Acclimation of Salix to metal stress

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  • New Phytol. (1997), 137, 303-314

    Acclimation of Salix to metal stress

    BY TRACY PUNSHON* AND NICHOLAS M. DICKINSONSchool of Biological & Earth Sciences, Liverpool John Moores University, Byrom Street,Liverpool L3 3AF, UK{Received 4 November 1996; accepted 28 May 1997)


    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 . 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.


    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 5C; r.h.55"o10"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^.H^O,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-^O 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:


    Short-term Cu pre-treatment


    Expt 2:


    Cu, Cd and Zn acclimation


    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 materialW'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 t^h. 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:


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


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