Plant Physiol. (1967) 42, 1427-1432

Trranslocation of Manganese, Iron, Cobalt, and Zinc in TomatoLee 0. Tiffin

United States Department of Agriculture, ARS, SWC, Mineral Nutrition Laboratory,Beltsville, Maryland 20705

Received Juine 22, 1967.

Summary. Tomato plants in solution ctulture vere treated with 0 to 50 jM Mn, Co,or Zn in the presence of 5 /LM Fe. Stem exudates were analyzed to determine quantitiesand forms of the metals translocated.

Mn had no effect on exudate volume. Co and Zn at 5 ,u. and above depressedexudate volumes. Nutrient Mn was concentrated 2 to 5 times in the exudates. Co andZn concentrations were between 1 and 3.

Tomato roots treated with 0 to 10 juM Mn released into the exudate an average of29 % of the Fe they absorbed. They released 21 % of the absorbed Fe when treatedwith 50 JuM Mn. On 50 FM Co or Zn, the roots released only about 1 % of the ab-sorbed Fe.

The exudates contained 12 to 47 ,a citrate, which was usually considerably in excessof Fe. Electrophoresis of exudate revealed Fe as the only metal in anionic form. Mn,Co, and Zn migrated as cations.

The concentration of Ca was >3 m\i and MIg >1.5 mm in the exudates. Estimatesbased on, metal-citrate equilibrium constants and constants of metal displacement causedby Ca and Mg confirmed that Mn, Co, and Zn wvere transported predominantly as inorganiccations in the stem exudates.

Most of the metal carr.ed upward in plants con-sists of inorganic cations (1, 9). Because Na, K,Ca, and Mg conmprise the bulk of transported metal,this is a reasonajble view. It is not true, however,that all metals are con,ducted in the xylem as cations.One exception presently known is Fe-citrate, theanion translocated in sunflower (10). Little informa-tion on the translocation of Mn, Co, and Zn is nowavailable. But the stability of these metals in organiccomplexes (5, 8) suggests an association and a pos-sible means of their translocation in plants.

Several investigators have studied the forimls ofmlicroelem-ents in plant stem exudates. Schmid aindGerloff (7) showed that Fe in tobacco exudate xvasnot a free ion, but was comibined with an unidentifiedorganic molecule. Work in this Laboratory (10)indicated that Fe in sunflower exudlate wNas conmplexedNvith citric acid. Exudate from cucumber and tomato(11) also contained Fe in aniolnic form, presunabIlyFe-citrate. Lingle, et al. (6) demonistrated by elec-trophor-esis that AMni and ZIn were catiolns in sovybeanexudate.

Citric acid strongly binds Fe, anid is usually inilmolar excess of Fe in stem exudates (11). Citratealso binds other metals (5, 8), but they are not helIas tiglhtly as Fe. The citric acid in excess of Fe,therefore, couldl serve to tranisport other metals inplants.

The exiperiments with tomato reported here focuson the quantities of AMn, Fe, Co, anid Zn absorbed

and translocated, and on the formsn of the metalstranslocated.

Materials and Methods

'rhe experimental plant was tomato, Lycopersiconcsciulentumnl Mill., var. Marglobe. General cuilturalantd sampling procedures have been reported (10).The age-treatment schedule was as folilows. Seedswere germinated 5 days, then wvere transferred tonutrient so'utions au(I growil under shading to elongatethe stems. At 9 days the seedl'ings were grouped inpairs and transferred into full light, 4 plants in each8 liters of nutrieint. At 21 days the nutrient solutionwas renewed. At 27 days the plants were placed inthe ex-perimental nutrient solutions and decapitatednear the first leaf node. Stem exudates were col-lected for 10 hours.

.Vuttriewt Soluttions. Plantsw;ere grown withstandard major e'ements (10), but xvitlh changes inthe minor elemnlents (noted below- ) and NaCl toprovide 70 uM chloride. The microelemenit stocksolution contained the following clhemicals per liter:2.47 g Mn,Cl2*4H.O, 1.86 g H3BO3, 0.34 g ZnCI2,0.1/7 g CuCl122H.,O, and 0.12 g Na2MoO4*2H. 0.Addition of 0.2 mli of this solution per liter of nutrientgave (OsO): 2.5 MIn, 6 B, 0.5 Zn, 0.2 Cu, and 0.1 Mo.

To provide control levels of Mln and Zn in theexperimental nutrients used in the exudation period,microelement stock solutions (above') were prepared

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PLANT PHYSIOLOGY

without Mn or Zn salt. Separate solutions (10 mM)were made up with MnCL9A4H20, ZnCl2, andCoCl2*6H,0. These were used to vary th- metalsas shown in the tables. Where metal-ch'oride treat-ments were low, NaCl was added to the nutrients tobring chloride to a,pproximately 100 ,M. In 1 case(100 ,M Mn treatment, table II) the chl'oride wasdouble the usual amount. All nutrient solutions wereadjusted to pH 5.5 after addition of isotopes.

Metal Isotopes and Assays. Isotopes wvere ob-tained in HCl solution from the New England NuclearCompany'. 54Mn and 57Co (carrier free') and65Zn (13.7 c/g Zn) were used in nutrients at20 fAc/l'. 59Fe (9.25 c/g, Fe) was bound to equiva-lent Ethvlenediamrine dl ( o-hydroxyphenylacetic acid)(EDDHA) and the mixture diluted with enough0.02 M FeEDDHA to give 5 ,xmoles of Fe and 20 /uc59Fe per liter of experimnental nutrient. The solutionwas sampled before and after the 10-hour absorptionperiod to determine the uptake of isotopes. Samplesof the nutrient solution and exudate were assayed ina gamma scintillation spectrometer. Ca, Mg, andMn were determined by atomic absorption analysis.

Electrophoresis Apparatus. The apparatus de-scribed previously (10) was used to separate metalsby filter paper electrophoresis. Another apparatuswas constructed to study the migration of metals insemifree solution without the filter paper support.The apparatus consisted of a rigid plastic tube into

1 The naming of companies and products is for thereader's benefit, and does not imply endorsement bythe United States Department of Agriculture.

which 12.5-mm lengths of polyethylene tubing (13-mm,inside diameter) were inserted to provide compart-ments between millipore discs. Holes in the tube,matchIng ho&es in the tubing sections, permitted addi-tion and recovery of buffer and metal solutions.After electrophoresis, the solutions were aspiratedinto glass tulbes and assayed.

Buffer Solutions. The principal buffer used forboth paper and tube electrophoresis was 20 mm sodiumacetate (pH 5.4). A second buffer (20 mM citrate,pH 5.4) contained 4 g Na3citrate*2H20 and 1.34 gcitric acd HO per liter. Appropriate dilutions ofthis with the acetate gave the buffers listed infigure 3.

Results

Metal Effects on Fe Uptake and Transport. Mnwas less drastic in its effects on Fe uptake than wereCo and Zn (table I). Tomato roots absonbed 16 %of the Fe from solutions containing 50 pM Mn. Theytook up between 40 and 60 % of the Fe supplied inthe lower Mn treatments. Co and Zn began to de-press Fe uptake when their concentrations were near1 jM. Mn had no effect on exudate volumes, butCo and Zn at 5 SM and above depressed exudatevolumes.

The quantities of nutrient Fe released into thetotal exudate (IR, tabtle I) seem remarkably constantfor treatments containinig up to 10 jM Mn. A breakin the Fe transport appeared between 10 and 50 FLMMn. Depression in Fe transport began at lower con-centrations of Co and Zn.

Table I. Effects of Manganese, Cobalt, aild Zinc on Iron Uptake antd Release intto Tomato ExudateTwo plants were placed in each liter of nutrient solution

5 jM FeEDDHA and 59Fe. The plants were decapitated, andcontaining the metal variable in the presence ofexudate was collected for 10 hours.

Exudate Ironvolume release

(IR)

ml32292426292533292214105

262828192211

,umole0.8480.7710.7100.7230.6900.1700.9270.8320.4200.1330.0480.0090.8480.8820.6130.2030.1320.009

IR/IU

30.330.827.330.127.621.332.030.824.77.83.21.0

30.335.327.913.510.21.5

Iron in exudate

R* T** R/TM26.526.629.627.823.86.8

28.128.719.19.54.81.8

32.631.521.910.76.00.8

M26.325.627.627.523.06.6

29.327.923.615.311.18.7

32.031.425.314.211.63.2

101104107

'101

-1.03103961038162432110210087755225

* From 59Fe analysis.** From total Fe analysis by orthophenanthrolinle.

Nutrientmetaltreatment

Ironuptake(IU)

IAAMn 0

0.51.05.0

10.050.0

Co 0

0.51.05.0

10.050.0

Zn 00.51.05.0

10.050.0

,umole2.82.52.62.42.50.82.92.71.71.71.50.92.82.52.21.51.30.6

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TIFFIN--TRANSPORT OF TRACE METALS IN TOMATO

Relation of Fe Uptake to Release. The absorbedFe that was released into the exudate (IR/IU)averaged 29 % for Mn treatments up to 10 um. Thevalue of 21 % on the 50 uM Mn indicates a relativelysmall depression in Fe transport when compared tothe lowv values of 1 and 1.5 % associated with thehigh Co and Zn treatments.

Specific Actizvity Relationships. The molarities ofFe in column R (table I) were calculated from the59Fe content of the exudates. The values in column Trepresent total Fe determined as ferrous phenanthro-line. The R and T values showed close agreement,with values for R/T be.ng around 100 % for all Mntreatments. This indicates that essentially all the Fein the exudate came from the nutr ent. Values above100 % obviously are in error, because the plants couldnot se'ect and concentrate the 59Fe.

The effects of Co and Zn were very different.At the highest concentrations of these metals, theR/T values were 21 and 25 %. Presumably the rootsreleased 4 or 5 atoms of Fe from internal stores foreverv atem of nutrient Fe they translocated to theexudate.

In summary of table I, it is ev.dent that Mn didnot inh lbit Fe uptake and transport or supress exudatevolumes nearly as much as the other metals.

Aln, Co, and Zni Uptake and Transport. Resultsin table II show that on the 0.5 and 5 0 ,,UM treatmentsthe plants absorbed all or nearly a'l the metal supplied.This was true also for the 25 pum Mn treatment. Onthe 25 and 50 FM treatments, however, the quantitiesof Mn absorbed were about double those of Co andZn. Low concentrations of Co and Zn depressedexudate volumes, but Mn had no effect, even at100 MM.

On all treatments except one (0 5 AM Co), theconcentration cf metal in the exudate was higher

than the initial supply of metal in the nutrient. Rootsconcentrated Mn, 2 to 5 tilmes the original nutrientlevel. Co and Zn concentrations were between 1 and3 times the nutrient levels.

Ratios of Metals in Exuidates. A comparison oftotal Fe (table II) with total Mn (table III) showsthat Mn/Fe in the exudates ranged between 0.4 and19. Metal concentrations (table III) ind:cate a rangeof from 333 to 14 for Ca/Mn ratios, and from 183to 7 for ratios of Mg/Mn. The importance of metalratios in determining the forms of metals translocatedin exudate will be discussed in a later section.

Relation of Metal Uptake to Transport. The per-centage of albsorbed metal that was released is givenby the ratio MR/MA. Generally a higher percentageof the Mn was released than was noted for Co andZn. The percentages were fairly constant except forlower values in the intermedate range of the Mnand Zn treatments. The retention of metal by theroots was greater in that range. On the lowest treat-ments, however, the roots released relativelv high

Table III. Calciumt, Magtesium, antd Manganese inlTomtato Exudate

This table presentsperiment in table II.

additional data for the Mn ex-

Nutrient Metals in exudatetreatment

MIn Ca Mg Mn

,uM /LM lM 'm

0.5 4000 2200 125.0 3850 1950 20

25.0 3650 2100 9850.0 4250 1850 154

100.0 3100 1550 214

Table II. Ulptakc anid Traiisport of Manganese, Cobalt, anid ZiWnc by TomlatoTwo planits were placed in eachi liter of nutrient solution containing the metal variable (with Mn, Co, or Zn

label) and 5 Au.i FeEDDHA. The plants were decapitated, and exudate was collected for 10 hours.

ExudateExudate metalvolume conc*

ml3432333135

371596

35242116

/M2.5

14.875.9128.7194.1

0.413.570.8

110.61.26.3

31.778.3

Totalmetalrelease(MR)

,umole0.0850.472.503.996.790.0150.200.640.660.0240.150.661.25

Exudate ExudateAIR/MA iron citrate

17.09.4

10.913.517.33.84.35.54.98.43.15.38.3

,aLm27.420.718.411.111.524.113.97.6

10.127.49.85.63.9

,aM4432271712

4732171942202623

Based oni radioactivity.

Nutrientmetaltreatment

Mnl 0.5

5.025.050.0

100.0Co 0.5

5.025.050.0

Zin 0.55.0

25.050.0

Totalmetal

absorbed(MA)

,umole0.55.0

23.029.539.20.44.6

11.713.50.54.8

12.515.0

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PLANT PHYSIOLOGY

C+)

0

.cm,

54Mn

1 2 3 4 5 1 2 3 4 1 2 3 4

FIG. 1. Radiograph of electrophoretically separated metals in toimato stenil exudlite. The exudates are charac-terizedl in tables II and IJI. The numbered paths (increasing in value) corresponid to increasing metal treatment,table II. X-ray films were exposed 30 days. Electrophoretic conditions: 20 yl exudate, \Vhatmiian No. 3 paper.

20 mni sodium acetate buffer pH 5.4, 18 mA (360 to 380 v) 45 miliniutes, rooimi temperature.

percentages of the absorbed metal, indicating a notableefficiency in metal transport.

Both Fe and citrate were lowered in the exudatesas Mn, Co, and Zn were increased in the externalmediu.m.

Forms of Translocated Mlletal. Thle exudates illtable II provided an opportunity to determine whetherthe citrate in, excess of Fe in stem exudate binds othermetals. The citrate exceeded Fe in all cases, and theexudates from the 0.5 and 5.0 jM treatments con-

tained, in most cases, enough citrate to bind (1:1)both the Fe and the competing metal. The radio-graphs (fig 1) show that alil the Mn migrated tovwardthe cathode. The Co and Zn migrated similarly.Strong binding by citrate would have resulted inanodic nm'gration. The metals apparently ran as in-organiic cations.

Tube Electrophoresis. Labeled exudates wsere ob-tained frcm experiments similar to those reported intables I aind II. For the electrophoretic analysis.buffer was withdrawn from the center compartmentand replaced by exudate. Figure 2 shows the peaksof radioactivity after electrophores3s. The distribu-tion identifies Mn, Co, and Zn as cations. Theseresults are simnilar to those obtained via paper clectro-phoresis. Fe was the only metal that traveled anod-icallv.

ANODECATHODE

65zn

57Co

-8 -4 0 4+ 8+

FIG. 2. Electrophoretic separationl of metals in to-mato stemii exudates. Patterins show relative distributionsof exud(late metal into the numbered compartments.Electrophoretic conditions: 20 mar so(lium acetate bufferPH 5.4 8 mnA (300 v). 20 minntes for Mln, Co, and(Z11, ani(I 35 m11intutes for Fe, rooil tem11perature.

57Co 6 5Zn

S

1430

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TIFFIN-TRANSPORT OF TRACE METALS IN TOMATO

Buffer Citrate Effects. The cathodIc position ofthe metals (fig 1) shows that they were not stronglybound to citric acid. Experiiments were thereforedesigned (fig 3) on the conjecture that citric acidadd-ed to the buffer might retain the metals longenough to carry them anodically or provide some

restrain,t on their cathodic migration. Buffer I was

without citrate, so that the only citrate in the systemwas in the 15 jul volumes of exudate spotted at theorigin. The citrate was about 45 KM in these exu-

dates (see the 0.5 FM treatments for Mn, Co, and Zn,table II). Previous work (10) showed that citratetravels anodically. This is opposite to the mgrationof the d'issociated m.etals. Thus, in buffer I therecould be no metal-citrate assoc.ation after the metalshad migrated a short distance toward the cathodle.

(+) iMn Co Zn Mn Co Zn

0

cm

Mn Co Zn

FIG. 3. RadIograph showing the effect of citratebuffer on the electrophoresis of metals in tomato stemexudate. Exudate samples (15 ,l) wvere from the 0.5

ALM treatments, table II. X-ray films were exposed20 days. Concentrations (mm) of acetate (a) andcitrate (c) in the buffers wcre as follows: I, 20 a; IIL19.96 a and 0.04 c; III, 19.6 a and 0.4 c. Other electro-phoretic conditions as in figure 1.

In buffer II, however, the meta's could not avo'dthe restraining effects of citrate because, as theymigrated cathodically, they were always runningagainst 40 FM citrate. Consequently, in buffer II

the m,etals did not migrate as far as they did inbuffer I.

In buffer III, the retention by citrate was enoughto cause a net movement of Co and Zn anodically.The final position of Mn, however, was still towardthe cathode. Citrate greater than 400 ,UM (probablynear mM) would be required to carry Mn anodlzally.

Discussion

Translocation of Inorganic Solutte. The bulk ofthe minerals in the xylem undoulbtedly exists as freeions (9). This has also been emphasized by Biddulph(1) who sees "rather universal agreement that theminerals which ascend the stem do so largely ininorganic form." In this regard, reference has oftenbeen made to Wormall (12) who discussed constitu-ents of grapevine sap. Although he found two-thirdsof the solute to be organic, and most of this composedof oxalic, tartaric, malic, and succinic acids, he didnot discuss metal bind.ng by the organic acids. Themetals in the sap vere Na, K, Ca, Mg, Mn, Fe, andAl which were reported as simple salts (chlorides,sulfates, nitrates, phosphates, etc.). Ap-parently hismethods gave no indication of metal-organic assoc:a-

tion.Hydrated Metal Cations. The hydrated metal.

M (IH90) x+, is considered the normal form of thedivalent transition metal in aqueous solution (2). Asufficient increase of OH- in such a solution resultsin the displacement of H+ from the water shell of themetal and precipitation of the metal hydrox'de. Anydegree of comnplexing between organic compounds andthe hvdrated metal displaces water mol'ecules andlessens the tendency to form metal hydroxide (2).Fe3+ begins to hydrolyze at albout pH 2.5. Thisaccounts for the difficulty in maintaining soluble in-

Table IV. Metal-citrate Equilibriunm Contstantts and Metal Displacemient Equilibrium ConstaittsThe equilibrium constant K for metal-citrate (MCit) is of the formn Kmcit = (MCit)/(M) (Cit). From

Chaberek and Martell (2), the displacement equilibrium constant K', resulting from the displacement of M from thechelate by another metal M', is given by K' = K-i'cit/K:icjt = (M'Cit) (M)/(M') (MCit).

DisplacementEquilibrium Equilibrium equilibrium Literature

Metal constant constants constant sources forK divided* K' K value

FeZnCoMn

MgCa

2.5 X 1097080067610467819501479

Fe/ZnZn/CaCo/CaMn/CaM\g/Ca

Mn/MgCo/mgZn/Mg

3500047.945.73.21.3

2.434.636.2

(2, 3)(5, 8)(5, 8)(5, 8)(5, 8)(4, 8)

* To get K' values.

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PLANT PhIYSIOLOGY

organic Fe in cul'ture solutions at neutral or slightlyacidic pH. On the other hand, Mn2+, Co2', and Zn2+are very stable in the nutrient solutions (,pH 5.5)and stem exudates (pH 5.4) used in the present study.

Based on the information above, the results infigure 3 represent a competition principally betweencitrate and water for the metals. In other experi-ments (unpublished) 100 jM citrate in the buffer heldCo and Zn at the origin. The failure to migrate doesnot indicate a neutral molecule, but is visualized asa balance in the migration distances of the hydratedand chelated forms of the metalls. Such a poisedsystemi obviously was not achieved in figure 3.

Translocation of Metal Cltelate. In contrast tothe macroelement transport, there is reason to expectsome of the trace elements to migrate in complexedfoenm. The stability constants of the citrate chelatesof Fe, Co, Zn, and Mn (tabVe IV) seem to warrantthis expectation. Metal b:nding, however, was dem-onstrated only for Fe (fgs 1, 2).

Metal Competition antd Displacement. The stabil-itv of Fe-citrate (2.5 X 109) indicates that Fe is outof the range of serious competition from Co, Zn, andother metals with lover affinities for citrate. Thecitrate bound to Fe is therefore sulbstracted in orderto determine citrate that is free to bind other metals(e.g., 44-27 = 17 jM citrate: see the 0.5 aM Mntreatment, tab!e II). By use of the displacementconstants (table IV) it is then possible to obtain someidea of the concentration of metal con1bined withcitrate in the exudates.

The displacement of iIn by Ca is calculated fromK' = (MnCitrate)( Ca) / (in) (CaCitrate), whereconcentrations are uM. Values entered from tablesIII and IV give 3.2 - (x)(4000)/(12)(17-x).From this calculation, the alue for MlnCitrate is0.16 FM.

This value, howvever, does not include any dis-placement of Mn by Mg. Because the K' for Mn/Cais similar to that of Mn/Mg, the concentrations ofCa and Mg (4000 and 2200 uI, table III) are com-

bined to further qualify the Mnl equilibrium. Entryof 6200 juM, instead of 4000, in the equation aboveindicates that MnCitrate is about 0.10 ptM. Fromthis it is evident that <1 % of the Mn is bouind tccitrate. Displacement by other metals would, of

course, result in a low er equilibrium value forMnCitrate.

The competition from Ca and Mg hel.ps explainthe apparent complete dissociation of Mn in figure 1.Althouigh not given, calculations of the effects of Ca

and AM (at the concentrations above) on Co and Znindicate that these metals also would be present pre-dominantly as cations in the stem exudate.

Acknowledgments

I thank Dr. S. B. Hendricks and Dr. J. C. Brownfor their interest and help in this work. Discussionswith Dr. J. E. Leggett and help with atomic absorptionanal)ysis bv Dr. E. V. Maas are appreciated.

Literature Cited

1. BIDDULPH, 0. 1959. Translocation of inorganicsolutes. In: Plant Physiology; A Treatise, Vol.II. F. C. Steward, cd. Academic Press, NewYork. p 553-603.

2. CHABEREK, S. AND A. E. MARTELL. 1959. C) rganicSequestering Agents. John Wiley and Sor s, NewYork.

3. HAMNI, R. E., C. WI. SHULL, JR., AND D. M. GRANT.1954. Citrate complexes with iron (II) and iron(III). J. Am. Chem. Soc. 76: 2111-14.

4. JOSEPH, N. R. 1946. The dissociation constantsof organic calcium complexes. J. Biol. Chemi.164: 52941.

5. Li, N. C., A. LiNDENBAUM, AND J. M. WHITE.1959. Some metal complexes of citric and tri-carballylic acids. J. Inorg. Nucl. Chem. 12:122-28.

6. LINGLE, J. C., L. 0. TIFFIN, AND J. C. BROWN.1963. Iron uptake-transport of soybeans as in-fluenced by other cations. Plant Physiol. 38:71-76.

7. SCHIMID, XV. E. AND G. C. GERLOFF. 1961. Anaturally occurring chelate of iron in xylem exu-date. Plant Physiol. 36: 226-31.

a SILLEN, L. G. AND A. E. MARTELL. 1964. Sta-bility Constants of MIetal-ion Complexes. 2ndedition. Special publication No. 17. The ChemicalSociety, London.

9. SUTCLIFFE, J. F. 1962. Mineral salts absorptionin plants. In: International Series of Monographsotn Pure and Applied Biology. Vol. I. PergamonPress, New York.

10. TIFFIN, L. 0. 1966. Iron Translocation. I. Plantculture, exudate sampling, iron-citrate analysis.Plant Physiol. 41: 510-14.

1 1. TIFFIx, L. 0. 1966. Iron Translocation. II. Ci-trate/iron ratios in plant stem exudates. PlantPhysiol. 41: 515_18.

12. WVORMALL, A. 1924. The constituenits of the sapof the vine (Vitis viiniferia L.). Biochem. J. 18:1187-1202.

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