Plant Physiol. (1971) 48, 273-277

Translocation of Nickel in Xylem Exudate of PlantsReceived for publication February 19, 1971

LEE 0. TIFFINUnited States Department of Agriculture, Agricultural Research Service, Soil and Water Conservation, UnitedStates Soils Laboratory, Plant Industry Station, Beltsville, Maryland 20705

ABSTRACT

Topped plants of tomato (Lycopersicon esculentum), cu-cumber (Cucumis sativus), corn (Zea mays), carrot (Daucuscarota), and peanut (Arachis hypogaea) were treated with 0.5 to50 micromolar Ni (containing 0'Ni) in nutrient solutions. Xylemexudate was collected for 10 hours or, in the case of corn, for20 hours at 5-hour intervals. Electrophoresis of nutrient solu-tion distributed all Ni cathodically as inorganic Ni2+. Low concen-trations of Ni in tomato exudate migrated anodically, presum-ably bound to organic anion (carrier). However, this carrierbecame saturated at about 2 micromolar Ni in exudate, and ex-cess Ni ran cathodically. Most of the Ni in cucumber, corn, car-rot, and peanut exudate ran anodically, and its migration ratewas identical for all exudates. Peanut root sap contained 14 to735 micromolar Ni. The anodic Ni carriers in root sap and exu-date appear identical. The carrier in root sap became saturatednear 100 micromolar Ni, as shown by cathodic streaking ofNi exceeding that concentration. It appears that all five spe-cies translocate low concentrations of Ni in the same anionicform.

Plants readily take up Ni, and small amounts are found inmost plants. Haselhoff (4) in 1893 demonstrated Ni toxicity incorn and beans grown in nutrient solution. Since then, workershave shown Ni uptake in many species grown in solution cul-ture and soils (see Vanselow [11] and references cited therein).The present study is concerned with quantities of Ni ab-

sorbed by plant roots and translocated in xylem exudate and,in particular, with electrophoretic forms of Ni in the exudate.

MATERIALS AND METHODS

Plant Culture and Harvest. Experimental plants, representingfive families, were: tomato, Lycopersicon esculentum Mill., var.Marglobe; cucumber, Cucumis sativus L., var. Burpee's Sunny-brook; corn, Zea mays L., WF9tms X 38-1 1; carrot, Daucuscarota L., var. Gold Pac; and peanut, Arachis hypogaea L., var.Florigiant.Four of the above species (tomato excluded) were grown in

standard nutrient solutions replaced every 4 days in earlygrowth and every 2 days for the last 6 days before harvest. Ele-ment concentrations (bLM) in the nutrient solution were: 500 Ca,100 Mg, 400 K, 1700 N, 20 P, 60 S, 3 B, 1.2 Mn, 0.25 Zn,0.10 Cu, 0.05 Mo, and 1 Fe (as FeEDDHA,1 except for corn

1Abbreviations: FeEDDHA: ferric ethylenediamine di(o-hy-droxyphenylacetate); FeHEDTA: ferric hydroxyethylethylenedi-aminetriacetate.

and carrot which received 10 /.M FeHEDTA). In the exudationperiod, plants received: 1 ,uM FeEDDHA, half-strength mac-ronutrients, and full-strength micronutrients (B, Mn, Zn, Cu,Mo) as shown above. A solution of NiCl2 was used for Nitreatments. Details of treatments are given in the tables.Tomato plants were grown according to a previous schedule

(9). The element concentrations in the nutrient solution wereapproximately double those given above.The number of plants grown in 8 liters of nutrient solution

were: four tomato, four corn, six cucumber, eight carrot, andeight peanut. Plant ages (days) at harvest were: tomato, 27;corn, 22; cucumber, 26; carrot, 30; and peanut, 26. Cucumber,tomato, and peanut stems were cut at the cotyledonary nodefor exudate collection. Corn plants were cut at the base of thestalk, leaving about a 2 mm thickness of the base attached tothe roots. The carrot root (fleshy part) was cut near the bottom,so that about 2 cm of it remained attached to the lower rootsystem. Exudates from cut surfaces were delivered by plastictubing into test tubes held in ice packs (7).

After peanut root systems had produced exudate for 10 hr,they were rinsed with water and frozen. The roots were thawedand root sap was pressed out in a Carver press. The sap wasfiltered (0.45 ,u Millipore) and used for radiometric and electro-phoretic analysis.

Electrophoresis Apparatus. Two types of apparatus were em-ployed. One was used for horizontal separations (7); anotherwas constructed for continuous electrophoresis. The latter issimilar to a commercial model (1) but accommodates 58 X 58cm Schleicher and Schuell No. 470-C filter paper. Heavy wicksof the same paper (2 x 5 cm in cross section) were used at thesides to improve current distribution. A syringe pump delivered2 ml of exudate or root sap per hour at the origin.

Isotope and Assays. The isotope, 'Ni (3.4 mc/mg Ni), wasused throughout. Fifty microcuries of 0Ni and unlabeled NiCI2were added to nutrient solutions to give treatments of 0.5 to 50tM Ni. This provided specific activities of 100 /c/ymole in thelow Ni treatment and 1 ,uc/fimole in the high treatment. Vol-umes of 0.1 ml of nutrient solution and stem exudate or 0.01ml of root sap were diluted with water and dried in planchetsfor assay in a proportional counter. Nickel absorption was cal-culated from changes in isotope concentration in the nutrientsolution.

RESULTS

Tab!e I (experiments 1 and 2) shows uptake and transloca-tion of Ni by tomato. The Ni concentrations in exudate weresimilar for comparable treatments of the two experiments. Theroots released into the exudate 2 to 6% of the Ni they absorbed(NiR/NiA).

Figure 1 shows electrophoretic patterns of the four nutrientsolutions and corresponding stem exudates for experiment 1(Table I). The uniform darkness of the radiographs for nutrientsolutions (paths 1 to 4) results necessLrily from the constant

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Plant Physiol. Vol. 48, 1971

Table I. Uptake and Translocation of Nickel in Tomatoand Cucumber

Two tomato plants or three cucumber plants were placed ineach liter of nutrient solution containing a Ni concentration asshown and 50,c 63Ni. Plants were topped at the start of treatment,and exudate was collected for 10 hr.

Experiment andDatum No.

Experiment 1

(tomato)1234

Experiment 2(tomato)

123

Experiment 3(cucumber)

123

Ni in Total NiNutrient AbsorbedSolution NiA

M

0.55.0

25.050.0

0.55.0

50.0

0.55.050.0

Mmoles

0.43.916.129.6

0.42.313.3

0.474.232.9

Exudate Ni inVolume Exudate

ml m

26271914

242312

26189

0.55.6

27.949.3

1.05.6

55.5

2.413.110.1

level of isotope (despite the decreasing specific activity) in thesesolutions. The radiograph shows all Ni running as inorganiccation toward the cathode. The paths for exudate, however,display a very different pattern. They show cationic Ni, butalso reveal significant quantities of the metal in anionic form.This is particularly noticeable for exudate 1 in which the spe-cific activity was 100 4tc/ 1tmole. Presumably Ni is chelatedby organic anion (carrier). The binding capacity of the carriercannot be deduced precisely from radiographs, but it is obviousthat the agent saturates at low concentrations of Ni.

Figure 2 gives quantitative electrophoretic distributions ofNi in tomato exudates. Exudate 1 of experiment 2 (Table I)contained 1 /zM Ni. The distribution in Figure 2 shows 85%of the Ni in anodic fractions 15 and 16. The remaining 15%ran cathodically. Distributions for exudate 2 were 35 and 65%and for exudate 3 were 5 and 95%. These distributions reflecta limited binding capacity (<3 jtM Ni) for the anodic carrier intomato exudate.Data for cucumber are shown in Table I, experiment 3. Cu-

cumber exudate 1 had a higher Ni concentration than tomatoexudates from comparable treatments. The opposite occurredon the 50 /tM treatment. Cucumber roots on this treatment ab-sorbed more Ni than tomato roots but released less of the metal(Nip) in the exudate. The 5 and 50 JlM Ni depressed exudateproduction more in cucumber than in tomato. Most of the Niin cucumber exudate migrated as a negatively charged com-pound (Fig. 3).

Table II gives data for Ni uptake and transport by corn,peanut, and carrot. The Ni level in corn exudate (experiment4) declined after the peak at hour 5. This apparently resultedfrom limited Ni supply, for a decline was not evident in ex-periment 5. Figure 4 shows electrophoretic patterns for corn,peanut, and carrot exudates. The six samples were run at thesame time on a single paper. The radiograph shows that mostof the Ni in the six exudates was in a negatively charged form.The migration rate of Ni appears identical for all samples.Radiographs for corn exudates of experiment 5 (not shown)

FIG. 1. Radiograph of electrophoretically distributed 63Ni in nu-trient solutions and tomato xylem exudates. Paths 1 to 4 (top) showinorganic Ni patterns for the four nutrient solutions (0.5, 5.0, 25,and 50 uM Ni treatments), experiment 1, Table I. Paths 1 to 4(bottom) show Ni patterns for the corresponding exudates. Electro-phoretic conditions: Whatman No. 3MM paper, 20 ,ul/spot, 20 mMsodium acetate at pH 5.4, 360 v, 40 min, room temp. X-ray filmexposure was 40 days.

>\}6001

r) 400-'(D -

1-2U 90

Ni treatments:0.5}iM5.0}iM

--- 50.0,}M

5 7 9 11 13 15 17 19

Effluent

FIG. 2. Continuous electrophoretic distribution of nickel in to-mato xylem exudate. The curves show effluent Ni distributions forthree exudates obtained on the 0.5, 5.0, and 50 ,uM Ni treatments,respectively, of experiment 2, Table I. Electrophoretic conditions:Schleicher and Schuell No. 470-C paper, 20 mm sodium acetate atpH 5.4, 800 v, 4 + 1C.

(i)'

1Total NiReleasedNiR

ismoles

0.0130.1510.5300.690

0.0240.1290.666

0.0620.2360.091

2

3

4

2 io A_v ........._ u

NiR/NiA

3.33.93.32.3

6.05.45.0

13.25.50.3

274 TIFFIN

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TRANSLOCATION OF NICKEL IN PLANTS

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1

2

3 I

FIG. 3. Electrophoretic distribution of nickel in cucumber xylem exudate. Paths 1, 2, 3 correspond to exudates 1, 2, 3 from 0.5, 5.0, and 50

,M Ni treatments, experiment 3, Table I. Electrophoretic conditions: Whatman No. 3MM paper, 50 Al/spot, 20 mm sodium acetate, pH 5.4, 500v, 1 hr, room temp. X-ray film exposure was 32 days.

Table II. Uptake and Translocation of Nickel in Cornz, Peanut,and Carrot

Two corn, four peanut, or four carrot plants were placed ineach liter of nutrient solution containing a Ni concentration asshown and 50uc 63Ni. Plants were topped and exudate was col-lected for 10 hr from peanut and carrot and for the designated timeintervals for corn.

Experiment and Datum No. Nutrient STimpe AbNibed Exolume ixudat

FM hr moles !ml|l MM

Experiment 4 (corn) 2.001 5 1.29 15.1 0.842 10 0.49 9.3 .683 15 0.10 5.7 .264 20 0.02 4.6 .14

Experiment S (corn) 20.001 5 6.01 12.3 11.972 10 2.88 7.1 13.733 15 1.51 5.1 11.184 20 1.68 3.9; 11.72

Experiment 6 (peanut)1 1.00 10 0.97 12.5 1.82 10.00 10 9.19 9.6 5.2

Experiment 7 (carrot) 20.00 10 11.01 5.9 25.2

also revealed anodic Ni, but significant amounts of the metalalso migrated toward the cathode, indicating that carrier wasbeing saturated at Ni concentrations in these exudates (11 to14 ,tM). Corn exudates collected at 20 hr (Table II) were tur-bid, and therefore not analyzed electrophoretically.

Table III shows uptake and translocation of Ni by peanutand concentrations of Ni in root sap. Figure 5 shows electro-phoretic patterns of exudates and root sap. Patterns for ex-udate indicate that essentially all Ni is bound to anodic carrierwhich holds an equivalent of up to 6 ,uM Ni. However, if Ni onpaths 4 and 5 is held by the same carrier present in exudate,then concentration of carrier is considerably higher in root sapthan it is in exudate. Root sap 1 contained 14 ,uM Ni; the dark

area on path 4 shows that most of the Ni migrated anodically.Root sap 2 had 102 1m Ni; it is obvious that most of the metalwas anodic. The slight streaking toward the cathode, however,indicates saturation of the carrier. The excess of Ni over car-rier is clearly shown on path 6. Relatively little of the total Ni(735 /tM) in the sap migrated anodically. The radiograph showsconsiderable streaking toward the cathode and a concentrationof Ni remaining at the origin.

Considerable quantities of Ni absorbed by the root systemsremained soluble and thus could be recovered in the root sap.Calculations from the volume and Ni concentration data forsaps 1, 2, and 3 (Table III) indicate a recovery of 0.27, 2.14,and 16.04 ,umoles of Ni, respectively. Comparing these val-ues with the quantities of Ni absorbed indicates that 52 to 84%of the absorbed Ni was recovered in the root sap.

DISCUSSIONConcepts of Metal Translocation in the Xylem. There is gen-

eral agreement that major cations are essentially free of organicassociates in translocation. On the other hand, stability con-stants of microelement chelates of organic and amino acids (2,6) suggest possible association in transport. Such an associationseems implicit in results of the present study, although theagent responsible for Ni transport is unknown.

Association between Fe and citrate in exudates of sunflower

Table III. Uptake and Translocation ofNickel by Peanut and Levelsof Nickel in Expressed Root Sap

Four plants were placed in each liter of nutrient solution con-taining a Ni concentration as shown and 50,c 63Ni. Plants weretopped and exudate was collected for 10 hr. Roots were rinsed,frozen, and thawed, and the sap was expressed.

Sample Ni Ni Exudate Ni in Root Sap Ni inNo. Treatment Absorbed Volume Exudate Volume Root Sap

AMM pmoles ml JM ml AMM1 0.5 0.46 6.7 1.9 18.5 142 5.0 I 4.22 12.5 6.0 21.0 1023 50.0 19.21 4.7 6.7 21.8 735

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276

(-)

Plant Physiol. Vol. 48, 1971

H-)

1

2

3

4

5

6

FIG. 4. Electrophoretic distribution of Ni in corn, peanut, and carrot exudate. The exudates are characterized in Table II. Corn exudates 12, 3 (experiment 4) are shown on paths 1, 2, 3. Peanut exudates 1 and 2 (experiment 6) are shown on paths 4 and 5. Carrot exudate (experiment7) is shown on path 6. Exudate volumes were 50 ,ul. X-ray film exposure was 90 days. Electrophoretic conditions were as in Figure 3.

(+)

FIG. 5. Radiograph of 63Ni after electrophoresis of peanut exudate and root sap. Paths 1, 2, 3 are for exudates from 0.5, 5.0, and 50AM Nitreatments (Table III). Paths 4, 5, 6 show patterns for corresponding root saps. Test volumes were 50 IAl. X-ray film exposure was 40 days.Electrophoretic conditions are given in Figure 3.

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TRANSLOCATION OF NICKEL IN PLANTS

(7) and soybean (10) has been demonstrated. Tomato and cu-cumber exudates also gave electrophoretic concentrations of Fein the position of Fe-citrate (8). The metals Mn, Co, and Znin tomato exudate appear from electrophoretic tests (9) to belargely dissociated, probably existing mostly as hydrated cat-ions. Calculations based on stability constants and metal dis-placement constants (9) suggest that Ca (>33 mM) and Mg(> 1.5 mM) in tomato exudates are among the factors that pre-vent effective chelation of Mn, Co, and Zn with citrate. Thecitrate binding and displacement equilibria were used as mod-els. The models do not tell how the metals were bound but sug-gest (from consideration of a few limiting factors) why theywere not bound to citrate. Other components in stem exudatessuch as chelating agents, metals, and H+ must also be consid-ered in any final statements about metal chelate equilibria inthese systems.

Nickel in Field-grown Plants. Vanselow (11) has tabulatedthe Ni content of field-grown crops and several species of nat-ural vegetation. A content of 0.05 to 5 feg/g dry matter brack-ets the range of Ni in most plant material. Tissues of a fewspecies have higher concentrations. Various grasses, includingfield-grown oats and wheat, contained 4 to 134 jug Ni/g drymaterial. The plant Alyssum bertolonii Desv. contained inleaves 4000 and in seeds 2500 jug Ni/g dry tissue (5). Gambi(3) has shown that large quantities of Ni in Alyssum bertolonjiDesv. accumulate in the epidermis of stems and in scleren-chyma tissue between vascular bundles. Apparently there areno published data concerning the form of Ni translocated inthese plants.

Translocation of Nickel in Xylem Exudates. Although Nicarriers have not been identified, the present study gives somegeneral properties of the Ni being translocated. It is evidentthat Ni in tomato exudate can be in at least two forms, therelative quantities of each depending on total Ni translocated.But at physiological levels of Ni (<3 uM) in the xylem it istranslocated as a negatively charged molecule.

Carrot, cucumber, and peanut apparently contain more Nicarrier in exudate than was observed for tomato. For example,the radiograph for carrot exudate (path 6, Figure 4) showsmost of the Ni as the anodic compound. The Ni equilibrium incorn exudate is probably more like that in tomato. At concen-trations of 1 t1M, the Ni migrated anodically (Figure 4). But

with 11 to 14 uM Ni (experiment 2, Table II) in the exudate,the radiographs after electrophoresis (not shown) revealed con-siderable Ni migrating cathodically.Compared to the electrophoretic behavior of Mn, Co, and

Zn in tomato exudate (9), the anodic Ni complex appears quitestable. The stability constant of the complex is unknown, butthe property that allowed it to be separated electrophoreticallyintact apparently is its limited (slow) exchangeability. In thesodium acetate system (pH 5.4), the components of an unstablecomplex (e.g., Mn-citrate) separate and migrate electrophoreti-cally toward opposite electrodes. Electrophoresis of the morestable ferric malate in the same system results in streaking ofthe metal from the origin to the anodic position of the metalchelate. Strong association of chelate components, however,gives compact spots such as those shown for Ni on most of theradiographs.

Acknowledgment-I have appreciated discussions of various aspects of metalchelate chemistry with Dr. R. L. Chaney.

LITERATURE CITED

1. BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG. 1958. A Manual of PaperChromatography and Paper Electrophoresis, Ed. 2. Academic Press, NewYork.

2. CHABEREK, S. AND A. E. MARTELL. 1959. Organic Sequestering Agents. JohnWiley & Sons. New York.

3. GAMBI, 0. V. 1967. Prima dati sulla localizzazione istologica del nichel inAlyssum bertolonii Desv. Giorn. Bot. Ital. 101: 5-60.

4. HASELHOFF, E. 1893. Versuche uber die schiidliche Wirkung von nickelhaltigemWasser auf Pflanzen. Landw. Jahrb. 22: 862-867.

5. 'MIN-GUZZI, C. AN-D 0. VERGNAN-O. 1948. Il contenuto di nichel nelle conceri diAlyssum bertolonii Desv. Considerazioni botaniche e geochimiche. Atti.Soc. Tosc. Sc. Nat., Mem. A., 55: 49-77.

6. SILLEN-, L. G. AND A. E. MARTELL. 1964. Stability Constants of Metal-ionComplexes, Ed. 2. Special Publication No. 17. The Chemical Society,London.

7. TIFFIN, L. 0. 1966. Iron translocation. I. Plant culture, exudate sampling,iron-citrate analysis. Plant Physiol. 41: 510-514.

8. TIFFIN, L. 0. 1966. Iron translocation. II. Citrate/iron ratios in plant stemexudates. Plant Physiol. 41: 515-518.

9. TIFFIN, L. 0. 1967. Translocation of manganese, iron, cobalt, and zinc intomato. Plant Physiol. 42: 1427-1432.

10. TIFFIN, L. 0. 1970. Translocation of iron citrate and phosphorus in xylemexudate of soybean. Plant Physiol. 45: 280-283.

11. VANSELOW, A. P. 1966. Nickel. In: H. D. Chapman, ed., Diagnostic Criteriafor Plants and Soils. University of California, Division of AgriculturalSciences. pp. 302-309.

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