absorption iron by in nutrient solutions 1,2 m. benedict

Absorption of Chelated Iron by Soybean Rootsin Nutrient Solutions 1,2

John N. Simons, Ronald Swidler, & Harris M. BenedictAgricultural Research Center, Stanford Research Institute, South Pasadena, California

During the past decade, many investigators havestudied the uptake of micronutrients when suppliedas chelates to plant roots. Considerable controversyhas arisen as to whether the metal is dissociated fromthe organic ligand at the root membrane and is takenup by itself, or whether the entire chelate is absorbedthrough the root nmembrane. Several investigators,notably Wallace and co-workers (1, 7), have offeredevidence that the entire chelate is taken up by theplant, whereas others, notably Tiffin and co-workers(3, 4, 5), have offered evidence that the metal is dif-ferentiallv absorbed from the chelate at the rootsurface.

During the past 18 months the Agricultural Re-search Center of Stanford Research Institute lhas beenconducting studies on the mineral nutrition of plantswith emphasis on iron uptake in soybeans. Certainresults were obtained which have a bearing on thegeneral question of absorption of chelated iron throughplant roots. These results are reported here in thebelief that they may contribute to the answver tothis question.

Materials & MethodsSoybean seeds (Glycine wviax L., var's. Hawkeye

or PI-54619-5-1) were germinated and grown insand for 1 week after which time they vere trans-planted to 1 gallon polyethylene pots containing anaerated nutrient solution (Hoagland No. 2) muain-tained at pH 6.0. The polyethylene containers wverepainted on the outside with aluminum paint. Fourplants were grown in each pot. Nutrient solutionswere nmade with distilled water and C.P. grade salts.Solutions were brought up to volume every 2 or 3days. The plants were grown for about five weeksin a complete nutrient solution; at the en(d of thisperiod they- were transferred to solutions containingall minerals except iron. In about two w-eeks theyoung leaves of the soybean plants show-ed typicaliron-chlorosis symptoms. At this time the plantswere transferred to new nutrient solutions containingvarious iron treatments, and recovery from the chloro-sis was induced. To obtain nmaximum uniformity ofinitial chlorosis in the plants, the two plants in eachpot whiclh were least uniform were removed beforesolutions with iron chelates were supplied.

1 Received Nov. 20, 1961.2 This work was sponsored by The James G. Boswell

Foundation as part of Stanford Research Institute ProjectNo. P-2990.

During periods of high light intensity leaves ofsome plants developed necrotic spots. These leaveswere not satisfactory for use in regreeming experi-ments. This condition was alleviated by placingSaran screens transmitting only 69 % of the incidentradiation outside the greenhouse (luring the summermonths.

The chelating chenmicals used in the studies andthe stability constants of the iron chelates are listedbelow:

Chelating comlpound Stability constant

1. Ferric nitrilotriacetic acid(FeNTA) 1015

2. Ferric N-hydroxyethylenedia-minietriacetic aci(l(FeHEEDTA) 1021

3. Ferric ethylenediaminetetra-acetic acid (FeEDTA) 1025

4. Ferric diethylenetriamine-pentaacetic acidl (FeDTPA) 10"

5. Ferric cyclohexanediaminetetra-acetic acid (FeCHDTA) 1029

6. Ferric ethylenebishvdrox--phenylglycine (FeEHPG ) > 1030

Results- Absorption of FeEHPG From Nutrienit Solutions:EHPG is a particularly useful chelating agent forexperimnenttal work on uptake of metal and ligandbecause of its optical activity. The ligan(l alone ah-sorbs liglht strongly at 276 m,u; the Fe chelate ab-sorbs miiaximally at 475 m,u an(l acidified FeEHPGahsorbs strongly at 280 mA. Up to 10 ppm the ab-sorbancy followNs Beer's Law. Thus it is possibleto determiiine the amiiount of uptake of iron and ligan(dor ligand alone by plant roots by measuring spectrallythe concentrations of FeEHPG and EHPG in nu-trient solution. In our studlies we usedl a BeckmanModel DK spectrophotometer for this purpose.

By use of the above described method, the (lif-ferential absorption of Fe froml FeEHPG lhas beenreported for sunflowers, zinnias, and soybeans (3, 5).In both studies large quantities of iron were foundin xylem exudate following the placenment of rootsfrom decapitate(d planits in nutrient solutions contain-ing FeEHPG. Relatively little loss of ligan(d fromlthe nutrieint solution was demolnstrated in these ex-periments (3, 5').

The present stu(dies on (lifferential uptake of Fefronm FeEHPG by sobeans (liffer froml this previous

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SIMONS ET AL.-ROOT ABSORPTION OF CHELATED FE

work (3, 4, 5) in that intact plants were used insteadof decapitated ones.

In initial experiments, the chlorotic soybeanplants (4 per container) were supplied with FeEHPGat concentrations of 0.75 and 1.50 X 10-5 M. Threereplications were used. Loss from the solution asa result of uptake by plants of either iron or ligandfrom solutions containing these relatively large con-centrations of chelated iron was too slight for con-clusions to be drawn. Consequently, the experi-mental procedure was changed in two ways: first,the cotyledons were removed from the young seedlingsat the time of transplanting to the pots to reduce thesupply of endogenous iron and, second, FeEHPGwas supplied at the lower concentration of approxi-mately 0.40 X 10-5 M. As in initial experiments,four plants were grown in each container and threereplications were used. Controls consisted of similarcontainers with aerated nutrient solutions containingFeEHPG or EHPG but without plants. The nu-trient solutions were sampled at weekly intervals andspectrophotometric determinations of FeEHPG andEHPG made. Prior to sampling, the water levelsin the containers were restored to their originalheights and the solutions stirred. The results of atypical experiment are shown in table I.

Table I

Absorption of Iron From FeEHPG by Soybean Plantsin Nutrient Solutions Over a 6-Week Period

Conc in M X 10-5

Date of No plantmeasure- Soybean solution ConNo plolntion controlment -F EHGFe EHPG EHPG

Fe EHPG ~onlyJune 13 0.39 0.43 0.38 0.42 0.19June 24 0.35 0.41 0.38 0.46 0.20July 1 0.27 0.38 0.36 0.46 0.19July 8 0.24 0.35 0.35 0.48 0.19July 15 0.19 0.35 0.33 0.42 0.19

Approximately half of the iron disappeared from

the nutrient solutions during the 32-day period ofmeasurement, whereas only a small percentage ofthe ligand disappeared. These results essentiallyconfirm the work of Tiffin et al. (5) on differentialuptake of Fe from FeEHPG.

- Effect of Excess Ligand in Nutrient Solution on

Growth of Iron-Deficient Soybean Plants: Althoughthe above results indicate strongly that the iron was

absorbed preferentially through the root membranes,certain observations on regrowth in other experimentsare difficult to explain on the basis of lack of absorp-tion of the ligand. We have had occasion to supplysix different iron chelates at low concentrations ofiron with varying quantities of ligand in the nutrientsolutions to chlorotic PI-54619-5-1 soybean plants.The iron was supplied at either 0.89 or 1.78 X 10-5 Aiand ligands (NTA, HEEDTA, FDTA. DTPA,

Table IIEffect on Regreening of Soybean Plants Supplied Iron

at Constant Concentration & Ligand at VaryingConcentrations in Nutrient Solutions

Conc of ligand in M X 10-5

Ligand 0.0 0.262 0.524 1.048 2.096 4.192No. plants that regreened when supplied

Fe at 0.89 X 10-5MNTA 0* 6 6 6 6 6HEEDTA 0 4 6 6 6 4EDTA 0 4 6 6 6 6DTPA 0 3 6 6 3 0CHDTA 0 2 4 3 3 0EHPG 0 2 6 6 2 0

No. plants that regreened when sutppliedFe at 1.78 X 10-5M

NTA 0 6 6 6 6 6HEEDTA 0 4 6 6 6 6EDTA 0 5 6 6 6 6DTPA 0 6 6 6 4 2CHDTA 0 0 6 6 3 0EHPG 0 3 6 6 4 0* Six plants were used in each treatment.

CHDTA, & EHPG) were given at concentrationsof 0.000, 0.262, 0.524, 1.048, 2.096, and 4.192 X10-5 M in these solutions. Three replications of twoplants per pot were used for each treatment. Re-sponses of the plants after 14 days were miost strik-ing (fig 1) both from the standpoints of regreening(table II) and root development (table III).

The striking effect on growth of the relative bind-ing tenacity of iron is shown in figure 1. As thestability constant of the Fe chelate increased from101' (FeNTA) to 1029 (FeCHDTA), there was acontinuous reduction in total growth of the plants.This reduction in growth was independent of the in-teractions between iron and ligand ratios. It wasnoted that where the higher concentration of ironwas used, this gradient of response was less apparent.

Where weaker chelates (FeNTA, FeHEEDTA,& FeEDTA) were used, the plants regreened rapidlyand completely, seemingly regardless of concentrationof iron or ligand supplied. This was not the case,however, for plants grown in nutrient solutions con-taining FeDTPA, FeCHDTA, or FeEHPG. Plantsgiven these stronger iron chelates (stability constantsrange from 1028 to >1030) usually showed severalresponses in common. First, the degree of regreen-ing of the plants was correlated with the level ofiron supplied. Second, the iron chelates were noteffective in promoting regreening at the lowest con-centration used. The only exception to this wasFeDTPA. This chelate differs from the others inthat it carries two negative charges instead of one.Possibly absorption of Fe by plants from this com-pound is less difficult than from chelates havingsimilar binding powers but which carry only onenegative charge. And, third, regreening was ham-pered by use of ligand in excess of that required

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waspp iea it0.8 X 0 Mi l ae. Noeta as oth stblty costn of th i Sronchlt inrae (FNT

FeCHDTA the growt Is reduced regardlessofeither iron origandconntratin..............._-_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~6

..V...................... Er9

F eH EED __=TA_ -_wu0S_ ~~~~~~~~~~~~~~~~._|.: .' 3~~ ~~~~~~~~~~~~~~~~~ ~~ ~~ ~~ ~~ ~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

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Fig. 1. Effect of verying levels of ligands oin growth, regreeninig, aind root development of soybean plants. Ironawas supplied at 0.89 X 10---> -m in all cases. Note that as the stability coiistant of the iron chelate increases (FeNTA->FeCHDTA) the growth is reduced regardless of either ironi or liganid concentration.

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Table III

Effect on Root Growth of Soybean Plants Supplied Ironat Constant Concentration & Ligand at Varying

Concentrations in Nutrient Solutions

Conc of ligand in M X 10-5Ligand 0.0 0.262 0.524 1.048 2.096 2.192

No. of plants that developed good root systemswhen supplied Fe at 0.89 X 10-5 M for 14 days

NTA 0* 6 6 6 6 6HEEDTA 0 6 6 6 6 6EDTA 0 0 6 6 6 6DTPA 0 3 6 6 4 0CHDTA 0 0 3 3 0 0EHPG 0 0 0 0 0 0

No. of plants that developed good root systemswhen supplied Fe at 1.78 X 10-5M

NTA 0 6 6 6 6 6HEEDTA 0 6 6 6 6 6EDTA 0 2 6 6 6 6DTPA 0 6 6 6 6 3CHDTA 0 0 6 6 4 0EHPG 0 0 6 3 0 0

* Six plants were used in each treatment.

to chelate all of the iron present in the solution. Infact, plants grown at the highest levels of DTPA,CHDTA, and EHPG almost always became mnorechlorotic than they were before treatment was started.

The effects on root growth of these various ironchelates were in most ways similar to the effects onregreening (table III). It is apparent that rootgrowth is particularly sensitive to iron supply andthat as iron availability increased, whether throughthe addition of more iron to the solutions or throughthe use of weaker chelating agents, root growth in-creased proportionately. As nmight be expected fromthe results on regreening, roots generally failed todevelop when placed in solutions of strong ligands(DTPA, CHDTA, or EHPG) containing quantitiesin excess of that required to chelate the iron present.

That these adverse effects on growth were theresult of the excess ligand in the solution was borneout by the following experiment. Chlorotic soybeanplants were grown in solutions containing 0, 20, 40,80, 160, and 320 X 10-5 M of FeEHPG. In addition,other plants were grown in solutions containing 160and 320 X 10-5 M EHPG without iron. Regreeningwas rapid and root growth excellent for the plantsgiven 20, 40, and 80 X 10-5 M of FeEHPG. At 160and 320 X 10-5 M of FeEHPG the plants turned redwithin 72 hours of treatment. The plants given 320x 10-5 M of FeEHPG subsequently died. Plantsgiven EHPG but no iron wilted and died within 48hours of being placed in the nutrient solutions. Itmust be concluded from these results that FeEHPGis not toxic to plants even at quite high concentrationsbut when the ligand is supplied without sufficientiron to chelate it, EHPG can be very deleterious.

Tiffin et al. (5) have reported the observationthat "colored exudates could be obtained from stems

of certain plants when their roots were inFeEDDHA" (FeEHPG). These exudates ab-sorbed light at 480 miu, thus indicating that FeEHPGwas present in the exudate. In our studies, we foundthat if FeEHPG were supplied in very high concen-trations (either to nutrient solutions at pH 6.0 oron a clay soil with a pH of 7.95), colored exudatescould be obtained from cut stems and the leaves ofsuch plants turned dark red within 48 hours. Ex-traction of leaf homogenate with butanol to removeanthocyanins left a pale red liquid which absorbedstrongly at 480 m,u and, upon acidification, absorbedstrongly at 276 mIA. There seems to be little doubtthat FeEHPG did exist in such plants. However,the concentration of FeEHPG required to producesuch results makes it highly questionable whethersuch uptake occurs under more usual circumstancesof application.

- Uptake of EHPG From Nutrient Solutions bySoybean Plants: In view of the toxicity of thestronger chelating agents when supplied in amountsgreater than that required to chelate all of the ironin the nutrient solution, more experimentation onthe uptake of strong ligands such as EHPG, DTPA,and CHDTA seemed advisable. The simplest hy-pothesis explaining the toxicity symptoms in the soy-bean plants was that an iron deficiency was causedby uptake of the non-iron chelated ligand. Both thesevere chlorosis induced in the foliage and the lackof root development in these plants are typical ofiron deficiency symptoms. If the symptoms were anexpression of iron deficiency, it was likely that theligand was being absorbed by the plant. Conceiv-ably, once the ligand was absorbed by the roots itwould compete for the iron in the plant. And be-cause of the extreme tenacity with which these ligandsbind iron, the net effect would be to rob the plantof its endogenous iron supply.

In order to test this hypothesis the following ex-periment was carried out. Large PI-54619-5-1 soy-bean plants with well developed root systems Wereused as test plants. The tops of the plants were re-

moved so that only the primary leaves and the twolowest lateral buds were left. These plants were

then placed (2 per pot) in 1-gallon polyethylenecontainers. The following treatments were used inthe nutrient solutions:

Treatment

123456

M X 10 ',

FeEHPG

0.00.150.150.150.00.0

M X 10 5

EHPG

0.00.00.300.600.300.60

Comparable solutions without plants were usedas controls. Three replications of each treatmentwere used. Solutions were sampled spectrally forFeEHPG, Fe, and EHPG content at the beginning

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Fig. 2A-F. Changes in concenitrations of Fe and EHPG in nutrient solutions in which soybean plants were grow-ing. Control solutions contained no plants but were aerated. Note that EHPG disappeared from the solutions onlywhen supplied in excess of that required to chelate the iron present. FeEHPG was supplied at 0.15 X 10-5 Mbut the relative chelating capacity of EHPG was such that 0.10 X 10-5 M of FeEHPG was in the solution. Notethat where excess ligand was supplied (c & e) the concentration of Fe rose to 0.15 X 10-5 m. The standard devia-tioIn of a single determination for EHPG was + 0.018 X 10-5 M; for a single determination of Fe, the value is +0.023 x 10-5Al.

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of the experiment and weekly for 3 weeks. Beforeeach sampling the water levels were restored to thetop of all pots and the solutions mixed thoroughly,figure 2.

It is apparent that where iron and EHPG weresupplied at about equimolar concentrations (fig 2b)no change in concentration of either component wasdetected. In fact, iron uptake through plant rootswas not demonstrated in any of the treatments (figs2b, c, e). However, where the concentration ofligand was in considerable excess of iron concentra-tion, there was a substantial decrease in ligand con-centration in the nutrient solutions (figs 2c, d, e, f)during the 3-week period of growth. Uptake ofligand was greater where 0.60 X 10-5 M was sup-plied than where 0.30 X 10-5 M was used. It is sig-nificant that where ligand without iron was supplied(figs 2e & 2f) a small but demonstrable amount ofFeEHPG was present in the nutrient solutions at theend of the growth period. The ligand had undoubted-ly extracted the iron from the roots of the plants.Competition between strong chelating agents and en-dogenous iron in roots has recently been reported byBrown et al. (1).

Table IV

Effects on Regrowth of Soybean Plants in NutrientSolutions Containing Various Levels of

FeEHPG & EHPG

Type of foliage growthTreatment**coninM x10-5 No. of No. of No. ofconcinmx ~~mildly very

green chlorotic chlorotic

1. 0.10 FeEHPG* 4 2 02. 0.15 FeEHPG plus 6 0 0

0.30 EHPG3. 0.15 FeEHPG plus 0 6 0

0.60 EHPG4. No iron or ligand 0 5 15. 0.30 EHPG 0 2 46. 0.60 EHPG 0 0 6

* FeEHPG was added at a concentration of 0.15 X10-5 M but the relative chelating capacity of EHPGwas such that 0.10 X 10-5 m of FeEHPG was inthe solution. Note that when excess ligand wassupplied (treatments 3 & 4) the concentration of Fewas 0.15 X 10-5M.

** Six plants were used in each treatment.

Growth responses of the plants were very strik-ing (table IV). In general, regreening was relatedto iron concentration in the solutions, but the presenceof EHPG in a non-iron chelated form also influencedplant response. Plants supplied with neither ironnor ligand had slightly chlorotic regrowth, but thosegiven no iron but either 0.30 or 0.60 X 10-5 MEHPG produced extremely chlorotic regrowth.Plants given 0.15 X 10-5 M FeEHPG or 0.15 X10-5 M FeEHPG plus 0.30 X 10-5 M EHPG grewwell and were not chlorotic. Those given 0.15 X10- M FeEHPG plus 0.60 X 10-5 M EHPG grewwell but were slightly chlorotic in appearance.

If the very chlorotic appearance of the plants sup-

plied EHPG but no iron was caused by iron de-ficiency, it should be possible to overcome this chloro-sis by spraying the leaves with inorganic iron. Thiswas done on one of the two plants in each potwhere treatments were supplied neither iron norEHPG and those given 0.30 or 0.60 X 10-5 MEHPG, respectively. Iron was applied as a ferroussulfate spray (0.1 g FeSO4/100 cc distilled H.,O)twice during a 10-day period. At the end of thistime the unsprayed plants were still chlorotic, where-as the sprayed plants all developed dark green leaves.It would appear that the acute chlorosis induced bygrowing the plants in nutrient solutions lacking ironbut containing EHPG was a result of the EHPGbeing taken up by the plant and chelating iron al-ready in the plant. This resulted in iron becomingunavailable to the plant and typical iron deficiencysymptoms ensued.

The most surprising observation made followingthe regreening of the FeSO,-sprayed plants concerneda color change in the nutrient solutions that containedEHPG. These solutions turned pink during the 10-day period. On the 10th day, the solutions containediron at the levels of 0.075 X 10- Ai FeEHPG in thepots that had originally contained no iron plus 0.30X 10-5 Ai EHPG and iron at a concentration of0.12 x 10-5 M FeEHPG in the pots that originallycontained no iron plus 0.60 X 10-5 M EHPG. Itwould appear that some of the FeSO4 sprayed onthe leaves had moved down into the roots and outinto the nutrient solutions where it was chelated intoFeEHPG.

DiscussionThe results of Tiffin and Brown and co-workers

indicated that when strongly chelated iron was sup-plied to decapitated plants in nutrient solutions (lur-ing a 22-hour period, the iron was preferentially ab-sorbed by roots with most (but probably not all) ofthe ligand remaining in the solution. The work de-scribed here substantiates their results. The resultsof the present study further suggest that when a

ligand is supplied in excess of that required to chelateiron in the solution, and hence chelates weakly withother cations-such as calcium and nmagnesium-inthe nutrient solution, the ligand (chelate ?) is readilytaken up through plant roots.

It is not known whether these weakly boundchelates are split at the root membrane (as is thecase for iron chelates) or whether the whole chelatemolecule is taken up by the plant. It would not besurprising if an analogous situation existed, but thepossibility of absorption of the entire chelate cannot beeliminated.

These results also suggest that the ligands whichbind iron most strongly on their entrance into theplant also bind endogenous iron so that it is no longeravailable for normal metabolic processes. Ironprobably exists in plants in the form of chelates. Itis interesting to note that when non-iron chelatedligands were present in the solutions, the PI-54619-5-1

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soybean plants were able to grow well so long as

the stability constant of the iron chelates of theseligands was 1025 or lower (FeNTA, FeHEEDTA, &FeEDTA). Apparently, the concentration of iron inthe plant, the concentration of the natural chelatesand the stability constant of the natural chelates or

the constant alone were all such that competition forthe endogenous iron supply by the synthetic ligandswas not sufficient to cause symptoms of iron deficien-cy to appear. Adverse effects on regrowth occurredwhen strong iron-chelating ligands (DTPA,

CHDTA, & EHPG) were supplied but usually only

when their concentration exceeded the iron concen-

tration in the solutions. Since non-iron chelatedEHPG was readily taken out of the nutrient solution

by soybean plants, it seems probable, in view of thesimilar growth effects caused by DTPA and CHDTA,that these ligands are also taken up by the plant.And, if this hypothesis is true, then all of the ligands

can be taken up. By working with different plantspecies andl a series of chelating agents, more infor-mation could probably be gained on this poinlt. To-bacco plants have recently been shown to possess a

natural chelating agent with a pK of between 10-17+ 10-21 (2).

It might be argued that some of the EHPG dlis-appeared from the nutrient solution because of a

breakdown of the compound through the activitv of

root exudates and that the remaining EHPG pulledenough iron from the plants to cause iron deficiencysymptoms to appear. However, such a hypothesis

cannot easily be reconciled with the observations thatwlhen EHPG was chelated with iron, very little of

the ligandl disappeared during several weeks of ex-

posure to plant roots, and the appearance of iron in

the solutions which contained EHPG (but no iron)was slight and appeared long after severe chlorosis

deficiency symptoms had been induced.The fact that plants can tolerate large concentra-

tions of a strongly bound chelated iron without a(d-verse effect points out the criticality of iron concen-

tration in the plant and the nutrient solution or thesolution alone. It appears that these strong ligandsact as competitive inhibitors in iron metabolism andit is only when the available iron supply in the plantis borderline that strong chelating agents will cause

iron deficiency symptoms. So long as strong ligandsare chelatedl with iron, uptake of iron by the plantappears to be limited to such an extent that insufficientligand is freed for the plant to be able to absorb theligand in harimiful amounts.

Summary

Absorption of Fe from ferric ethylene bishydroxyphenyl glycine (FeEHPG) in nutrient solutions was

denmonstrated in intact PI-54619-5-1 soybean plants.EHPG was taken up through roots only when itsconcentration exceeded that of the iron in the solu-tions.

The severe chlorosis that resulted from growingplants in solutions containing low levels of EHPGand no iron or solutions containing large excesses

of EHPG, cyclohexanediaminetetraacetic acid(CHDTA), or diethylenetriamlinepentaacetic acid(DTPA) over iron suggests that all of these ligands(when unchelated with iron) are picked up by theplant roots where they chelate iron which is in theplant. Spraying leaves of such plants with FeSO4resulted in a rapid regreening and, in the case ofEHPG, a substantial amlount of iron was miioved outof these plants and into the nutrient solution.

It appears that natural chelates in PI-54619-5-1soybean plants can compete favorably for iron withsynthetic chelates having stability constants of 10'or less [ferric nitrilotriacetic acid (FeNTA); ferricN-hydroxyethylenedianminetriacetic acid, (Fe-HEEDTA); & ferric ethylenediaminetetraacetic acid.(FeEDTA) ] but not with strongly bound clhelates(FeDTPA, FeCHDTA, or FeEHPG), all of whichhave stability constants >10'9. It is suggested thatthe principal reasons that EHPG from FeEHPG dloesnot enter plant roots more easily are: A, that theiron is so strongly bound to the ligand that the plantis not able to split the FeEHPG molecule and dif-ferentially take off the iron in large enough quanti-ties to make ligand available for chelationi with othermetals in the nutrient solutionl and B. rate of uptakeof non-iron chelate(d ligand is slow-er than rate ofuptake of iron by iron-deficient plants.

AcknowledgmentsThe authors gratefully acknowledge the assistance of

Dr. Robert W. Howell, Plant Physiologist, U. S. Region-al Soybean Laboratory, Urbana, Ill., in supplying soy-bean seeds used in these studies, and of Mr. HowardSallee, laboratory technician, for his valuable assistancein the greenhouse phases of the work. We also wishto thank the Dow Chemical Co. for generously makingavailable samples of NTA, HEEDTA, and EDTA, andthe Geigy Chemical Co. for supplying us wxith samples ofCHDTA, DTPA, and EHPG.

Literature Cited1. BROWN, J. C., L. 0. TIFFIN, & R. S. HOLUiES. 1960.

Competition between chelating agents & roots as

factor affecting absorption of iron & other ioIns byplant species. Plant Physiol. 35: 878-886.

2. SCH_MID, \W. E. & G. C. GERLOFF. 1961. Anaturally-occurring chelate of iron in xylem exu-

date. Planit Physiol. 36: 226-231.3. TIFFIN, L. 0. & J. C. BROWN'N. 1959. Absorption of

iron from iron chelate by suniflower roots. Science130: 274-275.

4. TIFFIN, L. 0. & J. C. BROWN. 1960. Differentialabsorption of metal chelate componenits by soybeanroots. Abst. Am. Soc. Platnt Physiol. meetings.

5. TIFFIN, L. O., J. C. BROWN. & R. W. KRAUSS. 1960.Differential absorption of metal chelate componientsby plant roots. Plant Physiol. 35: 362-367.

6. WALLACE, A., C. P. NORTH, R. T. MUELLER. L. M.SHANNON, & N. HEMAIDAN. 1955. Behavior ofchelating agents in plants. Proc. Am. Soc. Hort.Sci. 65: 9-16.

7. \WALLACE, A. 1956. Symposium on the use ofmetal chelates in plaint nutrition. Introduction:Metal chelates in agriculture. The National Press,Palo Alto, Cal. Pp 4-23.

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