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Influence of nickel stress on growth and some important physiological/biochemical attributes in some diverse canola ( Brassica napus L.) cultivars

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Influence of nickel stress on growth and some important physiological/biochemical attributes in some diverse canola ( Brassica napus L.) cultivars
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   Journal of Hazardous Materials 172 (2009) 964–969 Contents lists available at ScienceDirect  JournalofHazardousMaterials  journal homepage: www.elsevier.com/locate/jhazmat Influence of nickel stress on growth and some importantphysiological/biochemical attributes in some diversecanola ( Brassica napus  L.) cultivars M.A. Ali a , ∗ , M. Ashraf  a , H.R. Athar b a Department of Botany, University of Agriculture, Faisalabad, 38040, Pakistan b Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan a r t i c l e i n f o  Article history: Received 17 June 2009Received in revised form 21 July 2009Accepted 21 July 2009 Available online 28 July 2009 Keywords: Amino acids Brassica napus L. Osmotic potentialNi accumulationSoluble proteinsSoluble sugars a b s t r a c t Toassesstheeffectofnickelonsixcanolacultivarsaseriesofexperimentswereconducted.Onthebasisof shoot dry weight cvs. Shiralee and Range found to be nickel tolerant, Dunkeld and Ester as nickel sen-sitive,whiletheremainingcultivarsintermediate.Nickelaccumulationinshootswaslowerinthenickelsensitivecultivarsfollowedbythatinthetolerantones.Leafwaterandosmoticpotentialsdecreasedsig-nificantlyduetohighconcentrationofNi 2+ .Decreaseinosmoticpotentialwaspositivelyassociatedwithaccumulation of total free amino acids. By comparing accumulation of individual amino acids, pattern of accumulation of the amino acids was different in different cultivars. However, only histidine, serine andcysteine increased in appreciable amount in the xylem sap of different canola cultivars. Overall, nickeltolerantcultivarsShiraleeandRangeshowedhigherlevelsofhistidine,serineandcysteineundervaryinglevels of nickel than the others. This higher accumulation of histidine, serine and cysteine was positivelyrelated to nickel tolerance in all canola cultivars. Thus, differential nickel tolerance in canola cultivarsproposed to be associated with relative detoxification of Ni by developing complexes with histidine,serine and cysteine and can be used as potential indicators of nickel tolerance in canola. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Of many heavy metals known in nature, nickel (Ni) is essen-tial as trace element for normal plant growth and development,because it is constituent of some important enzymes such as ure-ase.However,highconcentrationofNiingrowthmediumcanleadtotoxicitysymptomsandreducedgrowthofplants[1].Toxiceffectsof high concentrations of Ni in growth medium on plants includealteration in uptake of essential nutrients, chlorosis, reduced CO 2 uptake, disturbances in gas exchange, alterations in water uptakeand generation of free radicals and reactive oxygen species thatproduce oxidative stress [1–4]. There are a variety of mechanisms by which plants can endurehigh concentration of heavy metals, including restricted uptakeand/ortranslocationofmetals,exclusionoftoxicheavymetalsfromcellsbyion-selectivemetaltransporters,excretionorcompartmen-tation of heavy metals, production of heavy metal binding factorssuch as proteins, peptides, amines, amino acids, and formation of complexes with these binding factors and metals to detoxify met-als [5–6]. For example, metal tolerant plants accumulated higher ∗ Corresponding author. Tel.: +92 0419200312; fax: +92 0419200764. E-mail address:  nudrataauaf@yahoo.com (M.A. Ali). prolineinresponsetoheavymetalstressascomparedtometalsen-sitiveplants,andthisaccumulationofprolineinstressedplantswasfound to be associated with reduced damage to membranes andproteins [7–8]. In another study, nicotianamine has been shown to chelate Ni and enhanced nickel tolerance in  Thlaspi goesingense [9]. From these reports it is evident that different amino acids canhave an important role in regulating metal toxicity in plants andthus it needs an extensive study. Furthermore, genetic variationand some degree of heritability for Ni stress tolerance have alsobeen reported in Ni hyperaccumulator  Thlaspi spp . [9–11]. In view of all these reports, the present study was aimed to assess vari-ability for Ni stress tolerance in some elite canola cultivars and toexamine up to what extent accumulation of different amino acidshas a role in Ni stress tolerance in canola cultivars. In general, thework reported in manuscript is to identify the potential indicatorsresponsibleforthetoleranceofnickelstress,whichcouldbeusedinfuture breeding programs to evolve high yielding canola varietieswith improved characters. 2. Materials and methods Four independent experiments were conducted to assess theresponse of six selected canola cultivars to varying nickel concen-trations. In the first experiment, LD 50  (Lethal dose at 50% growth) 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.07.077  M.A. Ali et al. / Journal of Hazardous Materials 172 (2009) 964–969 965 concentration of Ni was determined for canola cultivars. In thesecond experiment, screening of canola cultivars for Ni tolerancewas carried out and cultivars were grouped as tolerant, moder-ately tolerant and sensitive on the basis of shoot dry weight at theadultstage.Inthethirdexperiment,physiologicalandbiochemicalresponsesofcanolacultivarswereassessedundervaryingconcen-trationsofnickel.Inthefourthexperiment,accumulationofNiandaminoacidsinxylemsapwasdeterminedandparallelsweredrawnbetween them to assess their role in Ni tolerance. 3. Experiment 1: Optimization of LD 50  concentration of Nifor canola In order to determine LD 50  (nickel concentration where 50%growth was reduced), 20 seeds of each cultivar were allowed togerminate for 15 days under varying concentrations of Ni (0, 10,20, 30, 40  . . .  150mgL  − 1 ). After 15 days, germination percent-age, percent viable population size, shoot fresh and dry weightswererecorded.TheNiconcentration150mgL  − 1 wasthemosttoxicconcentration, which severely inhibited the growth. At Ni concen-tration100mgL  − 1 ,50%reductioninpercentviablepopulationsize,and shoot fresh and dry weights was observed and considered asLD 50 . This Ni concentration was used for further experimentationto screen the canola cultivars. 4. Experiment 2: Assessment of variation in tolerance of canola cultivars to Ni Seeds of 10 canola cultivars (Shiralee, Range, Torch, Rain-bow, Dunkeld, Ester, Con-I, Con-II, Tobin, Frontier) were obtainedfrom Ayub Agricultural Research Institute, Faisalabad, Pakistan.Twenty seeds were sown in earthen of 30cm diameter containing6.0kg homogenously mixed sun-dried sandy clay loam soil with acompletely randomized design (CRD) and four replications. Aftergermination the plants were thinned to maintain five seedlings ineach pot. After 3 weeks, all plants were subjected to 100mgL  − 1 NiasNiCl 2  for4weeks.Onthebasisofshootdryweightin100mgL  − 1 Ni,ShiraleeandRange,TorchandRain,andDunkeldandEsterwerecategorized as tolerant, moderately tolerant, and sensitive to Ni,respectively,whiletheothercultivarswerefoundtobeintermedi-ate in Ni tolerance. The cultivars so selected were further used toevaluateNitoleranceonthebasisofphysiologicalandbiochemicalattributes. 5. Experiment 3: Physiological and biochemical responsesof selected canola cultivars to Ni A pot experiment was conducted during the winter 2004–2005in a net-house at the Botanic Garden of the University of Agriculture, Faisalabad, Pakistan (latitude 31 ◦ 30 N, longitude73 ◦ 10 E and altitude 213m), with 10/14 light/dark period at800–1100  molm − 2 s − 1 PPFD, a day/night temperature cycle of 28/17 ◦ C and 65 ± 5% relative humidity. About 50 seeds of each of six canola cultivar were sown in each earthen pot (30cm diame-ter and 20cm in depth) filled with 6kg sandy clay loam soil (soilsaturation percentage 33%; pH 7.8; EC e  2.21mScm − 1 ). For deter-mining available Ni, soil was extracted in 1N ammonium acetatesolution (1:5) ratio following Allen et al. [16] and for total Ni inthe soil samples were digested in a mixture of sulphuric acid andhydrogen peroxide following Wolf  [17]. The mean available andtotalNiconcentrationlevelsinthesoilwere0.14and29.5mgkg − 1 ,respectively. After 1 week, the seedlings of comparable size grow-ing equidistantly were thinned to maintain five seedlings per pot.The experiment was arranged in a completely randomized designwithfourreplicates,fournickeltreatments(0,50,100,150mgL  − 1 )and six cultivars. The plants were irrigated with distilled water for3weeksbeforethestartofvaryingNitreatmentsasnickelchloride(NiCl 2 · 6H 2 O) for further 58 d after which time three plants out of five were harvested. Uprooted plants were washed with distilledwater and separated into shoots and roots, and then blotted drybefore recording their fresh weights. All plants parts were driedat 65 ◦ C until constant dry weight, and their dry weights recorded.Before harvesting the plants for determination of plant biomass,the following physiological parameters were measured: 5.1. Water relations The 2nd leaf from each plant was excised at 7.00 a.m., and theleaf water potential measurements were made with a Scholandertype pressure chamber (Arimad, UK). A proportion of the sameleaf used for water potential measurements, was frozen into 2cm 3 polypropylene tubes at  − 40 ◦ C in an ultra-low freezer for 2 weeks,after which time plant material was thawed and the frozen sapwas extracted by crushing the material with a glass rod. Aftercentrifugation (8000 ×  g  ) for 4min, the sap was directly used forosmoticpotentialdeterminationusingavaporpressureosmometer(Wescor 5500). Leaf turgor pressure was calculated as the dif-ference between leaf water potential and leaf osmotic potentialvalues. 5.2. Total soluble proteins Total soluble proteins were determined as described by Lowryet al. [12]. Fresh leaf material (0.2g) was homogenized in 4mL of  sodium phosphate buffer solution (pH 7.0) and centrifuged. Theextract was used for the estimation of soluble proteins and freeamino acids. The sample extracts were reacted with a Folin phenolreagent and the optical densities read at 620nm using a spec-trophotometer (Hitachi U-2000). 5.3. Total free amino acids Total free amino acids were determined following the proce-dure of Hamilton and Van Slyke [13]. For estimation of total freeamino acids, 1mL of each sample as extracted for soluble proteindeterminations was treated with 1mL of 10% pyridine and 1mL of 2% ninhydrin solution. The optical densities of the solutions wereread at 570nm using a spectrophotometer (Hitachi U-2000). 5.4. Total soluble sugars Totalsolublesugarswereestimatedfollowingtheproceduresof Malik and Srivastava [14]. Well ground dry leaf material (0.1g) of each sample was homogenized in 80% ethanol and centrifuged at3000 ×  g  .Theresiduewasretainedandrepeatedlywashedwith80%ethanol to remove all traces of soluble sugars. The resulted filtratewasdilutedupto100mLwithdistilleddeionizedwaterandreactedwithanthronereagent.Theaborbanceofthecoloredsolutionswasread at 625nm using a spectrophotometer (Hitachi U-2000). 5.5. Qualitative and quantitative estimation of individual aminoacids Amino acid profile was estimated according to the method of Braithwaite and Smith [15]. One gram fresh leaves were choppedin 2mL of 6N HCl in sealed test tubes and incubated at 125 ◦ C ina heating chamber for 24h. The resulting paste was centrifugedat 15,000rpm at 15 ◦ C for 10min. The supernatant was used forseparation of amino acid profile through paper chromatography.Individual amino acids were identified by comparing Rf values of   966  M.A. Ali et al. / Journal of Hazardous Materials 172 (2009) 964–969 Fig. 1.  Fresh and dry weights of shoots and roots of six canola cultivars when 21-day-oldplantsweresubjectedtovaryingconcentrationsofnickelfor58days( n =4). coloured spots and standards in a paper chromatogram. The col-ored spots of amino acids on paper chromatogram were cut witha scissors and eluted in 3mL of methanol and the optical densitiesof the solutions were read at 540nm using a spectrophotometer(Hitachi U-2000). Fig. 2.  Nickel (  g/gdwt) in shoots and roots, and shoot/root Ni ratio of six canolacultivarswhen21-day-oldplantsweresubjectedtovaryingconcentrationsofnickelfor 58 days ( n =4). 5.6. Determination of Ni  2+ in plant tissues Ni 2+ in leaves and roots were determined using the meth-ods described by Allen et al. [16]. Each ground dry plant samples(100mg) was digested in 2ml of sulfuric-peroxide digestion mix-ture until a clear and almost colorless solution was obtained. Afterdigestion, the volume of the sample was made to 10ml with dis-tilled de-ionized water. Nickel was determined with an atomicabsorption spectrometer (PerkinElmer Analyst 100). 6. Experiment 4: Role of amino acids in Ni tolerance In this experiment, seeds of all selected cultivars were germi-nated for 1 week, thereafter transferred to perforated polystyrenefoam placed in plastic tank (70 × 40 × 25cm) containing 30 L fullstrength Hoagland’s nutrient solution (pH 6.5). The hydroponicswas continuously aerated. Plants were further grown under vary-ingconcentrationsofNi(0,50,100,150mgL  − 1 )for6weeks.Shootswere excised at the base, and the cut surfaces were blotted withabsorbent tissue. The root pressure exudates were collected over8h period and injected into eppendorf tubes, and then stored at-70 ◦ C. In the xylem sap, amino acid profile and Ni concentrationwereestimatedandquantifiedfollowingthemethodofBraithwaiteand Smith [15] and Allen et al. [16], respectively. 6.1. Statistical analysis of data The data were subjected to analysis of variance using a COSTATcomputer package (Cohort Software, Berkeley, California). Themean values were compared with the least significance differencetest following Snedecor and Cochran [18]. Fig. 3.  Nickel (  g/gdwt) in shoots and roots, and xylem sap Ni 2+ of six canola cul-tivarswhen7-day-oldplantsweresubjectedtovaryingconcentrationsofnickelfor42 days ( n =4).  M.A. Ali et al. / Journal of Hazardous Materials 172 (2009) 964–969 967 7. Results Exogenous application of 100mgL  − 1 Ni (LD 50 ) proved to bevery useful in discriminating canola cultivars as tolerant, mod-erately tolerant, and sensitive in second experiment. Shoot freshand dry weight of all selected canola cultivars were consistently( P  <0.001) reduced with increase in Ni concentration in rootingmedium (Fig. 1). Shiralee and Range were the highest of all thecultivars in shoot dry weight, while Range and Rainbow exhib-ited intermediate performance. Overall, cv. Shiralee was the mosttolerant in terms of shoot fresh and dry weight at all external Nilevels.Nickel concentration in the xylem sap, shoots and roots of allthe canola cultivars was increased with increase in Ni supply inthe rooting medium. Furthermore, this accumulation was higherwhencanolacultivarsweregrowninhydroponics(Figs.2and3).At100mgL  − 1 Ni 2+ , moderately tolerant cultivars Torch and Rainbowwere higher, while sensitive cultivars Dunkled and Ester, lower inshoot Ni 2+ concentration. However, it was observed that root Ni 2+ concentration was maximum in cultivars Range and Shiralee andminimumincultivarsEsterandDunkled,particularlyat100mgL  − 1 ofNi 2+ appliedthroughrootingmedium.Inshoot/rootNi 2+ ratioNitolerant cultivars Shiralee and Range were lower at all levels of Ni 2+ as compared to the moderately tolerant or sensitive canolacultivars.Leaf water potential and osmotic potential ( P  <0.001) in allcanola cultivars decreased with increase in Ni concentration in Fig. 4.  Leaf water potential, leaf osmotic potential and leaf turgor potential of sixcanola cultivars when 21-day-old plants were subjected to varying concentrationsof nickel for 58 days ( n =4). growthmedium(Fig.4).Esterwasthehighestandshiraleethelow-estinleafwaterpotentialofallthecanolacultivarsatthehighestNiconcentration in growth medium, whereas at other Ni concentra-tions Dunkeld and Ester were intermediate in leaf water potential.Leaf osmotic potential was found to be the lowest in Ni tolerantcultivars (Shiralee and Range), whereas the highest was in NickelsensitiveDunkeldandEsterascomparedwithallothercultivarsatallconcentrationsofNi(Fig.4).Leafturgorpotentialwasfoundtobemaintained in Ni tolerant cultivars (Shiralee and Range), whereasit decreased at highest Ni concentrations (Fig. 4).LeaftotalsolubleproteinsofNisensitiveormoderatelytolerantline decreased when 150mgL  − 1 Ni was applied through the root-ing medium. In contrast, leaf total free amino acids were increasedin all canola cultivars. However, maximum increase in total freeaminoacidswasfoundinNitolerantcultivars(ShiraleeandRange),whereasNisensitivecultivars(DunkeldandEster)werethelowestin this biochemical attribute (Fig. 5). Leaf soluble sugars were con-sistently decreased with increase in Ni supply in rooting medium.However, this reduction was more in Ni sensitive cultivars com-pared with other cultivars (Fig. 5).Most of amino acids in leaf were increased in Ni tolerant cul-tivars with increased in Ni concentration to the growth mediumsuch as histidine, lysine, serine, glutamic acid, cysteine, aspartate,glycine, methionine, arginine etc (Data not shown). However, con-centrationofhistidine,cysteine,lysineandserineinxylemsapwerehighestinNitolerantandthelowestinNisensitivecanolacultivarsat higher concentrations Ni in rooting medium (Fig. 6). Fig. 5.  Total soluble proteins, total free amino acids and total soluble sugars of sixcanola cultivars when 21-day-old plants were subjected to varying concentrationsof nickel for 58 days ( n =4).  968  M.A. Ali et al. / Journal of Hazardous Materials 172 (2009) 964–969 Fig. 6.  Accumulation of amino acids in xylem sap of six canola cultivars when 7-day-old plants were subjected to varying concentrations of nickel for 42 days ( n =4). 8. Discussions In the present study, increasing supply of Ni 2+ in the root-ing medium reduced the growth of canola cultivars as has earlierbeen observed in wheat ( Triticum aestivum  L.) [2] and Matricariachamomilla [3] at varying concentrations of Ni and Cd. However,a considerable genetic variation in response to Ni stress has beenobserved in the set of six cultivars/lines of canola examined here.For example, Ni tolerant cv. Shiralee was higher in shoot fresh anddry weight at all levels of Ni 2+ , while other Ni 2+ tolerant cv. Rangewas more sensitive at higher levels of Ni 2+ . Such variability amongcanola cultivars to nickel stress may have been due to differencesin accumulation or distribution of Ni in shoots and roots [19]. Inthe present study, moderately Ni tolerant cultivars (Rainbow andTorch)accumulatedconsiderablyhigheramountofNi 2+ inallplantparts i.e. stem, leaves, and roots, compared to the other cultivars.However, all cultivars tended to partition more Ni 2+ in the roots(Fig. 2). Thus, cultivars having low shoot/root Ni ratio had betterability to retain Ni 2+ in the roots, possibly by binding and seques-tering it in the vacuoles [20], which might have contributed totolerance to Ni 2+ . It seems reasonable to propose that variation insensitivity to Ni stress among six canola cultivars was due to dif-ferential accumulation of Ni 2+ in shoots, contributing to cytosolicdetoxification.Generally, plant water relations of six canola cultivars wereadversely affected with increase in Ni 2+ concentration of thegrowth medium. If we draw parallels between leaf osmoticpotential and leaf nickel concentration, it is obvious that hyper-accumulation of Ni 2+ caused a decrease in leaf osmotic potential( r  =0.858***).InviewofKramerandBoyer[21]hyper-accumulationof heavy metals just like Ni 2+ and Zn 2+ is analogous to the processofosmoticadjustment,inwhichcompatibleorganicsolutesand/orinorganic ions (e.g. Na + , K + , Cl − ) are accumulated under water orsalt stress to lower osmotic potential in the cell to maintain turgorand cellular activity. Likewise, Baker and Walker [22] were of viewthat metals might be hyper-accumulated to increase osmolaritywithin the cell. In view of a large number of published reports, it isconceivablethatreductioninosmoticpotentialofplantssubjectedtoanystress(waterstress,heatstress,saltstressetc.)maybeduetoeither water loss or an increase in dissolved solutes (organic com-patible solutes or inorganic osmotica such as Na + , K + , Cl − , etc.) or acombination of both [23]. Among organic osmotica or compatibleosmolytes,solublesugarsandaminoacidsandtheirderivativesaremore important for maintaining osmoregulation in cells or tissuessubjectedtostresses.Ifwedrawrelationshipbetweensolublesug-ars or total free amino acids and leaf osmotic potential, it is clearthat leaf osmotic potential is positively related to free amino acids(OPvsaminoacid r  =0.646***)butnottosolublesugars(OPvssolu-blesugars r  =0.00057ns).Thus,differentialgrowthresponsesofsixcanola cultivars to nickel stress can be related to their differentialaccumulationoffreeaminoacids.Forexample,thelowestosmoticpotential recorded in nickel tolerant cultivars Shiralee and Range
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