The leaf tissue copper concentrations increased with the increase of copper concentration in the solution culture. No difference in pattern of copper uptake was detected between the copper-tolerant and nontolerant plants. The effects of excessive copper concentrations caused reduction of Ca uptake in the leaf tissue and P uptake in both the root and leaf tissues, and no difference was found between the copper-tolerant and nontolerant plants. Increased tissue copper concentration caused greater reduction of Fe, Mn. and Zn uptake in the nontolerant plants than in the tolerant plants; this difference may be important for the growth of the tolerant plants under conditions of excessive copper concentrations. Protein extracted from the roots and leaves of both the copper tolerant and nontolerant plants was subjected to Sephadex G-75 column separation. Two major peaks of protein fractions were detected. Under low (normal level) copper concentration treatment, the copper-tolerant and nontolerant plants had similar Cu/protein ratios. However. under higher copper concentration challenged conditions the coppertolerant plant had a considerably greater Cu/protein ratio (peak II protein) than the nontolerant plants. The amino acid composition of the copper-rich protein fraction (peak II) extracted from both the tolerant and nontolerant plants demonstrated a high asparate (about 25%) content. The contents of glutamate, cystine, and glycine were about I I, 2.5. and IO’%. respectively. and the rest of the amino acids were in a range of 2 to 6%. This pattern of amino acid composition is different from the amino acid composition of the phytochelatin metallothionein-like proteins found in copper-tolerant plants which are very high in cysteine. Instead. the copper rich protein found in the L. purshiamts resembles the copper-binding protein found in spinach which has high acidic amino acids and asparate content. More studies are needed to characterize this copperbinding protein and discover its possible role in copper tolerance of L. pttrs/tiamr.s. B 1994 A~~~,,IIc Pms, Inc.

INTRODUCTION Although copper is a required nutrient element for plant development and growth (Sommer, 193 l), a slightly excessive amount of copper in the soil or growth medium may produce effects detrimental to the plants. The uptake of copper in sugarcane leaf tissue was proved to be an energy requiring mechanism and the uptake of Cu*+ and Zn*+ was found to be competitive to other nutrient elements for the identical carrier

0147-65

I3194

$6.00

Copyright 0 1994 by Academic Pres. Inc. All rights of reproduction in any form reserved.

214

COPPER

TOLERANCE

OF

Lorus

purshianus

L.

215

site (Bowen, 1967). Toxic levels of metals affecting the uptake of nutrients were found to be common (Baker, 1987a,b; Veltrup, 1978; Godbold et al., 1988). Copper-induced iron deficiency is considered to be a typical copper toxicity symptom (Marchner, 1986). Excessive copper was found to depress iron uptake by bush bean in low iron solution but not in high iron solution (Wallace and Muller, 1980). Excessive amounts of copper in soil substantially reduced the uptake of phosphorus by citrus seedlings (Spencer, 1966). Turner and Marshall (1972) and Wu et al. (1975) suggested that in Agrostis sp. there is a copper-complexing mechanism in the cell wall and/or intercellular space protecting metabolic activities in the cell. Therefore, more copper is accumulated in the roots of the tolerant plants than in the nontolerant plants. A similar phenomenon was observed in Silene cucuba (Lolkema and Vooijs, 1986). However, in Lotus purshianus (Benth.) Clem. & Clem. no difference was found in the pattern of copper uptake between the tolerant and nontolerant plants (Wu and Lin, 1990). The objective of this study was to discover any discrepancy in nutrient uptake and copper accumulation in the protein between a copper-tolerant copper mine waste ecotype and a nontolerant pasture ecotype of L. purshiunus under long term growth conditions as affected by copper concentrations. MATERIALS

AND

METHODS

Copper Tolerance Tea L. purshiunus L. is an annual herbaceous plant. It is self-compatible but it can be outcrossed by insect pollination. One copper-tolerant (MT) and one copper-sensitive inbred line (MN) derived from the segregation of the copper mine waste population in Northern California and a nontolerant inbred line (FN) derived from a pasture field next to the copper mine waste were used for this study. These copper-tolerant and nontolerant lines were selected and established after seven generations of selfing (Wu and Lin, 1990). For the copper tolerance test, seeds of each line were scarified, placed on a wet filter paper in petridishes, and kept in a growth chamber at 25°C under 16 hr light. One-week-old seedlings of uniform size were used. Five seedlings, 2-3 cm in height, were transplanted into a supporting medium of black polystyrene beads. The beads were contained in a g-cm diameter hole in the center of a 12-cm diameter, 2.5cm-thick Styrafoam disc. A nylon mesh was attached to the base of the disk to contain the beads. The whole disk was floated on 2.5 liters of nutrient solution. The modified Hoagland nutrient solution (Hoagland and Amon, 1950) was used as a background culture solution which contained 0.5 M KN03, 0.5 A4 Ca(N03) - 4Hz0, 0.5 MNH4H2P04, and 0.25 M MgS04 - 7Hz0 as macronutrients. Micronutrients were provided as 25 mJ4 KCI, 12.5 mM H3B03, 1 mM MnS04 - H20, 1 mM ZnS04 - 7Hz0, 0.25 mM H2Mo04, 0.5 mM Cu as CuS04 - 7Hz0, and iron was given as 25 rnJ4 FeS04 - 7H20 [to avoid a chelate effect on copper toxicity which occurs if the EDTA form is used (Majumder and Dunn, 1985)]. For the copper tolerance test, 10 CLMCU as copper sulfate was added into the basal nutrient solution and the nutrient solution without a supplement of Cu was used as the control treatment. Three replicates were used for each inbred line and copper treatment. Nutrient solutions were replaced every 4 days. The plants for these and subsequent studies were kept in a temperature-controled greenhouse under conditions of 20°C during the day and 19°C at night with

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average daylight of 830 pmol mm2 s- ‘. Tolerance was measured following Wilkins (1978), based on the root length, root elongation rate, and root dry weight. At the end of the second week, the root elongation rate was measured, based on the increase of the longest root of each plant in 2 consecutive days. The root dry weight was measured after 24 days of growth. The tolerance index was calculated as the ratio of growth produced in the copper-containing solution to the growth produced in the control solution. Data were then log-transformed for the analysis of variance. Mineral Nutrient Uptake

For the study of effects of copper on mineral nutrient uptake, five plants were grown from seedlings in the basal solution supplemented with 3, 6, or 10 PM copper as CuS04 * 7H20. Each treatment was replicated three times. The 36 containers, including four copper treatment levels, three inbred lines, and three replications, were completely randomized. The plants were kept in a temperature-controled greenhouse under conditions of 20°C during the day and 19°C at night, with average daylight of 830 pmol mm2 s-r. Nutrient solutions were replaced every 4 days. The plants were grown for 24 days before harvest. At the end of the experiment, plants were harvested, rinsed with deionized water, and separated into root, stem, and leaf tissues. Five plants from the same container were pooled as one sample and dried at 60°C for 72 hr. The wet ashing method described by Zasoski and Burau (1977) was used for tissue digestion. Dried plant tissues were ground into powder and were digested in concentrated HN03 and HC104 at 2 10°C for 1 hr. The plant tissue digestants were dissolved and diluted with double distilled water before they were used for nutrient element analysis. Mineral concentrations of Cu, Fe, Zn, Mn, Mg, and Ca were measured with a Perkin-Elmer 28 atomic absorption (AA) spectrophotometer. For the Ca analysis, 1 cme3 of 5% La203 was added into 4 cme3 of the digestant before the AA analysis. Potassium was measured by flame emission. Phosphorus was measured by vanadatemolybdate yellow method (Champman and Pratt, 196 1). Within-laboratory accuracy and precision of mineral element analysis using spiked samples was described by Wu and Huang ( 1992). Protein and Copper Analysis

For protein and copper accumulation analysis, the copper-tolerant plant MT and the nontolerant plant FN were used. The seed germination and solution culture methods follow the previous experiments. Five plants were grown in each 2-liter container. The plants were grown either in the basal nutrient solution as the control treatment or in the basal solution supplemented with 10 PM copper as CuS04. Four replications were used for each treatment, and the nutrient solutions were replaced every 2 days. After 3 weeks of growth, the plants were harvested. To have a sufficient amount of plant tissue for the protein extraction, plant materials from two containers (replications) were combined. The protein extraction procedure followed was developed by Lolkema et al. ( 1984). One gram of fresh frozen root or leaf tissue was ground in liquid nitrogen into fine powder. Phosphate buffer (10 cm3 of 50 mM, pH 7.0) containing 5 mM mercaptoethanol was added and additional grinding was performed in a mortar. After filtration, the filtrates were centrifuged at 20,OOOgfor 15 min. The proteins were precipitated by 80% saturation of ammonium sulfate. After 1 hr of stirring, proteins were

COPPER

TOLERANCE

OF

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purshianus

217

L.

collected by centrifugation at 20,OOOg for 30 min. Proteins were redissolved in 1 cm3 extraction buffer and dialyzed against 50 mA4 phosphate buffer at pH 7.0 for 12 hr. Copper and protein remaining in the crude extracts and the redissolved ammonium precipitates were checked to determine the recovery rate. Protein assays were done by the protein-dye binding method (Bradford, 1976). The protein extraction processes were either held in a 2°C cold room or in an ice bath. A gel filtration column with a 28-cm length and 1S-cm inside diameter was packed with Sephadex G-75 (Pharmacia, Sweden) preswollen in 50 rnM of pH 7.0 phosphate buffer. An aliquot of 1 mg protein per sample was placed onto the column and was eluted with 50 mM phosphate buffer, pH 7.0, at an 18 cm3 per hour flow rate. The eluent was monitored at 254 nm absorbance (Casterline and Bamett, 1982) and collected into 40 fractions with 1.58 cm3 per fraction. Copper was measured by atomic absorption spectrophotometry (Perkin-Elmer 2380). Protein fractions of both the tolerant and nontolerant genotypes were examined for coordination between copper and protein concentrations. Amino Acid Analysis The protein fraction having the highest copper concentration in the peak I and peak II fractions of both the tolerant and nontolerant plants treated with copper were analyzed for amino acid composition. The protein fractions were first precipitated by 10% TCA (trichloroacetic acid). After centrifuge, the protein precipitate was washed with freezing cold acetone (-20°C). The dried protein samples were sent to the Protein Structure Laboratory at the University of California, Davis campus for amino acid analysis. The amino acid compositions were analyzed by a Backman 6300 analyzer with a post column Ninhydrin detection. RESULTS The results of the copper tolerance test are presented in Table 1. The tolerance ratios measured by the three growth parameters separated the three inbred lines of L. purshianzrs into tolerant and intolerant groups. The tolerant line had a root length tolerance ratio greater than 90%. In contrast, the mean tolerance ratios of both the copper mine and the field nontolerant lines were 35 and 45%, respectively. Tolerance TABLE COPPER

TOLERANCE

I

OF THREE INBRED LINES OF Lam purshinus BY THREE GROWTH PARAMETERS

L. MEASURED

Tolerance index (W) Inbred lines Copper mine tolerant line (MT) Copper mine nontolerant line (MN) Field nontolerant line (FN)

Root length 0.95 f 0.05” 0.45 f 0.08 0.35 f 0.05

Root elongation

Root dry weight

0.90 f 0.09” 0.42 r 0.08 0.28 f 0.06

1.34 + 0.15” 0.65 f 0.1 I 0.53 * 0.09

0 Tolerance ratios significantly different from those of the nontolerant plants (P < 0.001).

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ratios of the root elongation rate presented a result similar to the root length tolerance ratio. Tolerance ratios represented by root dry weights were higher and varied more than the tolerance ratios represented by root length and root elongation rate. The analysis of variance indicates that significant differences in copper tolerance exist between the tolerant and nontolerant inbred lines for all three growth parameters (P < 0.001). Copper Uptake

Patterns of copper uptake indicate that the plant tissue Cu concentration increases with the increase of copper concentration in the culture solution (Fig. 1). The roots accumulated more copper than the leaves. Under the 6 PM copper concentration treatment the roots had an average of 800 pg Cu g-’ dry weight, while the leaves contained only 50 pg Cu g-’ dry weight. The root tissue copper concentration reached a plateau under the 6 FM copper treatment. The leaf tissue copper concentrations increased with the increase of copper concentration in the culture solution, but the tissue concentrations were only 1% of the root tissue copper concentrations. The tolerant and nontolerant inbred lines differ in their copper tolerance, but are similar in their pattern of copper uptake and copper concentration in comparable organ tissues. Iron Uptake

Overall, the tissue iron concentrations decreased with the increase of copper concentrations in the culture solution (Fig. 1). The root tissue iron concentration of the tolerant line remained unchanged up to 6 @I copper treatment and was slightly reduced at the 10 PM copper treatment. However, the average root tissue Fe concentration of the nontolerant lines decreased from 450 pg g-’ dry weight under control the treatment to 200 wg g-’ dry weight under the 10 &I copper treatment. Zinc and Manganese Uptake

Zinc concentrations in the root tissue ranged from 300 pg g-’ dry weight for the control treatment to 200 pg g-’ for the 10 PM copper treatment, and in the leaves ranged from 100 to 200 pg g-’ dry weight. The magnitude of reduction of tissue Zn concentration was greater for the nontolerant than for the tolerant line (Fig. 1). Tissue Mn concentrations were also higher in the roots than in the leaves (Fig. 1). The root tissue Mn concentration of the nontolerant lines decreased from 400 pg g-’ dry weight under the control treatment to about 250 pg g-’ dry weight under 10 PM copper treatment, but the tissue Mn concentration of the tolerant line was only slightly reduced. Manganese concentrations in the leaf tissue exhibited a similar pattern as the root tissue, except the tissue Mn concentration was lower and ranged from 120 to 200 PLgg-’ dry weight. Calcium, Magnesium,

The uptake of Mg uptakes were nutrient solution. nontolerant lines

Phosphorus, and Potassium Uptake

the macronutrient elements is presented in Fig. 2. Potassium and not significantly affected by the increase of Cu concentration in the The root and shoot tissue K concentrations of both the tolerant and were similar and ranged from 0.9 to 1.O% of dry weight, and the Mg

COPPER TOLERANCE

219

OF Lorus pwsllianrrs L.

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FIG. I. Effects of copper concentration on micronutrient uptake of the copper-tolerant (-) and nontolerant (---and . . .) lines of Lotus pttrshiams L.

concentrations ranged from 0.5 to 3.0% dry weight. No apparent difference in Mg uptake between the copper tolerant and nontolerant lines was found. Tissue Ca concentrations in the root tissue were around 1% dry weight and were not affected by the copper concentrations (Fig. 2). However, the leaf tissue Ca concentration was reduced from 3.5% of the control treatment to about 2.5% under the

220

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AND

WU 4.00

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in culture soiution

(phQ

FIG. 2. Effects ofcopper concentration on macronutrient (--- and . . * ) lines of Lotus purshianm L.

I.-- 0.00 0.00

2.10 2.10

copper uptake

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4.20 4.20

6.30 6.30

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in cukure solution (PM)

of the copper-tolerant

(-)

and nontolerant

10 PM copper treatment. Calcium uptake was not different between copper-tolerant and nontolerant lines. Phosphorus concentrations were slightly higher in roots (about 2% of dry weight) and lower in leaves (about 1%) (Fig. 2). Both root tissue and leaf tissue P concentrations were significantly reduced by the increase of copper concentration in the solution culture. There was no significant difference between the tolerant and nontolerant lines.

COPPER

TOLERANCE

OF

Lo!us

p~~l~ia~~rrs

221

L.

Protein arld Copper Acctrinulalion The elution profiles of Sephadex G-75 chromatograms are presented in Figs. 3 and 4. Two major peaks of protein concentrations were found in the protein fractions (I and II) extracted from the roots of both the copper-tolerant and nontolerant plants. Protein fractions in the peak II were found to have greater copper concentrations than in the peak I protein fractions. Table 2 presents the average values of copper/protein ratios measured for the root cytosol protein extract before and after Sephadex G-75 column separation. Before the column separation, the average values of Cu/protein ratios of the root protein extracts of the control treatment were 8.0 and 4.0, and those for the 10 PM copper-treated plants were 2.0 and 2.5 for the tolerant and nontolerant lines, respectively. The Cu/protein ratios measured for the peak I protein fractions were low, about 1.O, and similar between the tolerant and nontolerant lines as well as between the copper-treated and control treatment plants. However, the Cu/protein ratio of the peak II protein fractions increased from 22 for the control treatment to 135 for the 10 PM copper treatment for the tolerant line and from 25 to 108 for the nontolerant line, which is a 70-fold increase. It is significantly different between the tolerant and nontolerant lines (P < 0.001). The separation profiles of the shoot protein extracts also demonstrated two major peaks, but the copper profiles were spread over a wide range of protein fractions, and the copper concentrations were much lower (Fig. 3 and 4).

Amino Acid Compositions The results of the amino acid composition analysis are presented in Table 3 along with a reference amino acid composition of a copper-binding protein found in a copper-

3.5 3 2.5 2 1.5 1 0.5 0

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FIG. 3. Sephadex G-75 gel filtration profiles of cytosol protein fractions and profiles of protein copper concentrations from roots and leaves of the copper-tolerant (MT) and nontolerant (FN) lines of Lofrcs p~shiunus L. grown in the nutrient solution culture.

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4. Sephadex G-75 gel filtration profiles of cytosol protein fractions and profiles of protein copper concentrations from roots and leaves of the copper-tolerant (MT) and nontolerant (FN) lines of Lo~trs purshianus L. grown in the nutrient solution culture supplemented with IO PM copper as copper sulfate. FIG.

tolerant spinach (Tukendorf et al., 1984). The protein fraction with the greatest Cu/ protein ratio found in the peak II protein profile revealed a high asparate (about 25%) composition. The contents of glutamate, cysteine, and glycine were about 1 1, 2.5, and lo%, respectively, and the rest of the amino acids were in the range of 6 to 2%. This pattern of amino acid composition of the protein fraction found in the peak II protein profile resembles that of the copper-binding protein found in spinach (Tukendorf et al., 1984). However, no distinct difference in amino acid compositions of the protein fraction in the peak I protein profile extracted from both the tolerant and nontolerant lines of L. purshianus displayed a similar pattern. It ranged from the highest of about 11.5% aspastic acid to the lowest of 1.5% cysteine (Table 3). DISCUSSION Heavy metal tolerance in plants may be achieved in two different ways, by avoidance and by tolerance. Avoidance is defined as an ability to prevent excessive metal uptake into its body (Levitt, 1980). Tolerance is an ability to cope with metals that are excessively accumulated within the plants. It is obviously an arbitrary definition to some extent. Tolerance mechanisms may be considered as avoidance mechanisms operating at integration levels lower than that of the individual organisms. Shoot/root partitioning ratios of tissue metal concentrations have been used as a measure of restriction of metal transport for comparing the behavior of metal-tolerant and less tolerant plant species and races (Lefebvre and Denaeyer-De Sme, 1985; Reilly and Reilly, 1973). In most cases, the plants from metal-contaminated soils have significantly lower ratios

COPPER TOLERANCE

OF LOWS purshianus

TABLE COPPER

223

L.

2

TO PROTEIN RATIOS DETECTED IN PROTEIN FRACTIONS AFTER SEPHADEX G-75 COLUMN SEPARATION

Cu/protein ratio Protein Control Before column separation After column separation Peak I Peak II Copper treatment Before column separation After column separation Peak I Peak II

Tolerant line

BEFORE AND

X

10-l

Nontolerant line

8.0

4.0NS

1.3 22.0

I.lNS 25.0NS

2.0

2SNS

I.0 135.0

0.9NS 108.0”

Nofe. The ratios were calculated based on data of duplicate sample analysis. NS, not significant. ’ Significantly different from the tolerant line at I%.

than the plants from uncontaminated soils, which suggests that metal tolerance in these species, such as is found in grass species, is associated with enhanced root accumulation and restriction of internal metal transport (Turner, 1970; Wu et al., 1975; Baker, 198 1). Resistant plants may exhibit an increased need for the metals to which they are resistant, as expressed by a less than maximum growth at normal availability levels (Ernst, 1983). However, there are few discoveries of restriction of metal uptake in metal-tolerant races of vascular plants. The Zambian “copper flower” Becium hombfei (De Wild) Duvign. Planke is very tolerant of copper, yet tissue analysis of plants reveals consistently low and similar copper concentrations in both shoots and roots, indicating restricted uptake of the metal (Reilly, 1969). In L. purshianus no difference in the pattern of copper uptake was detected between the copper-tolerant and nontolerant plants (Wu and Lin, 1990). This phenomenon implies that copper tolerance in this plant is not dependent on a mechanism of differential copper uptake. The copper concentrations used for this study are within the limit of biological threshold, because under the highest copper concentration (10 PM) the nontolerant plants were able to produce 30 to 40% of their normal growth. Therefore, for this study, any misinterpretation of the results of mineral nutrient uptake and copper accumulation can be minimized. The protein and copper accumulation analyses in this study have demonstrated that there is a quantitative difference in the accumulation of copper-binding protein between the tolerant and nontolerant lines. Copper-binding proteins have been found in Agrostis, Silene, and Mimulus (Rauser, 1984; Lolkema et af., 1984; Robinson and Thurman, 1986). The involvement of a metallothioneinlike copper complex in the mechanism of copper tolerance was found in Mimulus guttatus (Robinson and Thurman, 1986). These proteins are very high in cysteine, to which copper is bound much more firmly. The copper rich protein fraction detected

224

LIN

AND TABLE

WU 3

AMINO ACID COMPOSITIONS OF THE COPPER-BINDING PROTEIN FRACTIONS SEPARATED G-75 GEL FILTRATION FROM THE RENTS OF COPPER-TOLERANT (MT) AND NONTOLERANT shiunm

Amino

DEVELOPED

acid

Asp Thr Ser GIU GlY Ala Pro Val Met Ileu Leu Tyr Phe LYS His A% CYS

IN NUTRIENT

FN-I I I.5 6.1 7.4 10.6 9.3 8.4 4.5 6.9 0.6 5.1 8.7 3.1 4.2 6.4 1.8 3.9 I .4

SOLUTION

MT-I 11.7 6.0 7.5 10.9 9.3 8.5 4.7 6.5 I.1 5.0 8.5 2.9 4. I 6.2 I.6 3.9 I.5

CULTURE

SUPPLEMENTED

Amino

acid composition FN-II 23.2 5.0 5.6 9.6 10.6 6.6 4.2 5.1 0.6 3.5 5.8 2.9 3.6 4.9 1.7 5.0 2. I

WITH

BY SEPHADEX (FN)

L. prtr-

IO ptn4 COPPER

(“i mol) MT-II

Spinach”

29.6 4.0 7.1 II.4 9.6 5.4 3.2 3.7 0.8 2.6 4.4 2.2 2.6 4.7 2.2 4.2 2.3

Nofe. The Roman numeral following the genotype stands for the copper-binding protein been taken from the peak I (non-copper related) or peak II (copper related). ’ Data of spinach copper-binding protein amino acid from Tukendorf et al. (1984).

20.5 5.5 7.3 18.2 IO.9 9.3 5.3 5.7 0.8 3.7 5.0 I.3 2.6

0.6 3.3 fraction

having

from the roots of the L. pwshianzts does not reveal a high cysteine content. Instead, the amino acid composition of the copper-binding protein resembles the copper-binding protein found in a copper-tolerant spinach (Tuckendorf et al.. 1984) which has high acidic amino acids and a high asparate content. Copper-binding protein without high cysteine content was also reported in copper-tolerant oats (Tukendorf and Baszynski, 1985). The inducibility of metal-binding complexes in plants has been found to be very common (Grill ef al., 1987; Wagner and Trotter, 1982). This copper-binding protein quantitative difference was not found in the shoot tissue of the copper-tolerant and nontolerant plants of L. purshinus. Alternation of cellular metabolism and cell membrane structure have been suggested to play a role in metal tolerance in plants (Robinson and Thurman, 1986; Verkleij et al., 1988; Veltrup, 1978; Woolhouse and Walker, 198 1) and such mechanisms may not demonstrate any quantitative difference in copper accumulation. It is not clear whether this detected quantitative difference of copper-binding protein plays any major role in copper tolerance for this species. More research is needed to further characterize this protein and discover the mechanism of copper tolerance in the copper-tolerant

L. purshiunus. Iron, zinc, copper, and manganese are categorized as “borderline” ions. These ions compete with each other for the same binding sites. The displacement of Fe2+, Zn’+,

COPPER

TOLERANCE

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Lottrs

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or Mn*+ by Cu2+ is expected (Nieboer and Richardson, 1980). Copper-zinc and coppermanganese antagonistic effects were found in durum wheat (Morard, 1986). Bowen ( 1969) indicated that the absorption of Cu*+, Zn2+, and Mn*+ is coupled with oxidative phosphorylation, and Zn*+ and Cu*+ are absorbed through the same carrier site. Iron, zinc, and manganese concentrations in root tissue of bean plants were reduced by adding 0.5 mg liter-’ of copper into the solution culture, if iron was provided as Fetatrate not as Fe-EDTA (Daniels el al., 1972). The results of this study found that the tissue concentrations of these three ions in both the copper-tolerant and nontolerant L. pwshianus plants were generally reduced by an increased copper concentration in the culture solution. However, the copper-tolerant line was able to maintain a higher level of tissue concentration than the nontolerant plants. This difference might be a result of copper tolerance of the plants rather than a part of the tolerance mechanism, but it may be important for the plant growth under copper-stressed conditions. An excessive amount of copper in soils markedly reduced the uptake of P by citrus seedlings, and the application of P into the soil reduced the copper toxicity (Spencer, 1966). Copper-tolerant Silene maritima was found to be more efficient in P uptake and transport than nontolerant plants (Baker, 1987a,b). For L. pzrrshianus, excessive copper concentrations reduced P uptake in both the copper tolerant and nontolerant plants. This P uptake reduction may be due to its reaction with copper and reduced its availability to the plants. Calcium is a relatively large divalent cation. It readily enters the apoplasm and is bound in an exchangeable form to cell walls, at the exterior surface of the plasma membrane, and tends to be exchanged by other divalent ions of higher ionic strength (Van Steveninck, 1965). It is a nontoxic mineral nutrient, even in high concentration, and is very effective in detoxifying high concentrations of other mineral elements in plants (Hanson, 1984; Kirkby and Pilbeam, 1984). This may be the reason why increased copper concentration in the solution culture leads to a reduction of tissue calcium concentration because of the displacement of calcium in the cell wall and cell membrane. However, this pattern of tissue Ca concentration was revealed only in the leaf tissue. The root tissue had lower tissue Ca concentrations. The relative ion concentration of calcium in the external root environment was much higher than the copper concentrations used for this study. Therefore, the tissue calcium displacement by copper in the root tissue may not be detectable. In addition, no difference in the pattern of the effects of copper concentrations on Ca uptake was found between the tolerant and nontolerant plants of L. purshianzts. Excessive copper concentrations may damage cell membranes and cause leakage of K+ of plant roots (Wainwright and Woolhouse, 1975). However, the K concentration of both the tolerant and nontolerant lines of L. purshianus was not affected by the increased external copper concentration. This indicates that copper concentrations used for this study, even though they induced considerable growth reduction for the nontolerant plants, did not cause damage of the cellular structure of the plants and had little effect on K uptake. The effects of excessive copper concentrations may induce a reduction of mineral nutrient uptake in L. purshianzrs. However, the degrees of effect are different among the mineral nutrient elements. A difference in mineral nutrient uptake between the copper-tolerant and nontolerant plants was found only at the micronutrient level.

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This difference may be important for the growth of tolerant plants under conditions of excessive soil copper concentrations. CONCLUSIONS Copper-tolerant and copper-sensitive inbred lines of L. purshiunus L. were examined for the effects of excessive copper concentrations on mineral nutrient uptake and accumulation of copper in protein fractions. Calcium uptake in the leaf tissue and P uptake in both the root and leaf tissues were decreased by the increase of copper concentrations in the solution culture. No difference was found between the coppertolerant and nontolerant lines. Increased copper concentrations caused greater reduction of Fe, Mn, and Zn uptake in the nontolerant plants than in the tolerant plants: this difference may be important for the growth of the tolerant plants under conditions of excessive soil copper concentrations. Proteins extracted from the roots and leaves of both the copper-tolerant and nontolerant plants was subjected to Sephadex G-75 column separation. Under copperchallenged conditions the copper-rich protein fraction of the copper-tolerant plant had a considerably greater Cu/protein ratio than the nontolerant plants. The amino acid composition of the copper-rich protein fraction extracted from both the tolerant and nontolerant plants the amino-acid composition of the copperbinding protein resembles the copper-binding protein found in a copper-tolerant spinach, which has high acidic amino acids and a high asparate (Tuckendorf ef al., 1984), but different from the metallothionein-like copper complex found in plants which is very high in cysteine. More studies are needed to characterize this copper-binding protein and discover its possible role in copper tolerance in L. purshianus. REFERENCES BAKER, A. J. M. (1987a). Ecophysiological aspects of zinc tolerance in Silene marifima

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acid digestion method for multielement