Plant and Soil231: 81–90, 2001.© 2001Kluwer Academic Publishers. Printed in the Netherlands.

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Effects of nickel onFrankia and its symbiosis withAlnus glutinosa(L.)Gaertn

C. T. Wheeler1,3, L. T. Hughes1, J. Oldroyd1 & I. D. Pulford2

1Plant Science Group, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, GlasgowG12 8QQ, UK.2Environmental, Agricultural and Food Chemistry, Joseph Black Building, University of Glasgow,Glasgow G12 8QQ, UK.3Corresponding author∗

Received 7 August 2000. Accepted in revised form 24 December 2000

Key words: Alnus glutinosa, Frankia, histidine, nickel, nitrogen fixation, nodulation

Abstract

The tolerance of nickel byFrankia in culture and in symbiosis withAlnuswas determined. Yield of threeFrankiastrains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysedcasein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixednitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptakehydrogenases is presumed to be the cause of enhanced nitrogenase activity.

Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did notaffect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seed-lings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggestingthat effects of nickel on host plant processes rather thanFrankiaare the primary cause of inhibition of nodulation.This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 dayafter the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. Nonitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrationsequal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel tothe shoot.

Introduction

Nickel is an essential trace element for plants andfor many bacteria. In higher plants, it is a compon-ent of urease (Marschner, 1998), while in bacteriait is present in several enzymes in addition, such ashydrogenases, some superoxide dismutases, carbonmonoxide dehydrogenase and methyl-coenzyme M re-ductase (Hausinger, 1994; Ragsdale, 1998). Nickelis of ubiquitous occurrence in soils and is presentin particularly high concentrations in serpentine soils(Proctor and Baker, 1994). Application to soil of

∗ FAX NO: +44-141-330-4447.E-mail: C.Wheeler@bio.gla.ac.uk

sewage sludge or fly ash and emissions from smelterscan all give rise to phytotoxic concentrations of nickel(McGrath and Smith, 1990).

The major requirement for nickel in nitrogen-fixing prokaryotes is for synthesis of the uptake hy-drogenases which catalyse the oxidation of hydro-gen liberated by nitrogenase during the reduction ofdinitrogen (Klucas et al., 1983). Nickel supply inrhizobia is mediated by the accessory protein nick-elin, which has a dual role of mobilisation of nickelinto hydrogenase and of nickel storage (Olson andMaier, 2000). Increasing the supply of nickel to atbreshhold stimulates bacterial growth. For example,growth in culture of bradyrhizobia nodulating cowpeaand pigeon pea was increased by 1µg l−1 nickel as

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was nodulation, nitrogenase activity and growth ofthe host plants. Concentrations greater than 10µgl−1 inhibited these processes (Singh and Rao, 1997).Increasing the nickel content of solution cultures inwhich peas were grown elevated hydrogenase activitysubstantially (Brito et al., 1994).

Tolerance and adaptation of microbial strains toheavy metal contaminated environments is well doc-umented (Chaudri et al., l992a,b; DiazRavina andBaath, 1996; Nies, 1999). For rhizobia, effect-ive strains have been isolated from a range of soilscontaminated with heavy metals resulting from longterm applications of sewage sludge (Smith and Giller,1992). It is notable that all isolates and strains ofRhizobiumtested by Chaudri et al. (1993) toleratedhigher concentrations of heavy metals in bufferedsystems than in the soil solutions from which theyoriginated. Differences in toxicity thresholds can beascribed to variations in the tolerance of different bac-terial and plant species and to effects of buffers and pHon metal ion activity (Chaudri et al., 1993).

Because of their nitrogen fixing habit, actinorhizalplants, in particular alders, are used widely for re-clamation of derelict and heavy metal contaminatedland (Wheeler and Miller, 1990). However, inform-ation concerning the tolerance of host plant speciesand their symbionts to specific heavy metals, in par-ticular nickel, is sparse although inhibition of growth,nodulation and nitrogenase activity ofAlnus rubraandA. glutinosaby cadmium has been reported (Hensleyand Carpenter, 1987; Wickliff et al., 1980) The oc-currence of hydrogenases in both free living and sym-biotic Frankia has been described (Sellstedt, 1989;Sellstedt and Lindblad, 1990) and an essential re-quirement of nickel for hydrogenase activity noted forFrankia in culture (Sellstedt and Smith, 1990). Theseauthors found that increasing the concentration ofnickel in the growth medium to 1.0µM increased hy-drogenase activity substantially with indications thatgreater activity would result from further increases innickel concentrations.

In the present study, the tolerance of nickel byFrankia and the effects of nickel on nodulation,growth and nitrogen fixation inAlnus glutinosahavebeen determined in order to identify the symbiont mostsusceptible to inhibition by this heavy metal.

Materials and methods

Culture, harvesting and nickel tolerance ofFrankia

The three strains ofFrankia used were Ag1.1.8 Bu(UGL 010708), fromAlnus glutinosa(Hooker andWheeler, 1987); Ac4 (UFI 01010104), fromAl-nus cordata(Margheri et al., 1983) and E38 (UFI132738), fromElaeagnus angustifolia(Lumini andBosco, 1999).Frankia were cultured for approxim-ately 3 months at 25◦C, in darkness in 250 mlbottles with 150 ml BuCT medium (Malcolm etal., 1985) containing l−1 distilled water, pH 6.8:K2HPO4, 1.0 g; NaH2PO4.2H2O, 0.67 g; CaCl2.H2O,0.1 g; MgSO4.7H2O, 0.2 g; Na propionate, 0.5 g;Tween-80, 0.5 g; casamino acids, 1.2 g; FeNaEDTA,10 mg; H3BO3, 1.5 mg; ZnSO4.7H2O, 0.6 mg;MnSO4.7H2O, 0.8 mg; (NH4)6Mo7O24.4H2O, 0.2mg; CuSO4.7H2O, 0.1 mg; CoSO4.7H2O, 0.1 mg.

After 3-months growth, mycelium from the re-quired number of culture bottles was mixed, homogen-ised lightly and 10 ml aliquots transferred into 150 mlconical flasks containing 40 ml of either low mg BuCT(33 mg l−1 MgSO4.7H2O) or of low Mg BuT medium,which was of similar composition but without casa-mino acids. Magnesium concentrations were loweredto reduce ameliorating effects of this element on nickeltoxicity (Proctor and McGowan, 1976). Twenty flaskswere prepared for each combination of bacterial strainand medium. The bacteria were left in these flasks for1–10 weeks to adjust to the low Mg and to allow theslower-growing strains time to gain biomass. After thisperiod, NiSO4.6H2O was added to five replicate flasksper treatment giving concentrations of 0, 0.25, 0.75and 2.25 mM.

At harvest, the contents of the flasks were filteredthrough Whatman No. 1 filter paper which was washedwith a total of 12.5 ml of distilled H2O into vials thatwere stored temporarily at 2◦C. The mycelia werehomogenised thoroughly prior to assay of the turbidityof the suspension in 1 cm diameter glass cells at 500nm with cell free medium as reference.

Culture of plants

Seed ofAlnus glutinosafrom the area of the Allanderriver in Milngavie, Glasgow was germinated in traysof perlite (Silvaperl, UK), which had been soaked in asolution of 1 g l−1 Crones–N mixture and 0.25 ml l−1

Hoagland’s A–Z micronutrients (Wheeler and Miller,1990). Seedlings were grown in environment cham-bers with a 16 h photoperiod, irradiance 220 mol m−2

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s−1 from a mix of white and warm white fluorescenttubes (Omega, UK), and temperature of l8–20◦C darkand 21–23◦C light. When the seedlings began to dis-play chlorosis, they were watered once with 100 mlper tray of a solution of 0.5 ml l−1 Levington’s ‘Li-quinure’ (Fisons Ltd., UK), to provide a source ofcombined N. The manufacturer’s information for thisproduct indicates that it does not contain nickel.

Experimental treatments

Effects of nickel on seedling nodulationA period of 5–6 weeks after sowing, fiveA. glutinosaseedlings were suspended, with their roots suppor-ted by opaque plastic lids, in each of ten porcelainpots containing 2 l culture solution (0.5 g Crone’s–N nutrients, 0.25 ml A–Z Hoagland’s micronutrientsand 0.3 ml ‘Liquinure’ l−1). Seedlings were left for 2weeks to adapt to water culture conditions. The culturesolution was then changed to remove ‘Liquinure’ andNiSO4.6H2O solution added to duplicate pots to givefinal concentrations of 0, 0.025, 0.075, 0.225 or 0.5mM nickel. At this time, nodules (0.9 g fresh weight)were collected from 1 year old water culture plantswhich had been inoculated previously withFrankiaUGL 010708. The nodules were ground in a mortarand pestle with a small amount of distilled H2O, thepaste made to 100 ml with water and each seedlingroot inoculated with 2 ml suspension. The appear-ance of the seedlings was observed daily to monitorthe reaction of the plants to nickel. Culture solutioncontaining Crone’s nutrient was pH 6.2 initially andremained little changed during seedling growth. How-ever, it decreased to pH 5.2–5.5 over a 7 day periodwhen used to grow larger plants.

Four weeks after treatment with Ni and crushednodule suspension, the nitrogenase activity of the in-tact seedlings was determined by acetylene reductionassay (Hooker and Wheeler, 1987) following trans-fer to 30 ml glass bottles, closed with a ‘Subaseal’.Samples were taken for assay immediately and again1 and 2 h after addition of acetylene, for assay by gaschromatography. On completion of the assays, nodulenumber and size was assessed and the total chlorophyllcontent of 80% acetone extracts of the shoots of eachseedling determined by absorption spectrophotometryat 652 nm (Harborne, 1973). The shoot residues weredried in air to remove acetone, then dried to constantweight in an oven at 80◦C and weighed.

Effects of nickel on plants with established symbiosesExperiment 1 was designed to determine the effects ofnickel on leaf necrosis, nitrogenase activity and accu-mulation of nickel over a 5 day period. Plants wereinoculated as seedlings withFrankia UGL 010708and were grown in Crone’s–N solution with Hoag-land’s A–Z micronutrients for 10 months. They wereof approximately the same height with an average leafnumber of 22 when used. Prior to use, plants weretransferred into 500 ml water (controls) or 500 ml of0.59 mM NiSO4.6H2O in 1.3 l sealed jars fitted withports for injection of acetylene (to a final concentrationof 6.5%) and for gas sampling. Acetylene reductionassays and leaf necrosis assessments were carried outeach day for 5 days, when the plants were harvested.The roots were washed thoroughly to remove externalnickel and each plant was then divided into leaves,stem with buds, roots and nodules, before storage at−20 ◦C prior to analysis of nickel content.

A second experiment withA. glutinosaof similarage was carried out to determine daily changes innickel accumulation in relation to nitrogenase activ-ity and leaf necrosis. Three plants were harvestedfor nickel analysis immediately and nine plants trans-ferred to 2 l porcelain jars containing 850 ml 0.59mM NiSO4. Water levels in the porcelain pots wererestored daily. On the following 3 days, leaf necrosisof three treated plants was determined and the plantstransferred to 1.3 l jars for assay of acetylene redu-cing activity, as above. Plants were then harvested, asabove, for assay of nickel content.

Effects of histidine on nickel toxicityThe plant used for these experiments were nodu-lated by UGL 010708 and were 15- or 19-weeks-old.They were grown in water culture with Crone’s–Nnutrients+Hoagland’s A–Z micronutrients, as above.NiSO4.6H2O was added to pots (4 plants per treat-ment) to give concentrations of 1 mM and 2 mMnickel. Nickel, at the same concentrations, +3 mMhistidine, was added to two further sets of plants.Leaf necrosis of each plant was determined daily for7 days after treatment. The experiment was repeatedwith 19-week-old plants but treatments were with 1mM nickel+1 mM histidine and 2 mM nickel+1 mMhistidine.

Further sets of three plants were treated with com-binations of 1 mM NiSO4.6H2O, 3 mM histidineand 0.15 mM L-buthionine-[S,R]-sulfoximine, whichis proposed to be an inhibitor of phytochelatin pro-duction in cadmium treated birch (Gussarsson et al.,

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1996). Plants were harvested after 7 days, divided intothe major parts and analysed for their nickel content asdescribed below.

Assessment of leaf necrosis

Each leaf on a plant was assessed for necrosis or wilt-ing as below. The numbers allocated to each leaf weresummated and divided by the number of leaves perplant to give a whole plant ‘necrotic index’:<5% nec-rosis=0; leaf drooping, 5–20% necrosis=1; leaf droop-ing, 21–40% necrosis=2; leaf drooping and speckled,41–60% necrosis=3; leaf drooping and edges brown,61–80% necrosis=4; 81–95% necrosis=5; leaves com-pletely necrotic, dead=6.

Estimation of nickel content

The harvested plant material was oven dried at 80◦Cto constant weight before grinding to a fine powder ina porcelain mortar and pestle. Approximately 0.25 gof each sample was weighed out, with two replicatesfor each, into tall form 25 ml beakers, and coveredwith a watch glass. A total of 10 ml of 69% nitric acidwere added to each sample and to four empty beakersas analytical blanks. The beakers were covered witha watch glass and left overnight before digesting on ahot plate at 120◦C. On completion of digestion, thesamples were cooled and a small amount of deion-ised water added, the digest filtered through WhatmanNo. 50 hardened filter paper and filtrates and washingsmade to volume with deionised water prior to ana-lysis by atomic absorption spectroscopy (Perkin Elmer1100B).

Results

Effect of nickel onFrankiain culture

There was no significant effect on yield (turbidomet-ric assay) of addition of NiSO4 to cultures of threeFrankia strains (isolated from and effective onAl-nus glutinosa, UGL 010708; Alnus cordata, UFI01010104; andElaeagnus angustifolia, UFI 132738)when supplied with combined nitrogen. However,yield of UGL 010708 was increased almost four foldand that of UFI 01010104 doubled as the nickel con-centration of combined-nitrogen-free medium was in-creased from 0 to 2.25 mM (Figure 1). Yield of thehighly pigmented strain UFI 132738 increased sig-nificantly in both combined nitrogen and combined-nitrogen-free media containing 2.25 mM nickel. The

Figure 1. Effect of nickel on the growth ofFrankia strains UGL010708 (UGL0l), UFI 010708 (UFI01) and UFI 13270238 (UFI13).Strains were cultured in low magnesium propionate media with(+N) or without (0N) a combined nitrogen source (Casamino acids).Cultures were grown for 8 weeks with 0.0, 0.25 (�), 0.75 (�)and 2.25 (L) mM NiSO4.6H2O before harvest and spectrophoto-metric assay of the turbidity of the homogenised mycelium. Dataare means±standard error bars for five replicate cultures and areexpressed as % of control cultures (0 nickel) for which turbid-ity=100%.

basal growth medium used (Malcolm et al., 1985) wasnot supplemented with nickel, although the possibil-ity that trace amounts may have been present in thechemicals used cannot be eliminated.

Effect of nickel on growth and nodulation ofAlnusglutinosa

All control seedlings, which received 0.12µM nickelas a micro-nutrient in the culture solution, were nod-ulated effectively byFrankia UGL 010708. Visualobservation suggested that all nickel treatments re-duced root formation while dry weight measurementsof the residues remaining after acetone extractionof the shoots also indicated significant reductions ingrowth compared with controls, ranging from 35%in 0.025 mM nickel treatments to 85% in seedlingstreated with 0.5 mM nickel (Table 1).

The number of leaves per seedling at harvest de-clined with increasing nickel concentration so thatseedlings treated with 0.225 or 0.5 mM nickel formedno more than one new leaf during the growth period,whereas approximately five new leaves were formedon control seedlings. Differences in leaf formation andin leaf necrosis are reflected in seedling chlorophyllcontent, which in seedlings receiving 0.225 or 0.5 mMnickel, was approximately one sixth of control plants(Table 1).

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Table 1. Nodulation and growth of nickel treated seedlings. Either 9 or 10 eight-week-old seedlings, as indicated, were treatedwith nickel for 4 weeks prior to assessment. Data are means (S.E.) for the seedlings in each treatment. Shoot residues are thedry weights remaining after acetone extraction

Treatment No of Shoot residue Chlorophyll C2H4 (nmol) Nodule no. seedling−1

NiSO4 (mM) seedlings dry wt (g) content (mg seedling−1 h−1 <1 mm 1–5 mm >5 mm

nodulated/ seedling−1)

total

0 9/9 0.17 (0.012) 0.58 (0.13) 177 (59.1) 29.0 (5.8) 4.4 (1.1) 0.4 (0.43)

0.025 10/10 0.11 (0.009) 0.31 (0.05) 408 (111.0) 11.5 (3.4) 6.7 (2.07) 1.0 (0.28)

0.075 9/9 0.071 (0.005) 0.22 (0.04) 40.0 (26.4) 12.4 (1.57) 0.2 (1.2) 0.1 (0.1)

0.225 8/10 0.035 (0.004) 0.07 (0.016) 0 2.0 (0.82) 0.7 (0.22) 0

0.5 2/9 0.025 (0.004) 0.10 (0.04) 0 2.8 (1.95) 0 0

The numbers of larger nodules on seedlings receiv-ing 0.025 mM nickel 4 weeks after inoculation werenot significantly different from those on control plantsbut the number of small nodules (<1 mm diameter)was reduced by 60%. The nitrogenase activity of theseseedlings was more than double that of the controls,however (Table 1). With 0.075 mM nickel, all seed-lings were nodulated but most nodules were of smallsize (<1 mm diameter) and with greatly reduced ni-trogenase activity, which was 25% that of controls.With 0.225 mM nickel, 80% of the seedlings werenodulated but the nodules were small and there wasinsignificant nitrogenase activity. Leaves of seedlingsreceiving 0.5 mM nickel were completely necrotic 1week before harvest but 20% of these seedlings hadformed a few small nodules.

Effect of nickel on 10-month-old nodulatedA.glutinosa

Observation of leaf necrosis and concomitant estim-ates of plant nitrogenase activity showed that leavesof plants became progressively necrotic over a 4 dayperiod following transfer to culture solution contain-ing 0.59 mM nickel so that leaves were mostly brownand dead by Day 4 (Figure 2). Nitrogenase activ-ity of both control and nickel treated plants fell byone third 24 h after the first assay, probably due toa combination of the effects of transfer of plants towater (undertaken to minimise complexing betweensalts in Crone’s medium and nickel) and to handling(Wheeler et al., 1978). However, while nitrogenaseactivity of controls remained constant over the sub-sequent 3 days there was no detectable nitrogenaseactivity in the nickel treated plants at Day 2. By this

Figure 2. Effect of nickel on leaf necrosis and nitrogenase activ-ity (acetylene reduction) of 10-month-old alders in symbiosis withFrankia UGL 010708 (Experiment 1). Data are means±standarderror bars for 2 control (0 nickel,�) and 3 nickel treated (0.59mM NiSO4.6H2O, �) plants. The derivation of necrosis units isdescribed in the ‘Materials and methods’.

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Table 2. Changes with time in the distribution of nickel inA. glutinosatreated with 0.59 mM NiSO4.6H2O. In the first experiment,the plants utilised for daily assays of necrosis and nitrogenase activity (Figure 2) were harvested on Day 4. The parts from two(controls) or three (nickel treated) 10-month-old plants were harvested at each time, dried, combined, ground and mixed beforeanalysis of duplicate samples so that (S.E.) of the means represent analytical variability. In experiment 2, the parts from three10-month-old plants were harvested at each time and treated similarly prior to analysis for nickel. Necrosis units are defined in‘Materials and methods’

Time after Leaf Nitrogenase Nickel content of tissues (µg g−1 dry wt)

treatment necrosis, activity, C2H4 Leaves Stem Roots Nodules

with NiSO4 units µmol.plant−1h−1

Experiment 1

Control 6.7 (0.51) 5.3 (0.84) 26.6 (3.53) 24.3 (1.10)

Day 4 5.0 (1.01) 6.2 (1.33) 467 (15.7) 431 (1.47)

Experiment 2

Day 0 1.8 (0.336) Not determined 10.3 (1.42) 5.9 (0.25) 38.1 (5.01) 31.5 (0.64)

Day 1 2.2 (0.25) 1.15 (0.97) 8.2 (0.25) 3.6 (0.7) 384 (8.01) 300 (10)

Day 2 3.5 (0.48) 0.13 (0.10) 5.1 (0.06) 6.3 (5.37) 406 (40.2) 109 (2.7)

Day 3 5 (0) 0 4.07 (0.03) 2.6 (0.70) 418 (8.03) 238 (4.45)

time, approximately 45% of leaves of treated plantswere necrotic.

Distribution of nickel in treated plants

Analysis of plants from the experiment above, har-vested at the end of 4 days treatment with 0.59 mMnickel, showed that the nickel contents of leaves andstems of treated plants were not significantly different(Experiment 1 in Table 2). The concentration of nickelin roots and nodules of control plants was four to fivetimes higher than that of leaves or stems, but in treatedplants most nickel was retained in the roots and nod-ules which both showed concentrations that were 17times that of the shoot.

Analysis of plants from Experiment 2, that wereharvested on a daily basis following nickel treatment,showed that the roots and nodules accumulated highlevels of nickel by Day 1, when two of the three plantsstill retained significant nitrogenase activity (Table 2).By Day 2, nickel levels in the roots had not changedsignificantly from Day 1 but there was insignificantnitrogenase activity by Day 3 (Table 2). An apparentdecrease in the nickel content of nodules by Day 2 wasreversed by Day 3.

Effects of histidine on nickel toxicity

As shown in Figure 2 and in Table 3, there is a stronglinear correlation between necrosis and time in daysafter treatment of solution culture alders with nickel.

Table 3. Effects of simultaneous application of histidine onnickel toxicity. Leaf necrosis of 15-week-old (Experiment 1)and 19 week old alders (Experiment 2), grown in−N culturesolution, was assessed over a 7 day period following treatment.Necrosis units are defined in ‘Materials and methods’. Data aremeans of four plants for each treatment, assessed at mid-day.Linear regression analysis of the relationship between days aftertreatment and necrosis was carried out using Microsoft Excel

Treatment Rate of leaf Correlation

necrosis (necrosis coefficient

units d−1)

Experiment 1

Control 0.07 0.96

1 mM nickel 0.72 0.99

2 mM nickel 0.76 0.99

1 mM nickel+3 mM his 0.04 0.79

2 mM nickel+3 mM his 0.12 0.97

3 mM his 0.19 0.97

Experiment 2

1 mM nickel+1 mM his 0.10 0.98

2 mM nickel+1 mM his 1.04 0.93

Plants supplied with 1 mM and 2 mM nickel werecompletely necrotic 7 days after commencement oftreatment. However, only slight necrosis, which wasnot significantly different from controls, was observedin plants supplied concomitantly with nickel and 3mM histidine (Table 3). In a further experiment, plants

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Table 4. Effect of added histidine on nickel accumulation. Water culture alders were treated for 7 days with the combinationsshown of 1 mM NiSO4.6H2O, 3 mM histidine (his) and 0.15 mM L-buthionine-[S,R]-sulfoximine (BSO). Plants in Experiment1 were 30 weeks, in Experiment 2, 26 weeks and in Experiment 3, 18-week-old. Data are means of analyses of 3 replicatesamples from the combined, dried, ground tissue from 3 plants in each treatment. Analytical standard errors (n=3) are shownin parentheses after means

Nickel Nickel content,µg g−1 dry wt tissue

Expt. 1 Expt. 2 Expt. 3

Nickel Nickel+his Nickel Nickel+his Nickel+BSO Nickel+BSO+his

Leaf 28 (1.9) 135 (2.65) 32.3 (0.33) 41.3 (0.67) 20 (0.58) 49 (4.2)

Stem 276 (27.3) 75 (2.36) 173 (16.3) 133 (3.53) 128 (2.02) 287 (12.1)

Roots+nodules 4122 (183) 3341 (98.5) 2912 (80.5) 6508 (332) 2668 (40.4) 3804 (49)

were treated simultaneously with 1 mM histidine and0.5 mM, 1 mM and 2 mM nickel. Over a 21-dayperiod, necrosis significantly greater than the controlplants was observed only in plants receiving nickel inmolar concentrations twice that of histidine (Table 3).

The amount of nickel accumulated over a 7-dayperiod by plants treated with 1 mM nickel, 1 mMnickel+3 mM histidine and 1 mM nickel+3 mM his-tidine+BSO are shown in Table 4. BSO alone hadno significant effect on necrosis and there was noobvious effect of this compound on nickel accumula-tion. Comparison with the data of Table 2 shows thatthe concentrations of nickel in the plant parts weremuch higher overall in these experiments. However,the plants used in the experiments with histidine wereyounger, the concentration of nickel supplied in thesolution culture was double and exposure to nickel 4days longer than that experienced by plants in the timecourse experiment. Roots+nodules again accumulatedmost nickel, with between 93 and 97% of the totalplant content retained in the root system. Stems ac-cumulated between 2 and 7% compared with between0.6 and 3.8% of the total in leaves. There was no con-sistent effect of histidine on the distribution of nickelbetween plant parts.

Discussion

The absence of an inhibitory effect of 2.25 mM nickelon yield of three strains ofFrankia dependent onamino acids or nitrogen fixation as nitrogen source(Figure 1), indicates thatFrankia in culture is rel-atively tolerant of nickel, although the formation ofcomplexes with propionate or other components of

the culture medium may have reduced the availabilityof nickel. The increased yield of theFrankia strainswith added nickel when reliant on nitrogen fixationfor growth is presumably due to increased synthesis ofuptake hydrogenases (Maier et al., 1990; Sellstedt andSmith, 1990) which conserve energy by re-oxidisinghydrogen liberated by nitrogenase. For two strains, theabsence of a significant effect of increased nickel onyield when amino acids (hydrolysed casein) were thesource of nitrogen nutrition supports this interpreta-tion. Yield of the third strain,Frankia UFI 132738increased significantly with 2.25 mM nickel so thata positive effect of nickel on other bacterial enzymescannot be discounted. For example, nickel-containingsuperoxide dismutases, the synthesis of which mightbe stimulated by Ni, have been demonstrated inStrep-tomyces(Youn et al., 1996) but it is not knownwhether theFrankia enzymes contain nickel (Steeleand Stowers, 1986). Practically, it is clear that increas-ing the nickel content of many media currently usedfor culture of Frankia (Lechevalier and Lechevalier,1990) is likely to improve growth substantially, par-ticularly when cultures are reliant on nitrogen fixationfor growth.

Although it is not possible from ‘in vitro’ cul-ture experiments to predict accurately the toleranceof nickel by Frankia in soils, nevertheless the dataof Figure 1 suggest that the micro-organism is toler-ant of the upper range of nickel concentrations thatmay occur in contaminated and some natural soilse.g. Nriagu and Pacyna (1998) reported that sewagesludge and coal fly ash may release 25–110 and 15–75 µg nickel g−1 soil, respectively. Nickel occurs inserpentine soils at concentrations up to 0.5–10 mg g−1

soil (Proctor and Baker, 1994). It is notable that high

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diversity ofFrankiawas found in association with dif-ferent species ofGymnostomain serpentine areas ofNew Caledonia, with some strains confined to theseregions (Navarro et al., 1999).

By comparison with the tolerance of nickel byFrankia in culture, growth and nodulation ofAlnuswas inhibited by all concentrations of nickel tested,from 0.025 to 0.5 mM (Table 1). Chlorosis and inhibi-tion of root elongation are well documented symptomsof nickel toxicity (Woolhouse, 1983) and both leafnumber and chlorophyll content of alder seedlingsgrown in 0.025 mM nickel were reduced at harvest,by 24% and 47%, respectively. Effects on roots werenot quantified but by the conclusion of the experimentroots of all nickel treated plants appeared shorter anddiscoloured compared with controls. In seedlings re-ceiving 0.025 mM nickel, the number of small nodules(<1 mm diameter) was reduced whereas an appar-ent increase in the number of larger nodules (>1 mmdiameter) was not significantly different from controlseedlings. This suggests that infection and nodulationproceeded as normal immediately after exposure tonickel, so that the development of early formed nod-ules (>1 mm at harvest) was similar to that of thecontrols. However, reduction in the number of smallnodules indicates that nodulation was inhibited lateras inhibitory effects on plant growth developed. Incowpea, 4µM nickel stimulated nodule number, nitro-genase activity and plant dry weight (Singh and Rao,1997), although inhibitory effects were observed athigher concentrations.

The two fold increase, compared to controls, in ni-trogenase activity of seedlings with 0.025 mM nickelmay be due to stimulation of hydrogenase activity, assuggested above as an explanation for the enhancednitrogenase activity and growth ofFrankia receiv-ing elevated levels of nickel in the culture medium(Figure 1). The acetylene reduction assay is not aquantitative measure of nitrogen reduced to ammonia,however (Minchin et al., 1983). Because seedlingsreceiving 0.025 mM nickel showed reduced growth,the nitrogenous products of any increase in reductionof dinitrogen (as opposed to reduction of acetylene)must be stored or excreted rather than utilised to in-crease growth. Practically, it seems that the level ofnickel (0.12µM) supplied to alders in the A–Z traceelement solution utilised in the current experiment issub-optimal for growth of nodulated seedlings and thatthis concentration can be increased substantially tooptimise growth of plants reliant on fixed nitrogen.

Experiments with more mature (10-month-old)plants confirmed that the primary effects of nickeltoxicity are on the host plant rather than the sym-biont. Thus, in 10-month-old alders on which noduleswere well developed, there was a time lag follow-ing exposure to nickel before nitrogenase activity waseliminated (Figure 2). Nitrogenase activity was similarto that of control plants one day after commencementof treatment with 0.59 mM nickel when leaf necrosiswas not significantly different from that of controls.By day 2 following treatment, however, almost halfthe leaves on the plants were necrotic and there wasinsignificant nitrogenase activity. These findings wereconfirmed for a second, similar set of plants (Table2) that also retained levels of nitrogenase activity 24h after treatment that were not significantly differentfrom the controls. At this time, the nickel content ofboth roots and nodules was 10 times that of the controlplants, again demonstrating a high level of tolerance ofnickel byFrankia.

Almost complete necrosis of the leaves of theplants was observed after 3 days. However, the nickelcontent of the leaves and stem did not differ signi-ficantly from that of controls. Most nickel taken upwas retained in the roots and nodules which appar-ently became saturated with nickel by day 1 since thenickel content did not increase further at days 2 and3. The leaves of the nickel treated plants had wiltedand collapsed by day 2 after commencement of nickeltreatment so that the toxic effects observed on theshoot probably result primarily from degeneration ofthe root system.

Histidine is a known chelator of nickel, both as thefree amino acid and in nickel binding proteins (Krameret al., 1996; Wu, 1992). One possible use of aldersis for bioremediation and it was of interest to knowwhether application of histidine would increase nickelaccumulation in a bound form in the shoots or whetherchelation to histidine taken up from culture solutioncould reduce nickel toxicity. Increased histidine con-tent of the species conceivably could be engineeredto enhance nickel tolerance and/or accumulation inshoots that could be removed by coppicing. Provisionof histidine in excess of the toxic concentrations ofnickel supplied to the plants eliminated the develop-ment of leaf necrosis over a 7-day period (Table 3).However, there was no reproducible increase with timein the nickel content of stems or leaves of histidinetreated plants. Most nickel was still retained in theroot system although again the amount accumulateddid not increase reproducibly (Table 4). There was no

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effect of BSO on either necrosis or nickel accumula-tion showing that phytochelatins of the type suggestedto be involved in cadmium tolerance in birch and alder(Gussarsson et al., 1996; Wheeler, unpublished data)are unlikely to be involved in nickel tolerance in alder.

The results obtained suggest thatA. glutinosais moderately sensitive to nickel and does not pos-sess mechanisms which would allow for hyper-accumulation or exclusion of this element. The poten-tial for improving tolerance by selecting, or engineer-ing alders for high histidine content is apparent, but isunlikely to result in increased nickel uptake from thegrowth medium. In contrast to the host plant,Frankiais highly tolerant of nickel. Consequently,Frankiapopulations are likely to survive in highly pollutedsoils and to be available to form effective symbiosesif nickel tolerant actinorhizal host plant species areplanted for reclamation purposes.

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Section editor: C. Atkins