Environmental and Experimental Botany 70 (2011) 266–276
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Environmental and Experimental
Botany
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /e n v e x p b o
t
Silicon amelioration of aluminium toxicity and cell death in suspension cultures of Norway spruce (Picea abies (L.)Karst.)
Subramaniam Prabagar,Martin J.Hodson,David E.Evans ∗
School of Life Sciences,Oxford Brookes University,Headington Campus,Oxford OX30BP,UK
a r t i c l e i n f o Article history:
Received 16October 2009Received in revid form 29September 2010
Accepted 2October 2010Keywords:Aluminium Silicon
Suspension culture Norway spruce Toxicity
Ameliorationregret
a b s t r a c t
A role for silicon (Si)in the amelioration of aluminium (Al)toxicity in gymnosperms is suggested by their codeposition in planta ,including within needles.This study was designed to investigate Al/Si interactions at the cellular level using suspension cultures of Norway spruce.Toxic effects of Al were dependent on duration of Al exposure,concentration of Al,and pH.Toxicity was reduced when Si was prent,and the effect was enhanced at pH 5.0compared to pH 4.2.Study of the ultrastructure of Al-treated cells indicated that changes in cell wall thickening,degree of vacuolation,and the degeneration of mitochondria,Golgi bodies,ER and nucleus preceded cell death,and significant amelioration was noted when Si was also prent.When the fluorescent dye Morin was employed to locali free Al,cells treated with Al and Si in combination showed less fluorescence than the cells treated with Al alone.Intensity of fluorescence depended on the concentration of Al,duration of treatment and pH.Notably,prence of Si reduced the concentration of free Al in the cell wall in parallel with amelioration of Al toxicity.We therefore propo that formation of aluminosilicate complexes in the wall and apoplasm provide a significant barrier to Al penetration and cell damage in Norway spruce.entire
© 2010 Elvier B.V. All rights rerved.
1.Introduction
夏天晒黑了怎么变白Aluminium (Al)is an important component of many soil miner-als and only if soluble,mainly due to low pH,does it become toxic.Its availability depends on its chemical form (largely dependent on pH)and on the formation of complexes,which are of limited solu-bility and therefore unavailable.Its toxic effects have been reported in monocotyledonous and dicotyledonous angiosperms (Foy,1988)and in gymnosperms (Schaedle et al.,1989).Globally,Al toxicity is responsible for limiting crop production in tropical and sub-tropical soils (Foy,1992),and is a potential cau of forest dieback in tem-perate and boreal forests (Godbold et al.,1988and Taylor,1989).In many situations,mechanisms for the amelioration of toxicity are critical for plant growth.
It is now well recognid that silicon (Si)can have ameliorative effects on the phytotoxicity of a variety of metals (Doncheva et al.,2009;Vaculik et al.,2009).Previously,the effect of Si on Al availabil-ity in soils and in solution has been studied,together with its effects on plant growth (Hodson and Evans,1995).Studies have included plant growth in soil (Morikawa and Saigusa,2004)and hydro-ponic systems (Cocker et al.,1998a ).Most studies have involved monocotyledons (Galvez et al.,1987;Hodson and Sangster,1993;
∗Corresponding author.
E-mail address:deevans@brookes.ac.uk (D.E.Evans).
Hammond et al.,1995;Ma et al.,1997;Hara et al.,1999;Kidd et al.,2001;Kidd and Proctor,2001;Wang et al.,2004)and a few herba-ceous dicotyledons (Li et al.,1989;Baylis et al.,1994).In general,the prence of Si in the culture solution resulted in amelioration of Al toxicity,and only in a few cas was this not obrved (Li et al.,1989).
There is considerable evidence to suggest that Al/Si interac-tions are important in gymnosperms.Conifers transport some Al to their shoot tissues (Hodson and Sangster,1999).In needles,Al is imported and deposited via the transpiration stream for veral years before they nesce and abscind.Silicon is also transported through gymnosperms to reach the extremities of the needles.Most studies have involved X-ray microanalysis,especially of Al/Si co-deposits in needles (Hodson and Sangster,1999,2002).In white spruce,Al colocalid with Si in the needle epidermis (Hodson and Sangster,1998)and the tissues with the highest Si (in the tip,the mesophyll and transfusion cells)were also the tissues with the highest Al content (Hodson and Sangster,1999).A similar pattern was obrved in the needles of Eastern hemlock (Sangster et al.,2009).In Norway spruce,codeposition of Al and Si was shown to occur in the tissues interior to the endodermis of damaged nee-dles (Godde et al.,1988);however codeposition is rare in needles of Do
uglas fir (Sangster et al.,2007).Codeposition of Al and Si has also been obrved in the root cell walls of Al-tolerant Norway spruce edlings (Hodson and Wilkins,1991).In a study of Norway spruce edlings in hydroponic culture,Ryder et al.(2003)demonstrated
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S.Prabagar et al./Environmental and Experimental Botany70 (2011) 266–276267
that Si ameliorated the toxic effect of Al.Speciation analysis indi-cated that Al3+and hydroxyaluminium species were equally toxic and in both cas,Al toxicity was ameliorated by Si.The effect was pH-dependent with amelioration detected at pH4.75and5.0,but not at pH4.5or below.
The prent study was designed to explore Al/Si interactions in gymnosperms at the cell level.Cell suspension cultures have been ud previously to analy basic mechanisms involved in Al toxic-ity at a cellular Ojima et al.,1989;Yamamoto et al.,1994; Staßand Horst,1995;Minocha et al.,2001;Conner and Meredith, 1985a,b;Ono et al.,1995;Jones et al.,1998;Ramírez-Benítez et al.,2009;Pejchar et al.,2010).The studies reveal that suspen-sion cultures are more susceptible to
Al damage in the logarithmic growth pha than in the stationary pha(Yamamoto et al.,2000), and that Al affects cell growth,cell division and viability(Pan et al., 2002)with loss of plasma membrane integrity occurring prior to cell death(Ikegawa et al.,1998).
Suspension culture systems provide an opportunity to explore Al uptake at a cellular level.In carrot suspension cells,Al con-tent incread with increasing external Al concentration(Honda et al.,1997),and was accompanied by inhibition of respiration and decread ATP content.In tobacco cells,uptake was accompanied by a loss of viability(Yamamoto et al.,1997),while Chang et al. (1999)obrved that only about10%of the Al entered the cyto-plasm,the remainder being associated with the cell wall.A similar obrvation was also made by Schmohl and Horst(2000)in maize.
The subcellular effects of Al have also been studied in suspen-sion cultures,with effects on the cytoskeleton(Sivaguru et al.,1999; Grabski and Schindler,1995)and membranes(Deleers et al.,1986; Oteiza,1994;Ono et al.,1995;Ikegawa et al.,2000;Sivaguru et al.,2005).Studies on the effects of Al on gymnosperm suspen-sion cultures have revealed both growth inhibition and effects at a subcellular level.Minocha et al.(1992,1996,2001)obrved that the addition of AlCl3to3-day-old red spruce suspension cultures caud a significant increa in cellular putrescine concentration, incread vacuolar and total cell volume,and incread surface area of Golgi membranes and endoplasmic reti
culum.Succinate and oxalate were shown to be creted into the culture medium (Minocha and Long,2004).Organic acid cretion has been sug-gested to be involved in Al tolerance in a number of whole plant systems(Cocker et al.,1998a).
Conducting Al/Si interaction experiments in suspension culture allows the investigation of amelioration phenomena in the abnce of the organid tissues of the whole plant.One possible hypothesis is that such organisation is a prerequisite for amelioration to occur. Al/Si interactions have previously been studied in suspension cul-tures of rice and coffee(Rahman et al.,1999;Quintal-Tun et al., 2007).The prence of Si only marginally reduced the toxic effects of Al in a nsitive variety of rice and had no significant effect in a tolerant variety(Rahman et al.,1999).Quintal-Tun et al.(2007) did not consider cell growth in their coffee suspension cultures, but showed that the toxic effects of Al on the phospholipid signal transduction pathway were ameliorated by silicic acid.It is thus unclear whether amelioration occurs in suspension cultures.The work described in this paper was therefore carried out on Norway spruce suspension cultures to investigate whether Al/Si effects at a cellular level contribute to Al tolerance in this species.
2.Materials and methods
2.1.Cell cultures
Cell suspension cultures of Norway spruce(Picea abies(L.)Karst.) were initiated by agitating a fragment of in vitro grown embryo-genic callus in a volume of liquid medium which was prepared using modified half-strength Litvay’s medium(Litvay et al.,1985) containing1%sucro,2mg L−12,4-D,1mg L−1benzyladenine (BAP),500mg L−1cain hydrolysate,and250mg L−1glutamine, at pH5.7prior to autoclaving.Rapidly growing embryogenic callus was then transferred to a250ml conicalflask containing100ml of maintenance medium.Cells were subcultured at9-day intervals (10ml into50ml medium,250mlflask)and incubated in the dark at 25±2◦C on a New Brunswick orbital incubator shaker at120rpm.
2.2.Aluminium treatments
彩妆培训班Aliquots(10ml)of9-day-old cell suspensions from5culture flasks were transferred with shaking to250mlflasks containing 50ml of fresh medium(pH4.2)to which AlCl3had been added to final concentrations of0,0.2,0.5and1.0mM Al(effective concen-trations of monomeric Al=0.09,0.23and0.48mM,Minocha et al., 1996)in triplicate.They were then shaken in the dark(120rpm, 25◦C).At0–48h after Al(0.2–1.0mM)addition,10ml samples of cells were centrifuged and washed twice with sterile culture medium and then cultured in fresh medium without Al for19days before measurement of dry weight(DWT)and packed cell vol-ume(PCV).Data was prent
ed as relative growth(DWT or PCV of Al-treated cells/DWT or PCV of untreated control cells).
Cell viability,determined as loss of plasma membrane integrity, was evaluated by spectrophotometric measurement of retention of Evans blue dye(Baker and Mock,1994).Cell suspension cultures (10ml)were treated with0–1.0mM Al solution in250mlflasks containing50ml fresh medium(pH4.2or5.0).After0to48h,10ml samples of the cultures were collected and washed twice with fresh medium.Washed cells were suspended in2ml of0.05%aqueous Evans blue solution and stained for15min at room temperature. Cells were dimented by centrifugation and washedfive times with distilled water,after which the dye no longer eluted from the cells.Trapped dye was relead by suspending cells in2ml of 1%(w/v)aqueous sodium dodecyl sulphate(SDS)and disrupting at room temperature in a sonicator equipped with a microprobe (for2min at44W;model W-380;Heat Systems-Ultrasonics,Inc., NY,USA).The solution was transferred to an Eppendorf tube and centrifuged at13,500×g for10min.The optical density of the supernatant was determined at600nm.
Percentage of cell death was estimated by dye exclusion.Cells were treated with0–1.0mM Al as described above.One drop of Evans blue dye(1%(w/v)aqueous SDS)was mixed with0.5ml of cell suspension and incubated for15min at room temperature, before viewing by light microscopy.Areas chon for counting con-tained a minimum of25cells and the percentage of dead cells was calculate
d.
2.3.Silicon and Al/Si treatments
Silicon and Al/Si treatments were conducted as described above. However,solutions were made by adding various concentrations of AlCl3solutions(0.2,0.5and1.0mmol)to1.0mM Si at both pH4.2 and5.0and silicic acid was ud as Si source.The solutions were then left to diment for6h at room temperature before u.
2.4.Determination of Al and Si content
The Al,Si and Al/Si treated cells were centrifuged and washed twice with sterile culture medium.The cells were suspended in 0.5ml of acid mixture(H2SO4:HNO3,1:1,v/v)and were digested using microwave digestion method.Concentrations of Al and Si were determined using atomic absorption spectrophotometry.
268S.Prabagar et al./Environmental and Experimental Botany 70 (2011) 266–276
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Fig.1.A:Effects of Al and Al/Si on relative packed cell volume (PCV)of Norway spruce cells at pH 4.2.Cells which had been cultured in half-strength Litvay’s medium for 9days were treated with 0.2,0.5,1.0mM Al and 1.0mM Si for the length of time indicated.They were then grown for a further 19days and the PCV determined.B:As part A at pH 5.0.C:Effects of Al and Al/Si concentrations on relative dry weight of cells at pH 4.2grown as described in A.D:As part C at pH 5.0.In all cas data shows means ±SE (n =3).( )0.2mM Al,1.0mM Si ( )0.5mM Al,1.0mM Si ( )1.0mM Al,1.0mM Si ( )0.2mM Al (*)0.5mM Al ( )1.0mM Al (᭹)without Al and Si ( )1.0mM Si.wind万点
2.5.Electron microscopy
Cells were removed from the Al and Al/Si treatments as above at both pH 4.2and 5.0and fixed in 1%(w/v)paraformaldehyde and 1%(v/v)glutaraldehyde in 0.1M sodium cacodylate buffer (pH 6.9)for 1h at room temperature (Karnovsky,1961).They were washed in buffer and post-fixed in 1%aqueous os
mium tetroxide for 1h,before being stained in 0.5%uranyl acetate at 4◦C overnight.The cells were dehydrated in a graded ethanol ries (30min each)and then embedded through ethanol:Spurr resin mixtures (3:1,1:1,1:3)for 1h each,before embedding in 100%Spurr resin (Spurr,1969)and polymerisation at 60◦C for 10h.Ultra-thin (70–90nm)ctions of resin-embedded cells were cut using a Reichert Ultracut E microtome (Leica,Milton Keynes,UK),collected onto copper grids (300hexagonal mesh:Agar Scientific Ltd.,UK),stained with lead citrate (Reynolds,1963)and 1%(w/v)aqueous uranyl acetate and obrved with a JEOL 1200EXII transmission electron microscope at 100kV.
2.6.Confocal microscopy
Aluminium was localid in cells by confocal microscopy.A fresh 0.1mM Morin stock was prepared as described by Browne et al.(1990).The cells were rind in 1.5mM CaCl 2and washed in 5mM ammonium acetate at the treatment pH for 10min.The cells were stained with 50M Morin and then washed five times with 20ml of 5mM ammonium acetate and examined by confocal microscopy.All images were captured using a Zeiss lar scanning confocal
microscope (LSM 510)using a plan-Neofluor 25×/0.81mm cor-rection lens,the argon lar,and FITC narrow band filter.2.7.Statistical analysis
Data were analyd as a ries of analysis of variance (ANOVA)to determine whether statistically significant differences occurred between the concentrations of AlCl 3and duration of treatment.The ANOVA was performed with GraphPad InStat for Windows and a probability level of 0.05was ud for most tests unless specified otherwi.3.Results
Aluminium inhibited the growth of cultures when applied for up to 48h at pH 4.2and pH 5.0(Fig.1).This was obrved in a decrea in PCV and DWT.All subquent experiments were carried out using cells in logarithmic pha as the were shown to be more nsitive (data not shown).The decrea in dry weight was greatest at 1.0mM Al at pH 4.2and incread with time,with 70%inhibition over 48h (Fig.1A and C)compared with pH 5.0(60%inhibition;Fig.1B and D).
Addition of Si alone had a negligible effect on cell growth param-eters or packed cell volume (data not shown).However,addition of 1.0mM Si in the prence of Al significantly ameliorated the effect of Al,this being most pronounced at pH 5.0.At pH 4.2,signif-icant (P <0.05)amelioration of the effect of 1.0mM Al on PCV was obrved from 12to 24h (Fig.1A);effects on DWT were also signif-
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Fig.2.A:Effects of Al and Al/Si concentration on %dead Norway spruce cells at pH 4.2which had been cultured as described for Fig.1were treated with 0.2,0.5,1.0mM Al and 1.0mM Si for the length of time indicated.B:As part A,at pH 5.0.In all cas data shows means ±SE (n =3).( )0.2mM Al,1.0mM Si ( )0.5mM Al,1.0mM Si ( )1.0mM Al,1.0mM Si ( )0.2mM Al (*)0.5mM Al ( )1.0mM Al (᭹)without Al and Si ( )1.0mM Si.
icant for 1.0mM Al from 6to 48h (P <0.05)and at 0.2and 0.5mM Al at 48h (P <0.05)(Fig.1C).At pH 5.0,Si effects on PCV were sig-nificant at 0.2mM Al from 12h (P <0.1)and highly significant from 24h
(P <0.05)and from 6h for 0.5mM Al and 1.0mM Al (P <0.05)other than 12h at 0.5mM (Fig.1B).Silicon also had a significant effect on DWT at pH 5.0with all treatments showing a significant effect (P <0.1)at 12and 24h treatment (Fig.1D).
Cell death,as measured using the exclusion of Evans blue dye,resulted from treatment with all three concentrations of Al (0.2,0.5and 1.0mM)increasing rapidly from 6to 12h of treatment at pH 4.2(Fig.2A).After 24h,percentage cell death reached approximately 80%in all three concentrations of Al.At pH 5.0,percentage cell death incread in a more linear fashion with time,with a maximum of 60%in 1.0mM Al after 48h and 40%at 0.2mM Al (Fig.2B).The prence of 1.0mM Si reduced the effect of Al on cell death at all three Al concentrations at pH 4.2(Fig.2A)and pH 5.0(Fig.2B).At pH 4.2,maximum percentage cell death at 48h was reduced to 50%with 1.0mM Al and to 45%and 35%(from >70%)for 0.5and 0.2mM Al respectively.At pH 5.0,the equivalent effects were from 80%dead cells to 55%for 1.0mM Al and 1.0mM Si and a 20%and 15%decrea in percentage cell death for 0.5and 0.2mM Al.
In the cells that were only treated with Al in solution the Al concentration in the cells incread with both time and higher con-centration.The pH of the background solution made little difference at 0.2and 0.5mM external Al concentrations,but cells grown in 1.0mM Al showed significantly lower upt
ake at pH 5.0than at pH 4.2(3h P <0.01and 24h P <0.005).
In the treatments with both Al and Si prent in the culture solution the Al content of the cells also incread with both time and higher external Al concentrations.In all treatments apart from 1.0mM Al at pH 4.2,Al content of the cells was reduced by inclusion of Si in the media (Fig.3A).
In the abnce of Al,but with 1.0mM Si in the culture solution the Si content of the cells incread over time.Uptake was initially more rapid in cells grown at pH 5.0(Fig.3D)than at pH 4.2(Fig.3C),but by 48h there was no significant difference.Treatment of cells with both Al and Si caud an increa in Si content of the cells relative to tho that were only treated with 1.0mM Si (Fig.3C and D).This effect was more marked at pH 4.2,particularly at 1.0mM Al in the culture solution (P <0.005at 24h).
In order to investigate the effects of Al and Si further,ultra-thin ctions were prepared for transmission electron microscopy (Figs.4and 5).Cells,which had been taken from actively growing suspension cultures derived from Norway spruce embryos,showed the characteristic features of normal cells.A few small vacuoles contained small amounts of electron den material (Fig.4A)but not in the control cells.For the whole experiment 2200cells were obrved.With 1.0mM Al at pH 4.2,incread vacuolation was apparent at 3h with vacuoles aggregated around the nucleus at 6h,th
en forming a large central vacuole and plasma membrane paration at 12h treatment (Fig.4B).Cell death was also apparent at 12h (Fig.4C).Treatment with 0.2mM Al at pH 4.2resulted in incread cellular vacuolation commencing at 6h (Fig.4D);at 12h,vacuoles filled the entire cell (Fig.4E).In addition to the formation of vacuoles,nuclei appeared irregular in shape in some cells at 24h (Fig.4F).
In the prence of 0.2mM Al and 1.0mM Si at pH 4.2,vac-uolation was obrved,but this was much reduced compared to Al only treatment at the same pH (Fig.4G).In the prence of 1.0mM Al and 1.0mM Si,the cell walls appeared thickened and more electron den at 6h treatment and at 12h,electron den spots were obrved in the cell wall (Fig.4H).After 12h,vacuoles were prent with electron-den deposits (Fig.4H and I).After treatment with 1.0mM Al and 1.0mM Si,cell walls in contact with medium appeared more electron den and thicker at 3h,small vacuoles were formed at 6h (Fig.4G);at 24h,vacuolation was less than in the 1.0mM Al treatment and the nucleus appeared as normal and remained central with intact cytoplasm (Fig.4I).
At pH 5.0,numerous vacuoles were prent after 3h in the pres-ence of 0.2mM Al.Vacuolation rapidly incread up to 24h,with small vacuoles combining to form larger ones (Fig.5A,D and G).In addition,numerous Golgi bodies were obrved associated with vesicles (Fig.5A).In the prence of
1.0mM Al,Golgi apparati and associated vesiculation were apparent by 3h.By 6h,mitochon-dria were distorted and lacking internal membranes (Fig.5B)and endoplasmic reticulum and Golgi apparatus had become distorted (Fig.5E)and by 24h and in many cells the cytoplasm was distorted and excluded to the cell margin and large vacuoles were formed (Fig.5H).In the prence of 0.2mM Al and 1.0mM Si at pH 5.0,cells appeared similar to the control cells (Fig.5F).In addition,large electron-den deposits in vacuoles were found (Fig.5C).The cell wall also showed higher electron density from 6to 24h treatment with 0.2Al and 1.0mM Si (Fig.5C,F and I).
Uptake and localisation of Al was determined using the Morin reagent,and no fluorescence was obrved in control cells.In cells treated with 0.2mM Al for 3h at pH 4.2,much of the fluorescence was detected in the cell wall (Fig.6A).After 24h of Al exposure,fluorescence was also obrved in the cytoplasm (Fig.6B).Strong fluorescence in the cell was associated with damaged plasma mem-branes in most of the cells and with cell death (Fig.6B).After
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Fig.3.A:Uptake of Al by Norway spruce cells at pH 4.2.Cells which had been cultured as described for Fig.1were treated with 0.2,0.5and 1.0mM Al and 1.0mM Si for the length of time indicated.B:As part A at pH 5.0.C:Uptake of Si by Norway spruce cells at pH 4.2grown as described in A.D:As part C at pH 5.0.In all cas data shows means ±SE (n =3).( )0.2mM Al,1.0mM Si ( )0.5mM Al,1.0mM Si ( )1.0mM Al,1.0mM Si ( )0.2mM Al (*)0.5mM Al ( )1.0mM Al (᭹)without Al and Si ( )1.0mM Si.
treatment with 1.0mM Al at pH 4.2,fluorescence was detected in the cell wall after 3h (Fig.6C)but after 24h,it appeared in the vacuoles (Fig.6D).Aluminium and silicon treated cells did not accu-mulate Al as was obrved in cells treated with Al alone.At pH 4.2,fluorescence was not detectable after 3h at both 0.2and 1.0mM Al (Fig.6E and G).After 24h,fluorescence was obrved in the cell wall and nucleus (Fig.6F).
At pH 5.0,fluorescence was prent in the cell wall after 3h of Al exposure (Fig.7A);this was lower than at pH 4.2.After 24h of Al exposure,Al accumulated at the surface of the cells (Fig.7B and C,0.2mM Al and Fig.7E and F,1.0mM Al).At pH 5.0,fluorescence was low in dead cells and was not detected in living cells after 24h at 0.2mM Al and 1.0mM Si (Fig.7H and I).4.Discussion
Addition of aluminium to suspension cultures of Norway spruce resulted in reduced growth (measure
d by a number of parameters),cell death (Figs.1and 2)and changes in cellular ultrastructure,including incread formation of small vacuoles,possibly derived from the Golgi apparatus,which subquently fud to form a large vacuole (Fig.4).The effects were rapid,occurring within 24h and reaching a maximum within 48h of treatment.They are in agree-ment with previous studies of Al toxicity;for instance,Minocha et al.(2001),studying red spruce (Picea rubens )suspension cultures obrved loss of viability,inhibition of growth and decread mito-chondrial activity and incread vacuolation within 24–48h.Effects of Al were greater at pH 4.2,with 77.9%cell death in 1.0mM Al over 24h,while the equivalent at pH 5.0was 53.1%(Fig.2).Martinez-
Estevez et al.(2001)obrved maximal inhibition of growth of cell suspensions of coffee at pH 4.3compared with pH 5.8.However,Ryder et al.(2003)obrved no effect of pH on root elongation in Al-treated Norway spruce roots,suggesting differences in respon between intact tissue and cells in suspension.
四六级准考证打印入口2020In agreement with Minocha et al.(2001),our results show that aluminium was taken up by the suspension cultures but initially confined to the cell wall (Fig.6A–C);at high (1.0mM)concentra-tions,Al was subquently also detected in vacuoles.Tanoi et al.(2006)obrved Al at the plasma membrane and cell wall in tobacco suspension cultures using lumogallion staining.Using Morin stain-
ing,Vitorello and Haug (1996)did not detect Al in the cell wall of tobacco cells,but did detect it in a discrete zone of the cell periph-ery.By 24h at pH 4.2,Al was detected in the cell wall,at the plasma membrane,in organelles and the nucleus (Fig.6).By this stage,Al uptake is likely to have occurred via a damaged plasma membrane;such damage was also suggested by Evans blue dye exclusion assay.
While addition of Si to the medium did not result in any direct effect on any of the cell growth or viability parameters studied,it was found to be taken up by the cells (Fig.3C and D).This was in contrast to rice,in which Si was reported to be ‘very low’by Rahman et al.(1999).Rice is known to be a heavy Si accumulator,while Norway spruce plants are only moderate accumulators (Hodson et al.,2005).However,it may be that any Si uptake in the rice cells was below the nsitivity of the assay ud by Rahman et al.,or that the prence of an Si efflux pump in that species (Ma et al.,2007)results in reduced accumulation in suspension cells of this species.
Amelioration of Al toxicity by the prence of Si was clearly obrved for all parameters studied (Figs.1,4and 5),in a man-
