relationships between cadmium, zinc, cd-peptide, organic ... · for determination of k+, mg2+,...

Plant Physiol. (1989) 91, 780-7870032-0889/89/91 /0780/08/$01 .00/0

Received for publication January 18, 1989and in revised form May 31, 1989

Relationships between Cadmium, Zinc, Cd-Peptide, andOrganic Acid in Tobacco Suspension Cells

Rachel M. Krotz, Bill P. Evangelou, and George J. Wagner*

Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky,Lexington, Kentucky 40546-0091

ABSTRACT

Responses of tobacco (Nicotiana tabacum) suspension cellsto Cd and Zn were studied in the presence and absence of ligandof Cd-peptide in order to understand the role of this peptideversus other mechanisms in Cd and Zn accumulation and accom-modation in plants. With 45 micromolar Cd and 300 micromolarZn (non-growth-inhibiting levels), metals appeared rapidly withincells, and intracellular Cd and Zn reached medium concentrationsafter 6 to 10 hours. Cd-peptide was observed in response to Cdafter 2 hours, but this form only accounted for -30% of solubleCd after 24 hours. Peptide was not observed in cells exposed to300 micromolar Zn for up to 7 days. Organic acid-to-metal stoi-chiometry indicated that endogenous organic acid content of cellswas more than sufficient to complex absorbed metals and noevidence was found for stimulation of organic acid biosynthesisby Cd or Zn. Metal-complexing potential of organic acids for Cdand Zn versus endogenous cations is discussed as is vacuolar-extravacuolar distribution of metals. The absence of Cd-peptidedoes not limit Cd-accumulation in the system studied. Resultssuggest that tobacco suspension cells accommodate the pres-ence of non-growth-inhibiting and growth-inhibiting levels of Cdand Zn by sequestration in the vacuole as complexes with en-dogenous organic acids and that this may be a principal meansfor accommodation of Cd as well as Zn in the presence andabsence of Cd-peptide.

Recent interest in unique (-y-Glu-Cys), Gly Cd-bindingpeptides, also called cadystins, phytochelatins, etc., centers ontheir structure, biosynthesis, and possible role in tolerance ofplants and plant cultured cells to challenge with high levels ofthis metal (4, 6, 9, 16, 20, 21, 24, 31 and references therein).While the amino acid composition and primary structure ofthis family of peptides is becoming understood for severalhigher plant systems and certain yeasts, their function(s),selectivity in binding metals, aggregation potential, mode ofbiosynthesis, and metal selectivity and concentration depend-ence in their induction, are not understood (31). Cd appearsto be bound in acid labile mercaptide complexes (19, 29),perhaps, under certain conditions with participation ofsulfide (20).

Positive correlations between occurrence ofCd peptide andtolerance (lack of growth inhibition) have been documentedfor two plant tissue-culture systems, suggesting a role forpeptide in tolerance to high level Cd exposure (7, 18). It isnot known if Cd-peptide is formed in plants exposed to lowlevels of metal as occur in agriculture (31). Indeed, available

evidence suggests that it is not, and that this ligand is notconstitutive (31). Therefore, the question arises as to themechanism of Cd (and Zn) accommodation and accumula-tion in the absence of Cd-peptide. Zn binding to this peptidehas not been demonstrated (19), though peptide induction byvery high levels of Zn (possibly secondary response) has beenreported (3, 18).

Vacuolar accumulation of Cd after high level Cd exposureof plants and Dunaliella is suggested from several recentstudies (5, 17). Earlier, we reported the absence of evidencefor vacuolar Cd in plants exposed to very low levels (0.01 M)of this metal (27). While the issue of Cd concentration versusvacuolar Cd accumulation is unresolved, vacuolar accumu-lation of this metal after high level exposure appears to occur.This presents a dilemma in that extracts ofplants and culturedcells exposed to high levels of Cd contain most metal as Cd-peptide, yet presumably most of the intracellular Cd is in thevacuole. Vacuolar sap pH of most plants is in the range ofpH 5, and pH for 50% dissociation of Cd (in vitro) fromligand of tobacco cultured cells and leaf peptide is about pH5 (19)-compared to pH 3.0 for 50% dissociation of princi-pally cytosolic metallothionein of animal cells (19). Peptidefrom tobacco has the same amino acid composition as thatfrom several other systems ( 19), so it is likely that tobacco Cdpeptide represents typical (-y-Glu-Cys), Gly Cd-binding pep-tide. The above discussion raises the question of the form ofvacuolar Cd in cells containing, and also those not containing,ligand of Cd-peptide. The latter condition occurs in tobaccocultured cells exposed to relatively low levels (<1 uM) of Cd,cells exposed to <1000 uM Zn (18), immediately after expo-sure of nonacclimated cells to high levels of Cd (this study),and to cells grown in the presence of BSO,' an inhibitor ofpeptide formation (18). It is noteworthy that most plants innature and agricultural crops are probably exposed to verylow levels (50.1 uM) of this metal (31).

Prior to the demonstration that plants exposed to highlevels of Cd form Cd-peptide, models to explain the mecha-nisms of heavy metal tolerance in plants focused on Zn andCu and possible roles of the cell wall and vacuolar organicacids in metal sequestration (32). Ernst was first to postulatevacuolar accumulation of Zn organic acid complexes as amechanism for Zn tolerance in naturally tolerant ecotypes(32). Considerable, but not entirely consistent, evidence existsfor a correlation between Zn content and organic acid contentin various plants and cultured cells (2, 8, 13, 15, 25, 26, 32).

' Abbreviation: BSO, buthionine sulfoximine.

780

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MECHANISMS OF Cd AND Zn ACCUMULATION IN TOBACCO CELLS

Here, we examine the possible involvement of vacuolarorganic acids in accumulation of Cd and Zn in cultured cellsexposed to non-growth-inhibiting and growth-inhibiting levelsofthese metals and in the presence and absence ofCd-peptide.

MATERIALS AND METHODS

All chemicals were reagent grade or analytical grade. Ultra-pure ZnSO4 (<0.0001% Cd) was purchased from AldrichChemical Co.

Suspension cells of Nicotiana tabacum L. cv Wisconsin 38were maintained on a 7-d cycle in B5 medium as previouslydescribed (18). Cells were in log phase growth between 2 and4 d. Two days after subculturing, metal or metal plus [1-'4C]Na acetate (0.3 1Ci per 30 mL culture, 50 mCi/mmol, Du-pont, New England Nuclear) were added aseptically fromfilter sterilized solutions to begin exposures. Where used, BSO(Sigma Chemical Co.) was introduced along with metal to 3-d cultures. To harvest cells, cultures were transferred to cali-brated tubes and centrifuged at IOOOg and packed cell volumewas noted (18). Cells were washed twice with 3% (w/v)sucrose, 10 mM CaSO4, on a Buchner funnel using gentlesuction. For analysis of metal content, washed cells were

frozen, thawed, and homogenized in a mortar and pestle at4°C using 1 mL 25 mM Tris/HCl (pH 8) per fresh weight ofcells. For recovery of acids, cationic, and neutral fractions,washed cells were homogenized in 0.2% (w/v) oxalic acid at4°C using a polytron homogenizer (Brinkman Instruments).Other extraction media and methods were used as noted. Apreviously described procedure was used to determine Cd-peptide content (18). Homogenates were centrifuged (4°C) at16,000g for 5 min and the resulting supernatants were centri-fuged at 100,000g, 30 min. Pellets were combined and washedwith growth media less metal; insoluble fractions were driedand digested with 9:1 (v/v) HNO3:HClO4 and the digest wasevaporated and analyzed for Cd and Zn content in 1% HCIusing flame atomic absorption spectroscopy (with backgroundcorrection for Zn). For determination of K+, Mg2+, Ca2+,NO3-, C1-, S042-, PO34, and H+, water washed cells were

homogenized in freshly boiled, distilled, deionized H20. TheK+, Mg2+, and Ca2+ contents of 100,000g supernatants were

assayed directly by flame atomic absorption spectroscopy.Nitrate was measured using the procedure of Lowe and Gil-lespie (11). For analysis of C1-, S042-, and PO43- a Dionexseries 4000 I instrument and HPICAS 4 column were utilized.Buffer contained 2.2 mM Na2CO3, 2.8 mM NaHCO3 (pH 9.6)and 0.025 N H2SO4 was used as anion supressor.To monitor organic acids, cold Ca-acetate was added to

100,000g supernatants of 0.2% oxalic acid homogenates, thesuspensions were centrifuged at 16,000g for 5 min at 4°C tosediment Ca-oxalate, and the pellets were washed with coldwater and resedimented (28). Washed Ca-oxalate was sus-

pended with 6 N HCI and radioactivity was counted in a

quench-corrected liquid scintillation system. To separate an-

ionic, cationic and neutral components, supernatants (afterremoval of Ca-oxalate) were applied to Dowex 50 (H+), 1 x

8, 200-400 mesh and the effluent of Dowex 50 and waterwashes to Dowex 1 (formate) 1 x 8, 200-400 mesh. Thecationic fraction was recovered from Dowex 50 with 1 N HCIand the neutral fraction was that eluted and washed from

Dowex 1 (28). An aliquot of each fraction was monitored forradioactivity. The anionic fraction was eluted from Dowex 1with 4 N formic acid, evaporated, counted, resuspended in0.022 N H2SO4 and separated on an Aminex-87H HPLCcolumn (Bio-Rad) using 0.022 N H2SO4 as solvent at 0.6 mL/min. Organic acids were monitored at A210 and quantifiedusing a model 3393A Hewlett Packard integrator. A lineardetector response was used from 0.2 to 0.7 mg for malic andfrom 0.15 to 0.5 mg citric acid. Fractions were collected andmonitored for '4C as described above. Attempts to monitororganic acids directly from extracts using the Aminex columnwere unsuccessful due to interfering components. Standardaddition experiments showed that recovery of acids fromtissue was 285%. Malic acid concentration in aqueous ex-tracts oftobacco cells were confirmed using the Dionex systemdescribed above. This system did not afford resolution ofcitric acid.

Vacuolar-extravacuolar distribution of metals was esti-mated using cells of Datura inoxia (7-d culture cycle). Cellsexhibited log phase growth between 1 and 5 d (not shown).The tobacco cell line which was the principal tissue used inthis study did not yield sufficient vacuoles (purity and quan-tity) for study of vacuole-extravacuole distribution. For Da-tura cells, 3.5 g of 4-d-old cells exposed to 0.35 AM Cd (as'0Cd, 2.5 ,uCi/mg, ICN), 30 AM Cd, 45 AM Cd, 300 or 500AM Zn were filtered, washed and suspended with 10 mL 2.0%(w/v) Cellulysin (Calbiochem Co.), 0.5% pectinase (72 units/ml, Sigma Chemical Co. No. P-5146) which was desalted onSephadex G-25 and made 0.5 M with mannitol. Incubationwas for 4 h at 29°C with gentle shaking. Protoplasts weresedimented, suspended with 0.5 M mannitol, 25 mM Mes/Tris (pH 5.5) and one-fifth was centrifuged and the pellet wasfrozen and set aside for monitoring protoplasts. The remain-der was centrifuged and the pellet gently suspended in lysismedium containing 0.08 M K2HPO4/KH2PO4 (pH 8), 10 mMDTT (28). The suspension was gently stirred at room temper-ature for 10 min to liberate vacuoles and then filtered throughtwo layers of cheesecloth wet with lysis medium. The filteredlysate was made 17% (w/w) with sucrose with gentle mixingand overlayered with 8% (w/w) sucrose, 0.66 M mannitol, 20mM Hepes/Tris (pH 8). After centrifugation for 10 min at5,000g, 20°C, vacuoles floated to the surface of the overlayer.Gradients were fractionated from the bottom and vacuoleswere observed by light microscopy after positive staining withneutral red and exclusion staining with Evan's blue. Protoplastcontamination was estimated to be 5 to 10%. Fractions ofgradients and the aliquot of protoplasts were examined for a-mannosidase using 0.1 M citrate/NaOH, pH 5.0 and 25 mMp-nitrophenyl-a-D-mannopyranoside (Sigma Chemical Co.).After incubation for 30 min at 39°C, the assay was stoppedby addition of borate/NaOH (pH 9.8) (to 0.1 M) and read atA4w. Fractions of gradients and the protoplast aliquot weremade 1% with HCI and analyzed for Cd by atomic absorptionspectroscopy. Contents of '0Cd was determined directly bygamma scintillation spectroscopy.

RESULTS AND DISCUSSION

Occurrence of and Form of Cd in Cultured Cells

A time course of Cd accumulation in tobacco suspensioncells exposed to 45 AM Cd (non-growth-inhibiting) is shown

781



Plant Physiol. Vol. 91, 1989

in Figure 1. We observed soluble metal tcells as quickly as cells could be washemin). Chilling cells and wash media reduoccurrence of soluble metal, and incubaplus trace'09Cd followed by immediateMCdSO4 to remove adsorbed metal indicaindeed penetrate cells rapidly (not showinflux of metal into cells, accumulation10 h then was relatively unchanged. EvidCd-binding peptide was found after abouincreased linearly to account for about:Cd after 36 h. The sum of Cd recovered a,and that identified in the figure as freenbe Cd bound to organic acid in vivo,soluble Cd. When cultures contained 0.1binding peptide was low or absent as exi

soluble Cd and "free metal" profiles w

under these conditions (in the presence

substantial Cd occurred in cells and up

addition, Cd-peptide was not a significantresults were found using 180Mm Cd,tpeptide accounted for about 30% of tota24 h (NR Reese, GJ Wagner, unpublexposed to 300Mm Zn (non-growth-inhresponses; immediate uptake followed bybut no evidence was found for the occurr

peptide and no BSO effect was observedthe above experiments, ligand of Cd-pelas '09Cd binding species eluting in theSephadex G-50 (30,000-1,000 D) and(>300 D). In 45 AM Cd and 300Mm Zn tisoluble (25 mm Tris extracted) to insolitreatment were 0.29 ± 0.05 (N = 4) and Irespectively. After 4 h treatment with 9was 0.54 ± 0.09 (N = 4). With timeculture decreased, suggesting saturationsites (not shown).

Total recovery of Cd and Zn in mediaand insoluble fraction was 90 to 95%. WaKH2PO4 (pH 8); 50 mm Hepes (pH 8); a

a

al)

EL.~

-o0-(). 6 12 18

TIME (hrs)Figure 1. Occurrence of Cd with time in topeptide and free metal forms in tobaccotreatment with 45 jM Cd ±0.1 mm BSO.

;o be associated withZd and harvested (8Iced only slightly the[tion with 45gM Cdvashing with 1.0 mMLted that metal doesvn). After the initialincreased for aboutlence for presence ofit 3 h, and this form33% of total solubleCd-binding peptide

aetal, but thought to

8) were compared as extraction media for metal determina-tions and the last was found to represent the lowest ionicstrength, yet to maintain the pH of the homogenate. In an

earlier experiment, somewhat higher levels of Cd were foundto be associated with the soluble fraction of cells, particularlyup to1O h after exposure (Fig. 1)(31). In that experiment, 25mM K2HPO4/KH2PO4, pH 8 was used as homogenizationmedium. This medium was subsequently found to be inca-pable of maintaining pH 8 in the homogenate and may haveresulted in displacement and solubilization of a portion ofwall or membrane bound Cd.

Role of Organic Acids in Cd and Zn Accumulation

4pproxAima4 tUL-Lai In an effort to determine if endogenous organic acid content

mM BSO, Cd as Cd- of cells was sufficient to complex metals within the vacuole,)ected (18) and total we determined organic acid and metal content and calculated

tere similar. Clearly organic acid to metal stoichemetry in cells exposed to 45gMor absence of BSO) Cd after 0 time (8 min), 2, 4, and 12 h; 600Mm Cd, 4 h; 300

to 12 h after metal uM Zn 0 time (8 min), 2, 4, and 12 h; and 2000AM Zn, 4 h

L component. Similar (TableI). Malate was found to be the predominant organic)ut in this case Cd- acid, representing about 73% of A210 absorbing material elut-

soluble metal after ing from the Aminex-87H column. Citrate accounted for

lished results). Cells about 25%. One additional, very minor, unidentified peak

ibiting) gave similar was observed to elute after citric acid. Therefore, data of Table

(slow accumulation, I are expressed as malate plus citrate. Concentration of soluble

rence of ligand of Cd metal in cells was estimated assuming that 75% of packed cell

(data not shown). In volume represents the soluble portion of cells. With 45 gMptide was monitored and 600Mm Cd treatments, excesses of organic acid over metal

pre-salt fractions of were >700- and 60-fold, respectively. Cells treated with 300

subsequently G-15 and 2000 Mm Zn showed excesses of about 70- and 12-fold,reated cells, ratios of respectively. Clearly, even at very high, growth-inhibiting

ible metal after 4 h levels of Cd and Zn, sufficient organic acid is present (un-

0.45 ± 0.07 (N = 3), doubtedly, largely in the vacuole [14], and see below) to

100 AM Zn the ratio complex intercellular, soluble metal. In a single experiment,

insoluble metal per cells were extracted with 0.25% HCI to allow recovery and

of cell wall binding quantitation of endogenous oxalic acid and its separation

Aminex-87H along with malic and citric acids. Oxalate was,ell washes, soluble found to be a minor acid, its concentration being about one-ter; 25mMK2HPO4/ tenth that of citric. We did not observe metal-dependentmd 25 mM Tris (pH stimulation of malic, citric acid (or oxalic acid, not shown)

content between two and four days of culture (Fig. 2). Malicacid increased over this period at the same rate as did dryweight.Examination of the literature shows that the organic acid

A Cd (v) concentration of tobacco cells studied here is in the same

Cd,0.1mM BSO ) range as that reported in other plant tissues (fresh weightMetal_-- basis). If one attempts to compare from the literature thending Peptidle---ISoluble Cd organic acid content (malate, citrate, and/or oxalate) found

in tissues of plants and cultured cells exposed to high levels- of Zn, one finds in Silene (13) Zn-sensitive and Zn-resistant*~ leaves, 115 and 137 umol/gm fresh wt, respectively (predom-

inantly oxalate); in Nicotiana plumbaginifolia cultured cells(8), Zn-sensitive and insensitive, 2.15 and 22 ,umol/g fresh

___*weight, respectively (predominantly citrate); in Deschampsia2~4 330 36 root (2), sensitive and resistant, 10 and 30 ,mol/g fresh

weight, respectively (predominantly citrate); Deschampsiatal soluble, Cd-binding plants (25), sensitive and resistant, 11 and 23 ,mol/g freshsuspension cells after weight, respectively (predominantly citrate); and N. tabacum

cells (this study), considered sensitive (predominantly malate),

45julJ45mNlFreeCd BiTotal

o..Mt:2

782 KROTZ ET AL.

0




Table I. Organic Acid-to-Metal Stoichiometry in Cd and Zn Treated Tobacco Cultured Cells

Malate + Malate + Citrate/ Estimated MetalTreatment Time Citrate Metal Metals Concn. of Cells PCV

at Harvest"

h pmol AM

Cd (MM)45 0 92.0 0.096 958 16 845 2 84.9 0.108 786 18 845 4 127.6 0.138 925 23 845 4 142.9 0.186 769 31 8

600 4 143.3 2.36 60.6 394.0 845 12 108.4 0.252 430 42 8

Zn (MM)300 0 81.0 1.16 70 206 7.5300 2 87.9 1.17 75 208 7.5300 4 88.1 1.41 63 250 7.5300 4 120.6 1.52 79 271 7.5300 4 91.5 1.96 47 291 9.0

2000 4 135.3 11.41 11.9 2174 7.0300 12 96.0 1.91 50 340 7.5

a Concentration of metal in cells at harvest were estimated assuming that 75% of the packed cellvolume (PCV) represents soluble portion of cells. Metal concentration data was obtained from homog-enates made in 25 mm Tris/HCI (pH 8), while extraction of organic acids utilized 0.27% (v/v) oxalate.

I A Control B 45jiMCdC 3Lqum Lan

uy 24.

20Z5

164,4ep

x 12 fC3[

0~~~~~~~

0~~~~~~~~~~~~~~~~

E

2 3 4 2 3 4 2

Time (days)

Figure 2. Content of malic and citric acids in control, 45 M.M Cd, and

300 mM Zn treated cells. The dry weight change over the log phase

of growth is shown for comparison.

14 ,tmol/g fresh weight. In crop plants, malic and citric acids

are predominant acids in many cases and total organic acid

in leaves is in the range of 2 to 0 gtmol/g fresh weight

(calculated from Clark [1I]). Thus, organic acid levels observed

in this study and compared in this manner, are in the rangeof those found in leaves of crop plants and ecotypes eithersensitive or resistant to high Zn exposure.

Levels of citrate, and Zn-to-citrate ratios, in roots of Des-champsia were reported to increase with time (weeks) of Zntreatment in tolerant but not sensitive plants (2). We usedradiolabeled organic acid precursors to examine the possibilitythat organic acid biosynthesis might be rapidly stimulated bymetal exposure of tobacco cells. To our knowledge, this hasnot been done previously to study possible effects of metals.If cytoplasmic acid is critical for transport of cytoplasmicmetal to the vacuole as suggested in the early model of Ernst

( 13, 32), one might expect biosynthesis of organic acids to bestimulated by occurrence of high levels of soluble metal incells. On the other hand, if organic acid is sufficient tocomplex even high levels of intracellular metal (as foundhere), and organic acid used for transport is recycled (or someother mechanism is used for metal transport to the vacuole),then one would not expect to see stimulation of biosynthesis.We note that it is unlikely the Cd-peptide serves to shuttle Cdto the vacuole during the initial stages of accumulation (upto about 2 h) because it is either absent in this interval, or itis present at very low levels. Since it was necessary to purifythe anionic components of cells prior to HPLC, we separatedand examined cationic, neutral and anionic fractions of [1-'4C]acetate-labeled, control, and metal-treated cells and ex-amined all three fractions as well as acids subsequentiallypurified by HPLC and oxalate recovered as calcium oxalate.Label in cationic and neutral fractions was low and unchangedby metal addition from 0 to 12 h while that in anionicfractions increased from 0 to 4 h, then remained unchanged(10). Incorporation ofradiolabel from ['4C]acetate into malateand citrate was similar to control, 45 uM Cd, and 300 jAM Zntreated cells (Fig. 3). Similarly, no evidence was found forstimulation of organic acid biosynthesis using 600 ,uM Cd and2000 uM Zn (not shown). Label recovered as calcium oxalatewas about one half that recovered as citrate and was alsosimilar in controls and metal treated cells (not shown). Theminor peak of endogenous acid (retention time 8 min, Fig. 3)observed and noted earlier was not labeled. When [1-'4C]ascorbate (precursor of oxalate in plants) (28) was used as asource of label, results similar to those obtained using acetatewere found. These results show that, in the short term (up to12 h), non-growth-inhibiting and growth-inhibiting levels ofthese metals do not stimulate biosynthesis of predominantorganic acids.

783




A

B

C

D

.W V .- i=-:t:!m 0~~4

u7-4

Control [2.4

k!

45)jM Cd

3C

ALM

OO,uM Zn

8 16.8 13 18.5Retention Time (minutes)

C 170/o Sucrosem

xE

a

'.

'a_VU2.1 |

-1.4 °

-0.7 <

2A a)

2.1 11.4

0.7 0

,oXE

2.4 a-2.1 U

1.4

2

.2

.1

a-

zw

z0U

4

w

0.7

Figure 3. Separation of anionic fractions of cells after radiolabelingwith [1-14C]acetate. A, Acid standards; B, control; C, 45 gM Cdtreated; and D, 300 Mm Zn treated.

Vacuole/Extravacuole Distribution of Cd and Zn inCultured Cells

Assuming that the bulk of malic and citric acid in tobaccocells is in the vacuole (14, 22), the bulk ofaccumulated metalwould also be expected to be vacuolar if acid complexationand vacuolar sequestration is a mechanism for metal accom-modation in the presence or absence of ligand of Cd-peptide.Because we were unable to prepare satisfactory vacuoles fromtobacco suspension cells used in this study, even after applyingthe four basic methods for vacuole isolation (30), we estimatedthe vacuolar-extravacuolar distribution of Cd and Zn in Da-tura cultured cells treated with 0.35, 30, or 45 ,M Cd or with300 or 300 ,uM Zn for 3 to 4 d. As shown in Figure 4, evidencewas found for vacuolar Cd in all treatments except 0.35 ,uMCd. Enrichment in a-mannosidase at the top of vacuoleflotation gradients was in all cases coincident with the occur-rence of purified vacuoles. In control gradients where metalswere added to lysis buffer, metal did not move into flotationzones (not shown). Yields of vacuoles (10-15%) were deter-mined by comparing a-mannosidase recovered in vacuoleswith that in a protoplast aliquot-assuming 100% of protoplasta-mannosidase to be vacuolar (30). Contamination ofvacuolepreparations with residual protoplasts in experiments reportedwas 5 to 10% oftotal structures present. Vacuole/extravacuoledistribution of metals was computed by comparing in eachindividual experiment protoplast and vacuole metal and a-

mannosidase content after factoring vacuole yield and proto-

80/o Sucrose0.66M Mannitol

5 15 25

B30pM Cd (4d)

-O~~~~~~~~.>-

FRACTION NUMBER

.3

1 tu

04-

4)

2 {

w

*1

._

0z

z

4

-2 ain

Figure 4. Profiles of metal and a-mannosidase in gradients used topurify Datura cultured cell vacuoles. Exposures were: A, 0.35 Mm Cd,4 d; B, 30 MM Cd, 4 d; C, 45 uM Cd, 3 d, and 300 ,M Zn, 3 d.Schematic at the top of figure describes occurrence of purifiedvacuoles in gradients.

plast contamination of vacuole preparations. With the excep-

tion of the low Cd treatment case (0.35 ,uM Cd), 86 to 100%of metals in protoplasts was determined to be localized in thevacuole.

Recently, we have prepared highly pure vacuoles fromleaves of 20 uM Cd-treated tobacco seedlings and concludethat most of the Cd is contained in the vacuole (R Vogeli-Lange, GJ Wagner, unpublished results). Experiments are

underway to determine if metals rapidly accumulate in vac-uoles of protoplasts as would be expected from data presentedhere.

Potential of Vacuolar Organic Acids in MetalSequestration

Vacuolar organic acids are thought to occur as complexes,primarily with H, K, Ca, and Mg and to be principallyresponsible for charge balance in mature plant cells (14). Wemonitored the content of these ions, nitrate, chloride, phos-phate and sulfate in tobacco suspension cells exposed tovarious concentrations of Cd and Zn for 4 h to determine ifmetals influence the ion contents of cells and to estimate ionbalance. Also, pH was monitored in these cell extracts madein freshly boiled milli-Q water (ground in mortar and pestle)

0.35jiM 1ceCd (4d

I'

icir-

-

9 _1

19, A. .#, iv Ia o1r'o a d U

l

784 KROTZ ET AL.




Table II. Ion Contents of Tobacco Suspension Cells after 4 h Treatments'

Treatment K+ Mg2+ Ca2+ N03- Cl- Cd2+ Zn2, Total meqb Total meqcCation Anion

mM

No metal 54 ± 18 1.8 ± 0.4 1.0 ± 0.2 2.9 ± 1.5 7.3 ± 2.0 60 63Cd (AM)

45 45 ± 3 1.6 ± 0.4 1.0 ± 0.1 2.7 ± 0.9 7.8 ± 1.9 0.027 0.05d 50 64600 52 ± 11 1.7 ± 0.6 0.9 ± 0.2 3.3 ± 1.4 7.9 ± 1.3 0.398 0.05d 58 65

Zn (NiM)300 44 ± 6 1.4 ± 0.3 0.9 ± 0.1 2.5 ± 1.0 8.4 ± 1.1 0.27 50 53

2000 40 ± 4 1.0 ± 0.2 0.4 ± 0.1 2.1 ± 0.7 7.3 ± 1.2 2.17 46 64

aData for K+, Mg2+, Ca2 , Mg2+ NO3-, Cl-, So42-, P04 and pH were from homogenates preparedin degassed, deionized water, that for Cd2+ and Zn2+ were from homogenates made in 25 mm Tris-HCI(pH 8) and for quantitation of organic acid contents, 0.2% (v/v) oxalic acid was utilized. The pH ofaqueous extracts of all treatments was 5.0 indicating 0.01 mm HW. b Total meq cation equals sumof ions represented. C Total meq anion calculated using mean of values from Table I for 4 htreatments (2 meq/mmol malic, 3 meq/mmol citric) plus NO3-, Cl-, S04-, P04-. Phosphate concentrationwas about 1.1 mm, sulfate was about 0.1 mm, and both were similar for all treatments (data notshown). d From homogenates in 0.2% oxalic acid.

Table Ill. Comparison of Conditional Log Stability Constants of Selected Organic and Amino Acids"Ligand H+ Ca2+ MgC+ cu2+ Zn2+ cd2+

Malate 3.26 (pK1) 1.80 1.55 3.4 2.8 2.8 (estimated)4.68 (pK2)

Citrate 3.08(pK,) 3.20 3.20 5.2 4.85 3.924.39 (pK2)5.49 (pK3)

Oxalate 1.14 (pK1) 3.0 2.55 8.29 (pK,, pK2) 4.68 3.523.85 (pK2) 7.04 (pK,, pK2) 5.29 (pK,, pK2)

Glyceric 3.52 (pK,) 1.18 0.86 1.80

Glycolic 3.71 (pK,) 1.11 0.92 1.92Tartaric 2.90 (pK,) 1.80 1.36 2.68

4.01 (pK2)Glycine 9.78 (pK2) 1.43 3.44 8.62 5.52Aspartic 3.90 (pK2) 1.60 2.43 8.40 5.60aValues from Martell and Calvin (12) and Sillen and Martell (23) determined in 0.15 ionic strength

media at 25°C using a variety of methods.

to estimate if vacuole pH is influenced by metal treatment.As shown in Table II (results of 3 experiments), K+ was theprincipal cation. Whether or not its concentration is reducedby Zn2+ treatment requires further study. The cell aqueousextract pH was 5.0 regardless of metal treatments. Chargebalance was calculated as 95, 78, 89, 94, and 72%, totalcation-to-total anion, in control, 45 jM Cd, 600 ,uM Cd, 300AM Zn, and 2000 ,M Zn treated cells, respectively. Using thepK's of malate and citrate listed in Table III, calculation ofthe distribution of acid species indicates that 270% of malicand citric acids would exist as acid-2 species at a vacuole pHof 5.0. While data of Table II are somewhat preliminary andperhaps could be improved by more experimentation to refinedetermination of organic acid concentrations, they point outthe abundance of organic anion and inorganic cation contentrelative to metal concentration in treated cells and show thelack of influence of Cd and Zn on sap pH.

Literature values of stability constants oforganic acid-metalcomplexes versus those of complexes with Mg and Ca were

compared to predict the ability of low levels of metal todisplace relatively higher levels of endogenous Mg and Ca.Potassium, which forms weak outer shell complexes, wouldeasily be displaced by divalent metals. Values shown in TableIII predict that Cd and Zn could effectively compete with Caor Mg as well as K for malate and citrate ligands. Proteincontent of extracts were determined to estimate the complex-ation potential of vacuolar protein for vacuolar organic acid.Protein was found to account for about 0.8% of dry wt andto be about 0.4 AiM in vacuoles-assuming a mean proteinsize of 100,000 D, that vacuoles constitute 70% of PCV, andthat 10% of cellular protein is in the vacuole. This lowvacuolar protein level would not significantly compete withCd and Zn for vacuolar organic acid despite the affinity ofamino acid for metals (Table III) (12, 23). We also note thatsimilar protein levels were observed in cells between 0 and 10h after treatment with 45 lM Cd and 300 Mm Zn.We conclude from data presented regarding the large excess

of organic acid, vacuolar compartmentation of Cd and Zn

785




TIME (hrs)

Figure 5. Occurrence of Cd with time in soluble and insoluble frac-tions of cultured cells exposed to 250 Mm Cd in presence and absenceof 0.2 mm BSO which inhibits formation of Cd-peptide :85%.

after moderate to high level metal exposure, and from abovearguments based on stability constants, that in the absenceand presence of ligand ofCd peptide, tobacco suspension cellshave the capacity to accommodate and accumulate Cd andZn by sequestration as vacuolar, organic acid metalcomplexes.

Cd-Peptide Is Not Required for Cd and Zn Accumulationin Culture Cells

We were interested to determine if the absence of Cd-peptide would affect the capacity of the cell to accumulateCd. Cells were exposed to 250 Mm Cd in the presence andabsence of 0.2 mM BSO which was shown to inhibit synthesisby >85% (not shown) (18). As shown in Figure 5, solublemetal in the presence of BSO was about 83% of that in theabsence of BSO. A similar lack of a BSO effect on Cdaccumulation was found by Huang et al. (6) for tomato cells.Interestingly, Cd associated with the insoluble fraction of cellswas higher in the presence of BSO so that total Cd associatedwith cells was similar to that in the absence of inhibitor (Fig.5). These results suggest that the capacity of cells to accumu-

late this metal is largely independent of the occurrence ofpeptide. Cells grew slowly in the presence of this high level ofCd but did continue to accumulate metals. Since sustainedtobacco cell culture growth in the presence of >22 Mm Cd iscorrelated with the occurrence of Cd-peptide and the absenceof "free metal" (18) but peptide does not greatly influenceaccumulation (Fig. 5), we may conclude that in this system,Cd-peptide serves to protect some Cd-sensitive site and notto sequester total metal. Cd-peptide formation may be an

adaptation to this end or perhaps represent the result ofperturbed glutathione metabolism after high level Cdexposure.

Results presented are consistent with a model where Cd or

Zn accumulation from solution containing high levels ofmetal (>45 gM Cd, >300 Mm Zn) is largely into the vacuole,perhaps complexed with endogenous organic acids. Peptide,once formed, may afford protection from toxicity in extra-vacuolar compartments.

ACKNOWLEDGMENTS

We thank R. Yeargan for assistance with vacuole isolation experi-ments, and Iris Deaton for help in preparation of the manuscript.

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