Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

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<ul><li><p>Plant and Soil 231: 8190, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands. 81</p><p>Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.)Gaertn</p><p>C. T. Wheeler1,3, L. T. Hughes1, J. Oldroyd1 &amp; I. D. Pulford21Plant 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</p><p>Received 7 August 2000. Accepted in revised form 24 December 2000</p><p>Key words: Alnus glutinosa, Frankia, histidine, nickel, nitrogen fixation, nodulation</p><p>Abstract</p><p>The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankiastrains 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.</p><p>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 than Frankia are 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.</p><p>Introduction</p><p>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.</p><p>E-mail: C.Wheeler@bio.gla.ac.uk</p><p>sewage sludge or fly ash and emissions from smelterscan all give rise to phytotoxic concentrations of nickel(McGrath and Smith, 1990).</p><p>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 1 g l1 nickel as</p></li><li><p>82</p><p>was nodulation, nitrogenase activity and growth ofthe host plants. Concentrations greater than 10 gl1 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).</p><p>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 ofRhizobium tested 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).</p><p>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 of Alnus rubra andA. glutinosa by 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 1.0 M increased hy-drogenase activity substantially with indications thatgreater activity would result from further increases innickel concentrations.</p><p>In the present study, the tolerance of nickel byFrankia and the effects of nickel on nodulation,growth and nitrogen fixation in Alnus glutinosa havebeen determined in order to identify the symbiont mostsusceptible to inhibition by this heavy metal.</p><p>Materials and methods</p><p>Culture, harvesting and nickel tolerance of FrankiaThe three strains of Frankia used were Ag1.1.8 Bu(UGL 010708), from Alnus glutinosa (Hooker andWheeler, 1987); Ac4 (UFI 01010104), from Al-nus cordata (Margheri et al., 1983) and E38 (UFI132738), from Elaeagnus 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 l1 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.</p><p>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 l1 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 for110 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.</p><p>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.</p><p>Culture of plantsSeed of Alnus glutinosa from the area of the Allanderriver in Milngavie, Glasgow was germinated in traysof perlite (Silvaperl, UK), which had been soaked in asolution of 1 g l1 CronesN mixture and 0.25 ml l1Hoaglands AZ micronutrients (Wheeler and Miller,1990). Seedlings were grown in environment cham-bers with a 16 h photoperiod, irradiance 220 mol m2</p></li><li><p>83</p><p>s1 from a mix of white and warm white fluorescenttubes (Omega, UK), and temperature of l820 C darkand 2123 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 l1 Levingtons Li-quinure (Fisons Ltd., UK), to provide a source ofcombined N. The manufacturers information for thisproduct indicates that it does not contain nickel.</p><p>Experimental treatments</p><p>Effects of nickel on seedling nodulationA period of 56 weeks after sowing, five A. 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 CronesN nutrients, 0.25 ml AZ Hoaglands micronutrientsand 0.3 ml Liquinure l1). 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 with FrankiaUGL 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 Crones nutrient was pH 6.2 initially andremained little changed during seedling growth. How-ever, it decreased to pH 5.25.5 over a 7 day periodwhen used to grow larger plants.</p><p>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.</p><p>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 with Frankia UGL 010708and were grown in CronesN solution with Hoag-lands AZ 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 at20 C prior to analysis of nickel content.</p><p>A second experiment with A. glutinosa of 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.</p><p>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 CronesNnutrients+Hoaglands AZ 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.</p><p>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.,</p></li><li><p>84</p><p>1996). Plants were harvested after 7 days, divided intothe major parts and analysed for their nickel content asdescribed below.</p><p>Assessment of leaf necrosisEach 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:</p></li><li><p>85Table 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</p><p>Treatment No of Shoot residue Chlorophyll C2H4 (nmol) Nodule no. seedling1NiSO4 (mM) seedlings dry wt (g) content (mg seedling1 h1 5 mm</p><p>nodulated/ seedling1)total</p><p>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) 00.5 2/9 0.025 (0.004) 0.10 (0.04) 0 2.8 (1.95) 0 0</p><p>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 (</p></li><li><p>86</p><p>Table 2. Changes with time in the distribution of nickel in A. glutinosa treated 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. Ne...</p></li></ul>