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New Phytol. (1997), 137, 303-314

Acclimation of Salix to metal stress

BY TRACY PUNSHON* AND NICHOLAS M. DICKINSON

School of Biological & Earth Sciences, Liverpool John Moores University, Byrom Street,Liverpool L3 3AF, UK

{Received 4 November 1996; accepted 28 May 1997)

SUMMARY

Nine different clones of six species of Salix (Salix cordata Muhlenb. non Michaux, 5 . fragilis L., S. caprea L.,S. cinerea h., S. burjatica Nazarov. and 5 . viminalis L.) and one hybrid (S. x calodendron Wimm.) were exposedto heavy metals in solution culture in an attempt to increase innate metal resistance. Resistance was estimatedusing comparative root measurements, and metal uptake was also studied. The first experiment entailed pre-treatments with background nutrient solution, or 0-25 and 050 mg Cu 1" amendments, and re-exposureto each of the same concentrations. In a second experiment clones were exposed to sub-toxic concentrationsof single metals (0-15 mg Cu 1"', O-lSmgCdl" ' or 2-5mgZnl ' ' ) and dual-combination treatments(0-075mgCu|- '- l-0-O75mgCdl- ' , O'O75 mg Cu 1"'+ 1 25 mg Zn I"' or 0'075 mg Cd 1"'+ 1-25 mg Zn 1" = )' withconcentrations gradually raised 10-fold over 128 d. Plants tested in the first experiment, following pre-exposureto Cu, were no more resistant to subsequent exposure to this metal. In the second experiment, gradual cumulati^ edoses resulted in reduced phytotoxicity and increased resistance, most notably to Cd. There appeared to be aninverse relationship between metal uptake and resistance. Copper uptake was restricted to the roots, whereas Cdand Zn were more evenly distributed throughout the plant. Exposure to dual combinations of metals resulted inseveral interaction effects on uptake: increased root-bound Cu in all combinations, and the increase in uptake ofboth Cd and Zn into the root tissues when supplied with Cu. The implications of these results for the use ofwillows in phytoremediation programmes are discussed.

Key words: Salix, heavy metals (copper, cadmium and zinc), resistance, acclimation, phytoremediation.

I N T R O D I' C T 1 O N

Many human activities, including mining, smeltingand the disposal of sewage sludge, have increased therelease of heavy metals into the biosphere (Nriagu &Pacyna, 1988). Heavy metals are not readily removedor degraded by chemical or microbial processes, andtend to accumulate in soils and aquatic sediments.One natural response to environmental contami-nation has been the evolution of metal-resistantpopulations of plants (Bradshaw, 1952; Gregory- &Bradshaw, 1965; Denny & Wilkins, 1987o; Turner& Dickinson, 1993). The potential use of metal-resistant plants for stabilization and reclamation ofcontaminated soils has been subsequently realized(Gadgil. 1969; Smith & Bradshaw, 1972) and theremight be considerable benefits to be gained fromplanting trees (Glimmerveen, 1996). However, mostinformation available on metal resistance is based ononly a handful of herbaceous species and few studieshave investigated metal resistance in woody speciesfor the purpose of land reclamation (Antonovics,

* To whom correspondence should he addressed.E-mail: bes tpuns(^ l iv jm.ac . uk

Bradshaw & Turner, 1971; McCormack & Steiner,1978; Baker, 1987; Watmough, Gallivan & Dickin-son, 1995; Punshon & Dickinson, 1996, 1997).Although there are many examples of trees survivingon metal contaminated soils (Kahle, 1993; Turner &Dickinson, 1993; Watmough & Dickinson 1995A;Glimmerveen, 1996) no attempt has been made toderive resistant material for transplantation to othercontaminated sites for the purpose of bioremedi-ation.

The processes responsible for metal resistancegenerally involve strategic uptake or avoidance ofmetals (Baker, 1987), the compartmentalization ofmetals in less metal-sensitive tissues such as vacuolesor else their exclusion from the plant (Baker, 1981;Denny & Wilkins, 19876). Resistance to Cd isthought to involve metal-chelating complexes calledphytochelatins (Jackson et al., 1987) whereas Curesistance might involve root cell-wall selectivity(Taylor, 1987). These physiological adaptations canbe selected on exposure to contaminated soils, anddepend on the prior possession of the appropriategenetic variability within plant populations. Selec-tion of metal resistance in plants occurred both inresponse to atmospherically deposited metals (Al-

304 7". Punshon and N. M. Dickinson

Hiyaly et al., 1988; Dickinson. Watmough &Turner, 1996; Turner & Dickinson, 1993) and tolocalized high-level pollution episodes such asdumping of metalliferous mine waste (Bradshaw,1952; Baker, 1981). Howe\er, it has becotne clearthat the role of phenotypic plasticit^^ has beenunderstated (Sultan, 1987; Dickinson, Turner &Lepp, 1991a; Watmough & Dickinson, 1996) andthis is the focus of the present study. Before we canproperly understand the variability that exists betw-een and within species to withstand toxic metals, aclear picture is required of the ability of individualplants to acclimate to pollution stress (Dickinson etal., 1991a, 1992). Acclimation is defined as thegradual and reversible adjustment of physiology andmorphology to changes in environmental conditions(Crawford, 1990).

The genus Salix possesses a notably high level of\ ariation with a wide range of morphological t>'pes,including trees and shrubs (Meikle, 1992; Stott,1992). In evolutionary terms, Salix is unique becauseit is not only one of the youngest tree genera, but ina relatively short time has become specialized to awide range of ecological niches (Pohjonen, 1991).Further evidence of the dynamic evolution of thegenus is found in the ever increasing number ofhybrid species recognized by taxonomists (Meikle,1992). Recent research on the potential of shortrotation coppice (SRC) willow and poplar as bio-logical filters for waste w"ater and sludge disposal basled to preliminary studies on metal uptake in clonesof tbe fast-grow ing biomass species S. viminalis andS. dasyclados (Landberg & Greger, 1994; Ostman,1994). These species npically inhabit fertile soils,but other hardier species such as S. caprea and S.cinera (Stott, 1992) grow on nutrient-poor andindustrially-contaminated soils (Grime, Hodgson &Hunt, 1988; Eltrop et al., 1991; Kahle, 1993;Punshon & Dickinson, 1997). These characteristics,combined with widespread hybridization betweenspecies, suggest that willows might be of use inphytoremediation schemes; appropriate genetic vari-ability for survival on metal-contaminated soilsprobably already exists.

Before it is possible to identify potential phyto-remediation sbrubs by screening and selection of thenecessary genetic variation for metal resistance, aclear knowledge of how this variation can beexpressed and an understanding of the stability ofmetal resistance traits is needed. Several workershave successfully induced resistance to heavy metalsin herbaceous species by using low dose pre-treatments (Brown & Martin, 1981; Aniol, 1984;Baker et al., 1986), and gradual acclimation of treesassociated with long-term exposure to aerially de-posited metals has been identified (Cumming &Taylor, 1990; Outridge& Hutchinson, 1991; Dickin-son et al., 1991a, 1992, 1996; Dickinson, Turner &Lepp, 19916; Watmough & Dickinson, 1995a;

Watmough et al., 1995). The objective of the presentstudy was to investigate whether short-term pre-treatments or gradual acclimation to elevated metaltreatments increased the resistance of Salix tometals. Uptake of metals was also investigated todetermine where and to what extent metals arestored within the plant. It was hoped that thisinformation could then be used to contribute to aprogramme of production of plants suitable forphytoremediation of metal-contaminated sites.

M.ATERIALS AND METHODS

Salix cuttings were sampled for two experiments inearly spring and early autumn 1995, from clonalmaterial from the National W'illow Collection held atNess Botanic Gardens (Table 1). Following initialresistance screening experiments a range of commonspecies was sampled, including fast-growing biomassshrubs (e.g. Salix viminalis), hardy stress-resistantspecies (e.g. S. caprea) and hybrids of both species(Punshon, Lepp & Dickinson, 1995). The 18-cmcuttings were maintained in 3-5 1 black poly-propylene buckets containing 1 1 of glass-distilledwater in a controlled temperature glasshouse(19 °C with a diurnal fluctuation of 5°C; r.h.55"o±10"o) without artificial lighting for f. 14duntil root prinnordia became visible. Before anysubstantial root elongation, cuttings were trans-ferred to a suspension hydroponics system(Punshon et al., 1995) supplied with 0-25-strengthHoagland's solution containing ^

151-6 KNO3, 2361 CaNO,)2.4H.,O, 57 561 6MgSO, .7H,O, 0 93 k c ] , 008 MnSO^.HÔ,0 39 H,BO.,, 0-08 ZnSO^. 5H2O, 0-38 Cu SO^.5H,O, 0-08 H.,MoO, and 1 73 Fe-Na EDTA(Hoagland & Arnon, 1941). This formulation pro-vided background micronutrient concentrations of0-03 mg r ' Zn and 0-0008 mg 1" Cu in solution.Heavy metal amendments were added as follows: Cuas CuSO^.5H2O; Cd as ^CdSO^.llB-Ô and Zn asZnSO4.7H2O at concentrations described below.The solutions were continuously aerated andchanged every 7 d; solution pH was adjusted to 5-8using 0-1 M HCl and maintained within the range5-5-5-9.

Short term Cu pre-treatment

Six contiguous hydroponic units were set up con-sisting of duplicate blocks of three treatments (0,0-25 or 0-50 mg Cu T^) with 27 replicate cuttings ofeach of five species randomized within each unit(Table 2). Metal concentrations used in theseexperiments were chosen from published work oncritical metal concentrations in trees (Burton, King& Morgan, 1985; Turner & Dickinson, 1993) torepresent a range from non-phytotoxic through tosub-lethal. The length of longest root, total number

Acclimation of Salix to metal stress 305

Table 1. Salix species and clones used in pre-treatment and acclimation experiments

Expt 1:

Clone

Short-term Cu pre-treatment

Accession

Expt 2:

Clone

Cu, Cd and Zn acclimation

Accession

S. cordata cv. Purpurescans 3280S. fragilis L. cv. Russeliana Kew 32355. caprea L. cv. Sidelands (^) 32895. caprea L. cv. Sutton (?) 32855. cinerea ssp. oleifolia 3294Macreight. '{£)' (2)

S. caprea cv. Higher Green D. (2) 3287S. X calodendron Wimm.* (o) 3311S. burjatica Nazarov. cv. Aquatica 3349gigantea Pavainen E78995. viminalis L. cv. Ivy Bridge (?) 3369

Sex of clone indicated where known.* 5 X calodendron = caprea x cinerea x viminalis.

Table 2. Treatment schedule for the acclimation experiment

Time (d)

0-28 dInitial low dosepre-treatmentduring cuttingestablishnnent

29^2 d100% dose increase

43-56 d50 "o dose increase

57-85 d'rest' period86-99 d55-5 '^0 doseincrease based onlevels used on43-56 d

100-114 d42-8 % doseincrease

]15-128 d50 "/o dose increase

Tray number and metal concentration (mg 1 )

(a) Single metal treatment (b) Combination treatment

(1) B.S.(2) Cu(0-15)(3) Cd(0-15)(4) Zn (2-5)

(1) (B.S.)(2) Cu (0'3)(3) Cd (0-3)(4) Zn (5 0)

(1) (B.S.)(2) Cu (0-45)(3) Cd (0-45)(4) Zn (7'5)

* Background

(1) (B.S.)(2) Cu (O-O75) + Cd (0-075)(3) Cd (O-O75) + Zn (1-25)(4) Cu (0-075) + Zn (1-25)

(1) (B.S.)(2) Cu (O-15) + Cd (0-15)(3) Cd (O-15) + Zn (2-5)(4) Cu (0-15) +Zn (2-5)

(1) (B.S.)(2) Cu (O-225) + Cd (0-225)(3) Cd (O-225) + Zn (3-75)(4) Cu (0-225)+ Zn (375)

nutrient solution*Chlorosis observed - further treatment suspended(1) (B.S.)(2) Cu (0-7)(3) Cd (07)(4) Zn (11-5)

(]) (B.S.)(2) Cu (1-0)(3) Cd (1-0)(4) Zn (17'2)

(I) (B.S.)(2) Cu (1'5)(3) Cd( l '5 )(4) Zn (25 8)

(!) (B.S.)(2) Cu (O-35) + Cd (0-35)(3) Cd (O-35) + Zn (5-6)(4) Cu (O'35) + Zn (5-6)

(1) (B.S.)(2) Cu (O'5) + Cd (0'5)(3) Cd (O5) + Zn (8-4)(4) Cu(0-5) + Zn(8-4)

(1) (B.S.)(2) Cu (075) + Cd (0-75)(3) Cd (075) + Zn (126)(4) Cu (075) + Zn (12'6)

B.S., Background nutrient solution (25 "o Hoagland's solution).

of adventitious roots per cutting and percentageviability were monitored every 7 d for 28 d. Cuttingswere then removed from the units, washed carefullyand three cuttings from each species were removedfor metal determination. The remaining cuttingswere then divided into three sub-treatments and re-randomized within the units so that all pre-treatedplants were subsequently re-exposed to each of thethree Cu concentrations. Root length and numberwere monitored after 1 d and 28 d of treatment, thenagain removed randomly for metal analysis.

Acclimation to elevated Cu, Cd and Zn

A further eight contiguous hydroponic units wereset up, consisting of six treatments (Cu, Cd, Zn,Cu-I-Cd, Cd + Zn and Cu + Zn) and two controlunits (background nutrient solution). Fifty-fourreplicate cuttings of four Salix species (Table 1)were randomized within each unit. Treatmentincrements followed a schedule which was not pre-determined ; but was based on monitoring datacollected each week, with increments and rest

306 T. Punshon and N. M. Dickinson

periods assigned in consideration of the health of testplants (Table 2). The aim of this treatment schedulewas to expose plants gradually to increasing metalconcentrations, rather than to test for resistance aftera single exposure to a phytotoxic metal concen-tration, and therefore to enable acclimated plants totolerate metal concentrations which would otherwisebe lethal. Length of longest root, total number ofadventitious roots per cutting and viability weremonitored every 14 d throughout the course of theexperiment.

Metal analysis of plant material

W'ashed material was separated into leaf, secondarystem, wood (original cutting segment) and rootmaterial and dried in an air-circulation oven at 80 °Cuntil there was no further weight loss. It was thenground to a fine pow der (> 1 mm stainless steelsieve), and 05 g samples were digested in triplicatein 10 ml of HNOg using a microwave digestion oven(MDS-81D: CEM Corporation) in 120-mi Teflon*PFA vessels. Copper, Cd and Zn were quantifiedusing a Perkin Elmer 375 Atomic AbsorptionSpectrophotometer.

Data analysis

Due to inherent differences in the rooting viability ofindividual species (Pohjonen, 1991) root growth datawere zero-adjusted (i.e. all zero values were omitted).Zero data were used instead as an expression of testpopulation viability. This process normahzedskewed data, and presented a more accurate value ofmean root grow th. In both experiments a modi-fication of the Tolerance Index (7"/) (Wilkins, 1978)widely used in metal-resistance studies was em-ployed. The parameters used in the index werevaried for each experiment. In tbe first experimentTJ was calculated from the equation:

TL ^xlOO, (1)

where TI^^^ = tolerance index at metal concentrationM; i?L = rnean relative rate of root elongation(defined as the growth rate in test solution/rate inbackground solution) expressed as mmd~'; i?p, =mean relative rate of adventitious root production(defined as the production rate in test solution/ratein background solution) expressed as roots d^^ Inthe second experiment TI was calculated from theequation:

(2)

where I I = mean length of longest root in testsolution/the length in background solution; Ipj =mean number of adventitious roots per cutting intest solution/mean number in background solution.

Differences between growth responses and metalaccumulation of the various clones were tested usingthe ANOVA General Linearised Model (in Minitab).Significant differences in the text are P < 0-05 unlessstated otherwise.

RESULTS

Effect of short-term Cu pre-treatments on resistance

Tolerance indices were not increased by using eithera low or high short-term Cu pre-treatments (Fig. 1).The response of root length and number variedbetween species, although the general trend was thatroot length was inhibited by Cu treatment but rootnumber was not (Table 3). The only evidence of abeneficial effect of pre-treatment was seen in S.cordata; the TI of plants pre-treated with050 mg Cu 1~ and subsequently exposed to0-25 mg Cu 1~' was over 100°o niore than that ofcuttings without a pre-treatment. However, pre-treatment of the same species with 025 mg Cu 1"resulted in considerably reduced growth. Both thespecific clone under test and the sampling dateexerted significant influences upon root elongationand production data.

Effect of gradual acclimation to Cu, Cd and Zn onresistance

The TI of plants gradually exposed to single heavymetals remained stable over the duration of the testin most cases. This was despite a gradual 10-foldincrease in concentration to maximum concen-trations of l-5mgCur^, l - 5mgCdr ' and25-8 mg Zn 1 ^ (Table 1). A slight decrease in Curesistance was observed in S. viminalis (Fig. 2).Resistance to zinc was stable in S. caprea and5. X calodendron with sinnilar T/s to those observedfor Cu, although it was elevated in S. burjatica andS. viminalis. The values for "Q TI in Figure 2 arehighest in response to Cd, and there appeared to bean upward trend in S. x calodendron, S. burjatica andS. viminalis, whereas 5. caprea remained stable. S.caprea demonstrated a similar level of resistance toall of the metals supplied, there was a distinct orderof resistance in other clones: Cd > Zn > Cu.

Values of TI (eqn (2)) did not fall substantiallywith dual-combination treatments. However, the% TI of 5. caprea in response to all three metalcombinations was considerably higher than the otherwillow species (Fig. 3). The TI of S. x calodendronin response to Cu-i-Zn and Cd-t-Zn fell by c. 50%between 14 d and 128 d (from an initial level of150% to a final level of 100%), despite a 10-foldincrease in metal concentration. TI values forS. X calodendron to Cu -H Cd remained stablethroughout the experiment. The TI values for S.burjatica stabilized after 28 d of treatment, withvalues at 128 d remaining over 50%

Acclimation of Salix to metal stress

200

307

200

100 -

0-25 050 025 0-50

200

X

a

1 100

Salix caprea cv. Sidetands200

0 025 050

Cu pre-treatment (mg h )

100 -

0 0-25 0-50

Cu pre-treatment (mg I-"")

200Salix cinerea ssp. oleifolia

0 0-25 050

Cu pre-treatment (mg I"'')

Figure 1. Tolerance indices (1) of fi\'epre-treated Salix c\onts in response to subsequent growth in backgroundnutrient solution (O). 0-25 mg Cu I"'(0) or O'SO mg Cu 1"' (Q) for 28 d. (Means of zero-adjusted data; n = 72.)

77-5%, T/it,,,^j.^, = 60^0 and r/jed+zm = 63-5 "o). Asteady decrease in TI occurred in S. viminalis, forwhich the lowest values were in response to Cu + Zn(Fig. 3).

Metal uptake

In expt 1 (short-term pre-treatment) Cu accumu-lation occurred primarily in roots, with slightlyelevated concentrations in the aerial tissues (Fig. 4).The copper concentration of dried tissues of allclones showed the same trend; leaves and wood

accumulated Cu in the range of c. 25-100//g Cu g" d. wt. The only exception to this wasS. fragilis which was found to contain > 300 figCu g" d. wt when exposed to a high pre-treatment and a low subsequent treatment(0-50 mg Cu 1" followed by 025 mg Cu I"). Rootconcentrations were much higher; in the range300-1200/Ag Cu g"'d. wt; once again the highestconcentrations were detected in S. fragilis plantsgiven a high concentration pre-treatment. New stemtissue contained 25-150//g Cu g" hut the pattern ofCu accutnulation was more complex. The magnitude

T. Punshon and N. M. Dickinson

Table 3. F values from GLM analysis of root length and number data frompre-treated clones re-exposed to copper in nutrient solution (excluding Salixcinerea spp. oleifolia owing to the low viability of the test population)

Source df

Cu pre-treatment (2)Subsequent treatment (2)Willov\- clone (3)Sampling date (1)Duplicate hlock (1)Interactions:Pre-treatment x treatment (4)Pre-treatment x willow clone (6)Treatment x willow clone (6)

Rootelongation

9-46**18-89**25-39**

115-80**170 n.s.

1-18 n.s.1-15 n.s.4-90**

Rootproduction

0-39 n.s.2-55 n.s.

60'10**34-82**3-21 n.s.

1-50 n.s.075 n.s.116 n.s.

**P< 0-001; *P<0-05.n.s., no significant differences.

300

200 -

300

14 28 42 56 85 99 114 128 14 28

Measuring date

56 85 99 114 128

Figure 2. Tolerance indices (2) of four Salix clones exposed to gradually increasing concentrations of copper(D). cadmium (A) and zinc (O) over 128 d. The arrows indicate the dates on which metal concentrations wereincreased. (Means of zero-adjusted data; w = 54.)

of Cu accumulation in all plants was in the order:roots > wood > new stem > leaves. There appearedto be no relationship between Cu uptake and theduration of exposure; Cu concentrations in plantsanalysed after the 28 d pre-treatment w ere oftenhigher tban in plants subsequently re-exposed to Cu,for example in S. caprea cv. Sidelands (Fig. 4).

For expt 2 (acclimation to elevated Cu, Cd andZn) results are presented for both the single anddual-combination metal treatment. The uptake ofCu was again largely confined to the roots with300-400/ig Cu g'^ d. wt detected in all clones (Fig.5). Uptake of Cu into the aerial tissues was much

lower, with a maximum of 100/ igCug" ' d. wt inthe woody component and no more than50//g Cu g~ d. wt in stems and leaves. There wereno significant interclonal differences in Cu accumu-lation, but the extent of metal uptake differedsignificantly between tbe different plant tissues(Table 4). Cadmium concentrations did not exceed100 /ig Cd g ' ' in any of tbe tissues. Salix caprea andS. X calodendron accumulated less Cd in tbe woodycomponent of the cutting, with greater concen-trations in the roots, stem and leaves. In S. burjaticaand 5. viminalis Cd was accumulated in the orderleaves > stem > wood > roots. Tbese clonal differ-


300 300

200 -

100 -

300

200 -

100 -•

14 28 42 56 85 99 114 128 14 28 42 56 85 99 114 128

Measuring date

Figure 3, Tolerance indices of four Salix clones exposed to gradLial]\' increasing concentrations of dual-combination of heavy-metal treatments, consisting of Cu-I-Cd (D). Cu + Zn (A) and C d + ± Z n (O). Thearrows indicate the dates on which metal concentrations were increased. (Means of zero-adjusted data: n = 54.)

Ia.oo

1000

800 -

600

400 -

200

B L C D _ J X C D - H ^ ' ? ? T ! ^,c D D a c D - ^ - ' - J i ^ i

L H C D ^ x m - r i xm DD ni IJ —I I j

CD -I I

X n: ITreatments

Figure 4. Copper concentrations (/tg g"' d. wt) in tissues of Salix caprea cv. Sidelandstreatment (LHS of dotted line) and a further 35-day treatment (RHS dotted line). B,solution; L, 0-25mgCul- ' ; H. OSOmgCul" ' .

following a 28-d pre-background nutrient


Control Control Zn

r

C

o

o

700

600

500

400

300

200

100

0

700

600

Copper Cadmium Zinc

11Control Cu Control Cd Control

Copper Cadmium Zinc

Control Cu Control Cd

TreatmentControl

Zn

Cu Control Cd Control

Figure 5. Concentrations of Cu, Cd and Zn (fig g" d. wt) in roots ( • ) , wood (B), stem (US) and leaves (0) offour Salix clones after 12S d exposure to background nutrient solution (control) and cumulative single metaltreatments. (Means and SE, n = 3.)

ences are supported by statistical analyses (Table5). Interclonal differences in zinc accumulation weremuch greater than either Cu or Cd. S. x calodendroncontained the highest leaf-bound Zn concentrationof > 600//g g-* d. wt (Table 6).

The most obvious effect of supplying combi-nations of metals on tissue concentration in expt 2

was increased Cu in the roots of all clones (Figure 6).Cadmium uptake was restricted in comparison tothat of Cu, but although the levels were still abovecritical concentrations (Table 6) they did not causeany visible signs of phytotoxicity. Concentrations ofCd in the root tissues of all clones were elevatedwhen Cd was supplied with Cu, but not when it was


Table 4. F values from GLM analysis of metal concentration data from thesingle metal treatment acclimation plant

Test value

Source (df) Copper

592-17**603-76**

l ] 6 n . s .280-46**

0-78 n.s.1-93 n.s.

Cadmium

1532-02**21-21**6-52*2-28**7-24**649**

Zinc

552-32**15-32**18'35**33-13**23-96**6-07**

Metal treatment (1)Tissue compartment (3)Willow clone (3)Treatment x tissue compartment (3)Treatment x willow clone (3)Tissue compartment x clone (9)

* » P < 0-001, * P < 0 - 0 5 .n.s., no significant differences.

Table 5. F values from GLM analysis of metal concentration data from themixed metal treatment acclimation plant

Test value

Source (df) Copper

85-79**587'52**

2.74*82-73**

1-61 n.s.1-88 n.s.

Cadmium

287-88**114'50**

2-92*40-33**

5-87**7-47**

Zinc

42-55**9'32**

13-97**4-42**3-97*5-62**

Metal treatment (1)Tissue compartment (3)Willow clone (3)Treatment X tissue compartment (3)Treatment x willow clone (3)Tissue compartment x clone (9)

**P< 0-001, *P<0-05.n.s., no significant differences.

Table 6. Concentrations of heavy metals tn plants*

Element

CopperCadmiumZinc

Normal rangein plants

5-2001-2-41-^00

Critical

a

20-1005-30

100-^00

concentrations in plants-f

b

5-642-18

100-900

*Data from Alloway (1995).+ Critical concentration in plants is the level above which toxicity efFects are

likely; a, data from Kabata-Pendias & Pendias (1992); b. values likely to causea 10* ,, depression in yield, data from McNichol & Beckett (1985).

supplied with Zn. A significant increase in the Znconcentration within leaf tissues of S. caprea wasobserved when Zn was supplied with Cu, but thiswas not seen in the other clones.

DISCUSSION

The results of the present study suggest thatacclimation to toxic metals in willows can be achievedby acclimation to gradually increasing concen-trations of heavy metals rather than by a short-termpre-treatment. The first experiment showed thatcuttings established in unamended nutrient solutionwere more able to continue growing in the presenceof elevated metals than were pre-treated plants.

Small increases in resistance were observed in someclones, although these mainly resulted from in-creased root production, and no overall beneficialeffect was observed. Metal toxicity at the two pre-treatment doses was more significant than anyinduction effect. Anomalous results such as theincrease in TI of S. cordata by > 100 "0 following ahigh pre-treatmenT and a low treatment did notindicate a convincing trend, as all other pre-treat-ments increased "oT'- by no more than 20°(I (Fig. 1).

Single doses of pre-treatments have been usedwith marginal success for herbaceous species, suchas wheat (Aniol, 1984) and Holcus lanatus L. (Bakeret al., 1986), but the present study suggests that it isnot suitable for woody species. A well documented


Io

cou

800

600 -

400 -

200

Copper Cadmium Zinc

Control Cu + Cd Cu + Zn Control Cu + Cd Cu + Zn Control Cu + Cd Cu + Zn

800Copper Cadmium Zinc


800


800

600 -

.§ 400

CO 2 0 0

Copper Cadmium Zinc


Treatment

Figure 6. Concentrations of Cu, Cd and Zn {/ig g^^ d. wt) in roots ( • ) , wood (fl), stem ( • ) and leaves(0) of four Salix clones after 128 d exposure to background nutrient solution (control) and cumulative dual-combination metal treatments. (Means and SE, W = 3.)

example of metal resistance induction in woodyspecies has been presented by Turner & Dickinson(1993), and is summarized by Dickinson et al.(1996). The work details the impact of 100 yr ofmetal processing at a rod-rolling plant in Prescot,Merseyside, where aerial deposition of Cu, Cd andZn from the chimney stack contaminated surround-

ing vegetation. They found that the trees at this sitehad become acclimated to elevated metal levels andthat the cumulative nature of aerial depositioncontributed to this effect. Long-lived woody speciesmust adjust to changing environmental conditions inorder to survive and it is quite feasible they respondby acclimation, to avoid short-term exposure to toxic


metals which might be damaging and ineffective.The toxicity of heavy metals can differ according towhether exposure is acute or chronic, and for woodyspecies such as willows, chronic exposure appears tobe more effective at eliciting a resistance response.

In this study, cuttings exposed to graduallyincreasing concentrations of metals, either single orin combination, responded by maintaining hightolerance indices while concentrations increased.The hardy species, S. caprea was most amenable toresistance acclimation; growth increased in thepresence of dual-metal treatments, and the toleranceindex continued to rise in response to all treatmentsthroughout the duration of the test (Fig. 3). Copperuptake into aerial tissues was restricted and most Cutaken up was located on or within the roots, inagreement with the findings of other workers(Borgegard & Rydin, 1986; Punshon et al., 1995;Arduini, Godbold & Onnis, 1996). Copper concen-trations in leaves of acclimated plants in expt 2 wasgenerally no greater than 50//g Cu g~ d. wt al-though pre-treated plants in expt 1 contained up to300 /tg Cu g~' d. wt in the leaves despite a shorterexposure time (Figs 4, 5). The Cu concentration ofall tissues measured in the first 28 d of pre-treatmentwere greater in some cases than the subsequentconcentrations measured after a 64d. This trend,coupled with the observation that initial treatmentwith background nutrient solution increased re-sistance, suggests that the establishment of cuttingsis the most sensitive period of development, and onein which metal uptake is the least restricted. Haissig(1986) notes that there is considerable structural anddevelopmental instability of cutting material duringthe initial stages of growth, and the findings of thepresent study support this. The concentration of Cuin the woody component of the cutting increasedwith the duration of the metal concentration,indicating that Cu might have been strongly ad-sorbed to the surface of the cutting.

The resistance of all clones to Cd is also par-ticularly notable in this study and corresponded to alow level of accumulation even when combined withother metals (Fig. 6). Plants sur\ ived toxic Cdconcentrations without visible signs of phytotoxicit>-and had lower tissue concentrations of Cd than ofeither Cu or Zn. Accumulation of Zn was affected bycombination with other metals, and uptake of Zninto tbe lea\es of Salix caprea was doubled whensupplied with Cu, although Cd did not effect Znuptake (Watmough & Dickinson, 1995(7). Furtherstudy is required to clarify the nature of these metalinteractions, as contaminated sites rarely containonly one metal pollutant.

Reasons why cumulative treatment enabled certainclones of Salix to grow in concentrations which werepreviously lethal clearly lie in the pbenotypicplasticity of willows. The rapid acclimation ofwillows to the local environment has been docu-

mented in response to environmental stresses such asshading (Meikle, 1984); under reduced light con-ditions certain species of willow altered their leafmorphology to maximize light capture. Such pheno-typic changes can occur within the lifetime of anindividual clone, and the processes responsible forsuch morphological alterations might conceivablyact upon physiological processes such as mineraluptake. The results strongly suggest that increasedresistance (especially to Cd) is attributable toreduced uptake, and tissue metal concentrationappeared connected with cutting age and metalconcentration. Cumulati\-e treatment might allowplants to become acclimated by exerting a non-lethalselection pressure, and might be particularly suc-cessful with willows because of tbe ease by whichthey adapt to their environment.

The implication of these findings for phyto-remediation is that valuable woody plant species maybe selected on the basis of their innate resistance toheavy metals and nutrient-poor soils. The studysuggests that the metal resistance of species withsufficient genetic variability can be manipulatedthrough acclimation, and that acclimating recla-mation shrubs before planting on contaminated siteswill greatly increase their growth and survival. Thefinding has emphasized that full account must alsobe taken of the phenotypic variation (acclimationresponse) that exists within woody plants. Data onmetal uptake show that willows can take up fromnutrient solution levels of metals w-hich are abovenormal critical concentrations, yet continue to sur-\ i \e and grow. It is suggested tbat amelioration ofthe plant-available beavy-metal load (especially Cd)of contaminated soils might be decreased by phyto-remediation shrubs without necessarily losing pro-ductivity.

ACKNOWLEDGEMENTS

The authors would like to thank Liverpool John MooresUniversity for funding this work, and Dr Hugh McAllisterat Ness Botanic Gardens for the supply of viillow materialand helpful taxonomic advice.

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