Iron Transport in Pea Plants"-2Daniel Branton8 4 & Louis Jacobson

Department of Soils & Plant Nutrition, College of. Agriculture,University of California, Berkeley

Iron has long been recognized as an essentialelement for plant growth, but there has been littleeffort to determine how iron moves in normal, greenplants. Attention has focused on chlorosis, which hasfrequently been ascribed to iron immobilization orinactivation rather than to iron deficiency (5,6,30,31, 32). However, recent evidence shows a clearrelationship between total iron content and chlorophyllcontent (2, 20, 21, 29). Previous evidence to thecontrary can often be explained as a failure to washleaves prior to analysis (20), or a failure to main-tain a constant supply of iron (21). Viewing ironchlorosis as an iron deficiency emphasizes the needto study iron uptake and transport rather than ironinactivation.

Ion uptake from a nutrient solution into the rootsand to the shoots of a plant has been described as anactive process, dependent upon the metabolic activityof the root cells (4, 7, 34), and as a passive processdependent upon mass flow in the transpiration stream(11, 17, 18, 19, 22). The opposing viewpoints wererecently reviewed by Russell and Barber (33).Many of the relations between transpiration and iontransport were elucidated by Broyer and Hoagland(7). The important feature of their conclusion isthat it postulates an active secretion into the xylem-possibly against a concentration gradient. On theother hand, Hylmo's (17) extensive experimentsshowed a close correlation between transpiration andsalt uptake, and led him to believe that such metabolicprocesses are of little importance, especially for poly-valent ions. The evidence that Ca+ + is very slowlyaccumulated by excised barley roots (26) appearedto support this contention and suggested the pos-sibility that iron moves passively across the root inthe transpiration stream.

Schmid and Gerloff (35) have shown that anaturally occurring iron chelate could account toriron movement in the xylem at pH's which wouldotherwise cause it to precipitate. But how does iron,

1 Received Jan. 22, 1962.2 This paper is based on work performed in part under

contract AT (11-1)-34, project 5, with the Atomic Ener-gy Commission.

^ This investigation was carried out during the tenureof a Predoctural Fellowship from the National CancerInstitute, United States Public Health Service.

4 Present Address: Inst. fur Allgemeine Botanik,Eidg. Tech. Hochschule, Zurich, Switzerland.

at these same pH's, get into the plant in the firstplace? The unique problems which are posed byiron's insolubility at biological pH's have led manyinvestigators to conclude that plants growing in thesoil may take up iron from insoluble particles (9,12,15). A similar phenomenon may occur in solution-grown plants, though in this- case it has generallybeen implied that iron is taken up from a solublephase. For example, Glauser and jenny (12), whoare strong proponents of a contact mechanism fornutrient uptake from soils, stated that their percolat-ing nutrient solution at pH 6.1 contained enough dis-solved iron to satisfy the needs of their plants.Rather than dissolved iron, it is possible they weredealing with colloidal iron which would also havepassed through their percolating system. In othercases, the passage of nutrient solution-iron throughfilter paper (28, 38) has encouraged the belief thatmore iron remains in solution than calculations basedon the solubility product of Fe (OH)3 predict.However, there is no evidence to substantiate thisbelief.

This study was undertaken to answer two of thebasic questions concerning iron transport in greenplants: A, Is the passage of iron from a nutrientsolution to the shoots a passive process, or is it de-pendent upon the metabolic activity of the root?B, Do plants growing in inorganic nutrient solutionstake up iron from insoluble iron particles?

Materials & MethodsPea plants (Pisutm sativum, L., var. Alaska) were

used in all of the experiments. The pea seeds weregerminated in the dark at 25 C by soaking for 24hours in aerated, glass-distilled water. The seedswere then placed on a screen over aerated 1/10strength Hoagland solution. The plants remainedin the dark at 25 C for 2 more days. They werethen removed from the dark and planted in beakersby threading their roots through polyethylene tops.In the first experiment described below, 40 plantswere placed in each of sixteen 150-ml beakers. Insubsequent experiments, 16 plants were placed ineach of twelve 50-ml beakers. The beakers werewrapped in aluminum foil, filled with half strengthHoagland solution and placed in a circle under a750 w incandescent bulb. The plants continued togrow in half strength Hoagland solution until they

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PLANT PHYSIOLOGY

were 9 days old. All nutrient solutions were purifiedby the method of Stout and Arnon (37). Constantaeration was provided, and the solutions were

changed daily. The 750 w bulb was turned on andoff to provide a 12 hour light-dark cycle. Ambienttenmperature varied daily from 21 C to 28 C.

For an experiment, the groups of 16 (or 40) nine-day old plants wrere transferred, in their polyethylenebeaker-tops, to a 30 second distilled water rinse andthen to the experimental solution. Besides Fe59 or

in some cases Rb86, the experimental solutions con-

tained only CaSO, (1 meq/1) and KCl. The amountof K+ varied from 1 to 2 meq/liter, depending upon

how- muclh KOH was required to neutralize the HClin whiclh the radioactive materials were supplied.

Transpiration was calculated from the total weightloss of a beaker and its contents. The weight lossfrom identical beakers containing no plants provideda measure of loss by evaporation. Transpiration isexpressed as grams of H.0 lost per beaker. It

amounted to about one kilogram of H.,O in threehours per kilogram fresh weight of the shoots. Insome cases transpiration was reduced by placing theplants in a dark, humid atmosphere. A large num-

ber of preliminary experiments indicated that thisprocedlure was a reliable and reproducible means ofeffecting a tenfold decrease in the transpiration rate.

The plant tops were harvested by cutting the stemjust above the second trifid bract. Total iron was

determined by a modified bathophenanthroline method(36). For Fe59 or RbV6 determinations, the freshshoots or aliquots of the digested shoots were placedin counting vials and counted in a Tracerlab well-

type scintillation counter. In most cases, results ofthese determinations are given as relative activity, a

figure representing the average counts 'minute in 1

kg fresh weight of the tops divided by the counts/minute in 1 ml of the nutrient solution. This ex-

pression allows rough comparison between the tablesto follow, though exact quantitative comparison be-

tween two different experiments should not be made.In some experiments, decorticatedl plants were

used. These plants were prepared 24 hours beforethe start of an experiment by han(d stripping thecortical tissue off the central vascular column of thetap root. Decortication severed the lateral roots andtore the radial walls of the endodermal cells, leavingportions of their suberized walls haniginig from thedecorticated stele. Thus, the vascular column ofwhat remained of the root was not covered by anintact endodermal layer, and the xylem elements ofwhat had been lateral roots opened directly into the

nutrient solution. These plants could remain aliveand growT slowly for at least twN-o wveeks after decor-tication.

Results

A preliminary experiment was performed to de-

termine if isotopic dilution in the roots would impugnthe reliability of conclusions based on radioactivitymeasurements alone. The experimenital treatments

and results are indicated in table I. WVith intactseedlings much of the iron reaching the shoots camefrom the cotyledons; removing them markedly re-duced the total iron transported but barely affectedthe movement of labelled iron from the solution tothe shoots. WVith the cotyledons removed, the totaliron reaching the shoots coincided wvithin experi-mental error with the transport of an equivalentamount of labelled iron. The results suggest thatisotopic dilution in the roots was insufficient to biasconclusions based on activity measuremiients.

Table I also shows that prior growth in iron-freemedia decreased the subsequent movement of solution-iron to the shoots during the experimental treatment.Typical of these observations are the results of an-other experiment, recorded in figure 1. Groups of

Table ITotal & Labelled Fe Transport After

Different Ironi Pretreatments

Ironpretreat-ment*

Experimentaltreatment**

Increase of Feits shoots(,ug/plant)

Total*** Labelledt

+Fe Initact plants 0.36+-0.04 0.077+-0.005+ Fe Cotyledons removed 0.08±0.02 0.061+(0.005-Fe Intact plants 0.12±0.02 0.011+0.001

* All plants grown at pH 5.5 in half strength Hoag-land solution; +Fe: received 0.5 ppm Fe as FeCl3for 48 hour pretreatment; -Fe: received no ironduring pretreatment.

** All treatments at 0.5 ppm labelled Fe (8 juc/mg Fe)added as FeEDTA; initial pH of all solutions 5.5;uptake period 3 hours. Where indicated, cotyledonswere removed just prior to the uptake period.

*** Determined chemically. Each figure represenits thedifference between the average of nine replicatesamples (9 plants per sample) taken at the startand at the end of the uptake period. The statndarddeviation is shown.

± Each figure represents the average of niine replicatesamples (9 plants per sample) takeni at the end ofthe uptake period. The standard deviationi is shoxn.

plants wlhiclh had grown in nutrient solutions free ofiron Nwere pretreated for varying time periods innutrient solutions containing 0.5 ppm Fe added asFeCl3. Then all the plants were placed for 4 hoursin a solution containing 0.5 ppm Fe as FeCl3 labelledwith Fe59. As figure 1 shows, the least Fe59 movedto the tops of plants receiving no Fe pretreatment.There were no visually observable differences be-tween plants pretreated in FeCl, and those wlhiclh re-mained in iron-free solutions. None of the plantsshowed signs of chlorosis and all had made equallyrapid growth.

The results of figure 1 may be explained by as-suming that rapid, active accumulation of iron bypreviously iron-starved root cells competes with thepassive flowv of iron to the shoots. If this model iscorrect, it follow\s that for short timiie intervals:

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BRANTON & JACOBSON-IRON TRANSPORT IN PEA PLANTS

-8-C00c4-

c

'.

CO

CP

L._

0"01-

Hrs. in Fe Cl3 Before Beginning Fe59 Uptake

4.0 5.0 6.0

pH

Fig. 1 (left). The effect of iron pretreatment on subsequent iron transport. Plants grown at pH 5.0 in theabsence of iron were pretreated for varying time periods in half-strength Hoagland solutions, pH 5.0, containing 0.5ppm Fe as FeCl3. After pretreatment, the plants were placed in experimental solutions labelled with Fe59 (10 AIc,/mgFe) containing 0.5 ppm Fe added as FeCl3, initial pH 5.0. Uptake period 4 hours.

Fig. 2 (right). Iron concentration as a function of pH in supernatant of centrifuged solutions to which 0.5 ppmFe was added as FeCl3 labelled with Fe59. The curves show Fe added to: *- Half-strength Hoagland solutioncentrifuged 3 hours, 0 Half-strength Hoagland solution centrifuged 30 minutes, and A /A distilled watercentrifuged 30 minutes. Continued precipitation during successively longer centrifugation periods should not be in-terpreted as demonstrating slow hydrolysis; solutions stored in the laboratory for equivalent time periods were pre-cipitated at the same rate when later placed in the centrifuge.

I. Slowing the transpiration rate should decreaseiron translocation to the shoots, and

II. Preventing the active accumulation of ironin previously iron-starved root cells should increaseiron translocation to the shoots. Transpiration was

decreased by placing some of the plants in a dark,humid atmosphere. Active accumulation of iron bythe root cells was modified in two ways: addition of2,4-dinitrophenol (DNP) to one group of plants andcomplete removal of the cortex in another group ofplants. The results are given in table II. Reducingtranspiration substantially decreased iron movementto the tops. But preventing active accumulation inthe root cells did not increase translocation to theshoots. Neither the addition of DNP nor the re-

moval of the cortex increased iron movement inrapidly transpiring plants regardless of priorgrowth conditions. Less labelled iron was trans-located in DNP-treated plants than in the controls.Virtually no Fe59 was translocated in decorticatedplants. This was particularly surprising since move-

ment of Rb86, used here for comparison, was not so

affected by decortication.The striking effect of decortication on iron

movement suggested a careful re-examination of thenutrient solutions used. Calculations based on thedissociation constant of Fe(OH) 3 show that lessthan 10-6 ppm Fe+++ remains in solution at pH5.5. Since such calculations assume, among otherthings, equilibrium conditions, experiments were con-

ducted to verify their applicability. Solutions con-

taining 0.5 ppm Fe added as FeCl8 were freshlyprepared and adjusted to the desired pH with KOHor HCl. Tracer amounts of Fe59 were included.The solutions were centrifuged at 2 X 104 X g at20 C for varying time periods. The supernatant was

sampled and the percentage original iron remainingwas calculated. Typical results given in figure 2indicate that hydrolysis occurred rapidly and thatvery little iron remained in solution at pH 5.5.Furthermore, examination of our dilute, freshlyprepared FeCl3 "solutions" in the electron micro-

541

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05-z

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Table IIEffect of DNP, Decortication, & Transpiration on Fe Transport

Iron pretreatment*

+ FeFe

+ Fe

+ Fe- Fe

+ FeFe

+ Fe

+ Fe

Experimental treatment**

F&C3HgFe59C13Fe59C13

Fe59CI3

Fe59C13 + DNPFe"9Cl3 + DNP

Fe39C13, decorticatedFe59Cl3, decorticated

Rb86CIRb86Cl, decorticated

Transpiration

Conditions

HighHigh

Low

ml/beaker

4.74.2

HighHigh

HighHigh

HighHigh

Relative activityin shoots***

14738

38

4.74.2

91

0

4.54.8

4.64.2

521545

* All plants grown at pH 5.5 in half strength Hoaglandl solution; +Fe: reccdved 0.5 lpm Fe as FeCI3 for 48hour pretreatment; -Fe: received no iron during pretreatment.

** Labelled Fe (20 /.tc/mg Fe) added at 0.5 ppm as FeCl3; labelled Rb (about 0.1 ,uc mg Rh- at 1 meq liter; DNP,where indicated, added at 5 x 10- . Initial pH of all solutioins 5.5; uptake period 3 hours.

*** Relative activity = counts/minute in 1 kg fresh weight of shoots divided by counts /miinute in 1 ml nutrientsolution. Each figure is the average of three replicate samples, three plants per sample.

scope revealed particles similar to Grunes & Jenny's(14) Fe(OH)3 preparedl by heating andI long ripen-ing of concentrated FeCl:, solutions.

These observations showed that lack of iron trans-port in decorticated plants could be related to the ab-sence of any significant amounts of dissolved ironin the nutrient solution used. To test this assump-

tion plants were placed in solutions of iron solubil-ized by chelation with ethylenediamine tetraacetate(EDTA) (table III). In this case, iron readilymoved to the tops of decorticated plants. In normal

plants, DNP depressedl labelled iron movement fromFeEDTA solutions, as it had from FeCl3 "solutions."In contrast, DNP ha(l no effect on iron movement indecorticated plants in FeEDTA solutions. ThoughloNw transpiration decreased iron translocation in

normal plants, this effect was not as pronounced as

in decorticated plants. Rb86 movement, included intable III for conmparative purposes, showed a similarrelation between normal and decorticated plants.

The striking effect of chelation on iron movementinto (lecorticated plants suggestedl that (lecorticate(l

Table IIIEffect of DNP, Decortication, & Transpiration on Fe & Rb Transport*

Experimental treatment**

Fe59EDTA, decorticated plantsFe"9EDTA, decorticated plantsFe59EDTA, decorticated plants + DNP

Fe59EDTA, normal plantsFe59EDTA, normal plantsFe59EDTA, normal plants + DNPFe59EDTA, normal plants + DNP

Rb86Cl, decorticated plantsRb86Cl, decorticated plants

Rb86Cl, normal plantsRb86Cl, normal plantsRb86Cl, normal plants + DNPRb86Cl, normal plants + DNP

'rranspiration

C'onditions ml/beaker

HighLowHigh

HighLcowHighLow

HighLow

HighLowHighLow

4.0,0.5

4.3,0.54.20.6

Relative activityin shoots***

77372

808

286737312

40645

47214615561

* All plants grown at pH 5.5 in half strength Hoagland solution in the ab.sence of Fe.** Labelled Fe (3 Ac/mg Fe) added at 0.5 ppm as FeEDTA; labelled Rb (about 0.1 gc/mg Rb) at 1 meq/liter.

Initial pH of all solutions 5.5; uptake period 3 hours.*** Relative activity = counts/minute in 1 kg fresh weight of shoots divided by counts/minute in 1 ml nutrient solu-

tion. Each figure is the average of three replicate samples, three plants per sample.

542 PLANT PHYSIOLOGY

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BRANTON & JACOBSON-IRON TRANSPORT IN PEA PLANTS

plants could be used to test the hypothesis that rootsexcrete organic materials which complex iron in thesurrounding medium (16). Decorticated plantswere placed together with normal plants in one beak-er. Though the roots of the normal plants were inthe same solution as, and intertwined with, the vas-cular tissues of the decorticated plants, no labellediron moved to the tops of the latter. In another ex-periment, the excised cortex from one group of plantswas placed in a beaker containing another group ofdecorticated plants. Again, no solution iron movedto the tops of decorticated plants.

In all of the experiments described above, theconcentration of iron added to the nutrient solutionwas kept constant. In the next experiment, plantsgrown in the absence of iron were placed in nutrientsolutions which contained varying amounts of FeCl3,as indicated in table IV. In this case the activitydeterminations were used to calculate the amount ofiron reaching the shoots from the roots. Table IVindicates that decreasing the concentration of ironfrom 5 ppm to 0.5 ppm did not result in concomitantdecreases in the amount of iron reaching the shoots.Similar data were obtained using plants which hadgrown in the presence of iron.

Table IVEffect of Fe Concentration on Fe Transport*

Experimental treatment** Increase of Fe in shoots***(Aglkg fr wt)5.0 ppm Fe 181.0 ppm Fe 210.5 ppm Fe 19

* All plants grown at pH 5.5 in half strength Hoag-land solution in the absence of Fe.

** Labelled Fe (2, 10, or 20 ,uc/mg Fe, respectively)added as FeCl3 at the concentration indicated. Ini-tial pH of all solutions 5.5; uptake period 3 hours.

*** Calculated from activity in shoots and specific ac-tivity of solutions. The calculation reflects onlythe iron which is coming from the solution. Eachfigure is the average of three replicate samples,three plants per sample.

DiscussionHylmo (17), Epstein (11), and Kramer (22)

have described ion uptake and translocation in termsof two parallel processes: active accumulation bythe roots and passive movement of ions through freespace to the xylem and thence to the shoots in thetranspiration stream. If (fig 1) active accumulationof iron by previously iron-starved root parenchymais assumed to compete with passive flow of iron tothe xylem, inhibiting this accumulation should haveincreased iron translocation to the shoots. It did not(table II). Less labelled iron was translocated inDNP-treated plants than in the controls-regardlessof whether or not the plants had previously receivediron. In normal plants, DNP also inhibited iron

transport from FeEDTA solutions (table III). Inall normal plants, iron transport was reduced in thepresence of DNP; transpiration was not. Similarresults were observed by Butler (8) and Brouwer(4) in their studies of chloride uptake in wheat andbroad bean plants. However, Hylmo (18) claimedthat DNP eliminated only the active component("active bleeding") of transport and that the saltuptake in the presence of DNP was still proportionalto water uptake. He further suggested that transportof the active component also is partly dependent uponwater movement. But the data of tables II and IIIshow that DNP reduced the transport of labellediron in normal plants by 40 to 80 %, depending uponthe particular treatment and history of the plant.Though it is likely the conditions under which theDNP was used, i.e. pH, concentration, etc., causedonly partial inhibition, at least a substantial part(more than 75 % in most cases) of the iron transportwas related to the cellular metabolism of the rootsand could be inhibited by DNP. Similar considera-tions and conclusions apply to the transport of Rbfrom RbCl solutions (table III).

Though both table II and table III show tran-spiration affected the rate of Fe and Rb transport,comparison of normal and decorticated plants (tableIII) suggests transpiration exerted only an indirecteffect in normal plants. Decorticated plants were anexcellent model for study of passive transport. Ironmovement in decorticated plants seemed to be com-pletely passive since, in such plants, DNP had noeffect on FeEDTA translocation. Movement of bothFe (from FeEDTA solutions) and Rb to the tops ofdecorticated plants was decreased tenfold by conditionscausing a tenfold decrease in transpiration; in normalplants similar changes in transpiration conditionscaused smaller decreases in Fe or Rb translocation.

Neither the DNP effects nor the transpirationrelations which were observed in this study areadequately explained by the passive flow hypothesis.On the other hand, all of the data are consistent withthe view that salt transfer across the root to thestele is an active process. Figure 1 may be explainedby assuming that sufficient amounts of iron had tobe accumulated in root parenchyma before transferto the xylem occurred, an assumption which was sub-stantiated by radioautographic evidence to be pre-sented in a subsequent paper (3).

Broyer and Hoagland's-(7) hypothesis that tran-spiration promotes salt excretion into the xylem pre-dicts the observations in table III. Decorticatedplants responded as a wick to changes in transpira-tion and were insensitive to DNP. In normal plantsactive transfer of salts to the xylem partly counter-acted low transpiration conditions by increasing con-centrations of Fe (or Rb) in the transpirationstream. Normal plants were sensitive to DNP.Radioautographs (3) showed that DNP inhibitediron absorption in the roots; tables II and III showthat DNP inhibited iron transport to the shoots,suggesting that the two processes of iron absorptionand iron transport are mutually interdependent.

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PLANT PHYSIOLOGY

Ironi fromi FeCl3 'solutions" at pH 5.5 was nottranslocate(d to the sshoots of decorticated plants(table II). Since chelated iron and Rb were readilytranslocated in decorticated plants, the easiest ex-planation for lack of FeCl3 transport is that an in-significant amount of iron was in solution at pH 5.5.Centrifugation experiments bear out this conclusionand suggest that plants take up ironi from insolubleparticles. At pH 5.5 less than 2 % of the originalironl remlained in the supernatant of centrifugedFeCl, preparations (fig 2). Though concentratedsolutions of FeCl3 are knowin to hydrolyze slowly(23, 24), the experinments reported here bear outthe conclusions of Lanmb and Jacques (23. 24) thatiron hydrolyzes rapidly whein in dilute concentrations.

Concentration experiments (table III) supportthe lhpothesis that iron is taken up from solid par-ticles. Despite tenfold changes (from 5-0. 5 ppim1 Fe)in the nutrient, no change in the anmount of irontransported to the shoots was observed. Otlher in-vestigators (1, 31) also noted that the concentrationof inorganic iron supply in their nutrient did not lhavea proportional effect on Fe uptake. If the iron werein solution, one might anticipate changes in concen-tration to cause changes in the rate of iron transport.If. on the other hand, uptake of iron from FeCl3,occurred from iron particles which completely coveredthe surface of the root at 0.5 ppm, further addition ofFeCl3, would not be expected to cause any increasein iron uptake.

Several hypotheses to accounlt for iron uptake havebeen advanced. Hutner et al. (16) suggeste(d thatroots excrete metal-solubilizing substances. Tllissuggestion has received little experimental support(28), and our results using (lecorticated plants as a

bioassay for such compounds also failed to detecttheir presence. Jenny and his co-workers (10, 12,13) proposed that plants take up iron by direct inter-action of the root surface with iron particles ini tllesoil. According to their scheme, carboxyl gr1oupsassociated with cell wall material at the root surfacemay acquire iron by a contact decomposition processinvolving exchange of hydrogen ions for iron. If.lhow-ever, colloidal particles of iron were able to dif-fuse through the loose-textured, hydrated primarywalls of the root epidermis (27), direct interactionand complex formation between the plasmalema con-

stituents and the colloidal iron particles could occur.

On the other hand. entry of large protein moleculesinto barley root cells, presumably by pinocytosis, hasbeen observed by McLaren et al. (25), and a similarnmechaniism might allow direct entry of iron particlesinito the cytoplasm.

Summary

Ironi transport in intact pea plants was studied.The rate of iron movemient froiml a lnutrient solutionto the slhoots was nmeasured under various coinlditionsof transpiration, metabolic inhibition, and ironi con-centration. Chemlical ani(l ra(diochleiical determina-

tions were used to measure the rate of iron transport.In short term experiments, less solution iron was

transferred to the shoots of plants grown in the ab-sence of iron than to the shoots of plants grown inthe presence of iron. These differences were ex-plained by assuming the root cells of the iron-starvedplants absorbed iron to meet their own requiremlentsbefore transferring it to the transpiration streaml.

Decorticated plants servedl as a miiodlel system inwhich passive flow in the transpiration streaml couldlbe observed. Comparisoni of decorticated an(l niormiialplants indicated that iron transport from the nutrienitsolution to the shoots w,as depen(lent upon the nmeta-bolic activity of the root cells.

Normal, intact plants were able to take ul) irofrom colloidal particles on the root surface.

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BRANTON & JACOBSON-IRON TRANSPORT IN PEA PLANTS

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