Localization Ca-Mediated Apparent Ion Selectivity in … · This relation,ship was established from coIn-sideraltion of rates and extent of ion albsorption and ion interaotion's during

Plant P-'hysiol. ('1967) 42, 1658-1664

Localization of the Ca-Mediated Apparent Ion Selectivityin the Cross-Sectional Volume of Soybean Roots

J. E. Leggett and W. A. GilbertMineral Nutrition Laboratory, United States Department of Agriculture,

Agriculture Research Service, Soil and Water Conservation Research Division,Plant Industry Station, Beltsville, Maryland 20705

Received April 27. 1967.

Summntary. A major portioni o;f the calcium in a soybean root appears to be inless than 10 % of 'the root volume. Specifically, Ca is considered to be almost entirelylocalized in the epidermal cell layer. This relation,ship was established from coIn-sideraltion of rates and extent of ion albsorption and ion interaotion's during theabsorption process.

Presence of calcium at the root-solution interface was associated with a changein the apparent selectivity of K over Mg by soybean roots. Accumulation of calciumby soybean roots was negligible.

Essentiality of Ca for cellular growth and sur-vival has long been recognized. The range inobservations includes the Ca reversal of KCl-in-duced paralysis in Tubifex rioulorum (24), a Ca-induced increase of potential differences acrossmuscle (4), a decrease in ithe yeast cell volume inthe presence of Ca (13), Ca or Mg associatedshrinkage of smooth m-uscle (3), and increasedlrate of cation (8, 20, 23, 26), and anion (10,15)absorption in plant tissue, as we!ll as an increasedpreference for a particular cation (8) andl changesin the internail pH of mitochonidria (5).

Calcium is often considered to react with theplant root near the periphery of individutal rootcells boith in its absorption and its influence onother ions (18, 19). These deductions rest solelyon the necessity for the presence of Ca in theexternal solution ini order to obtain an increasedK aibsorpttion. However, the influence of Ca isnot fuilly recognized as to its ftunction in transportas is evident by use of single salt in experimentswith varying salt concentrationis or ones incluldinga constant amount of Ca. In either case, the inter-action of ions, dependent on their ratios and con-centrations, can l)e observel. Dnlring the courseof recent absorption stuidies, we have deduced( thelocalization of the root Ca and a Ca influence onabsorption of ions. Observations were made onthe levels and rate of change of salt compositionof soybeani roots in variouis solutions. T'he dataindicate that a large percentage of the Ca is locatedin less than 10 % of the root volume occulpied byK. Althouigh K appears to be untiformly distributedin the root, the Ca is localized in 10 % of the rootvolume near the external soluition-root interface.Calcitum is capable of rapid modification of thisinterface ancd resuilts in quick changes in both

individual and relative iOn absorption rates as wellas in ion retention levels.

Methods

Excised Roots. Soybean seeds (150 g ofGlycine 1n1x L. Merr., var. Hawkeye) were soakedin aerated water for 6 hours andl distribuited oncheesecloth supported on a stainless steel screen.The screen was placed on top of a polyethylenetray (13 X 11 X 5 inches) contajining 2 X 10-4 MCaSO4 solution at a level 1 cm below the screen.A stainless steel screen covered with cheeseclothwas placed over the seeds. Th'e tray was coveredwith a sheet of plastic and placed in a constanttemperature chamber (27°) wiith solution beingcontinually aerated. Forty-eight hours after plant-ing, the top cheesecloth and steel cover were re-move(l. Four (ladys after planting, the roots were ex-cised, cut to a 6 cm lenigth, measured from the rootapex and stuspended in a 2 X 10-4 M CaSO4 solution.

The excised roots were removed from the solu-bion and blotted on absorben't tissue to removeadhering so,lution. Root samples of 0.5 g portionwere weighed, rinsed 3 times with water and trans-ferred to 1-iliter voilumes of test soilution (chloridesalts). At the end of the absorption period, theroots were removed, rinsed 3 *times with water anidtransferred to 30 ml pyrex beakers. The sampleswere placed in a muffle oven and heated to 4800for 2 hours. After cooling, the ash was dissolvedin 20 ml of a solution containiing 0.1 N HNO3 and10 % (v/v) acetic acid.

Intorganic Ai(nlvsis. Potassium, sodium, cal-cium, magnesium, and chloride were determined inboth root samples and absorption solutions. Thecations were determined by usulal techniques of

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LEGGETT AND GILBERT-Ca LOCALIZATION IN SOYBEAN ROOTS

flame emission for K and Na (11), or by atomicabsorption for K, Na, Ca, and Mg (7). Chiloridecontent of root samples and absorption solutionswas measured by conductometric titration (6).So,lution's were analyzed at the conclusion of theabsorption periiod to verify any excess removal ofions and as a check on the original composition.

The pH was determined at the beginning andat the end of an absorption period on a smallalliquo,t of the solution. In the observations re-

S~~~~~~~~~

6 a C

4& 10~~~~IT N KCI4 <

be O2

s0O

3 _____---20- i

I20 -

2 4 6 8 10. 12 14 16 18 20 22 24H ours

FIG. 1. Influence of 10-2 N KC1 on the net uptakeof K and Cl and the loss of Ca by soybean roots as afunction of time.

ported here, the difference between initial and finalpH values were less than 0.2 unit. This changefrom pH 5.5 was noit considered to be significantfor bhe ion absorption under study and we, there-fore, did not make periodic adjustments.

Results

The interaction of a plant root with its environ-ment can be evaluated fro'm observations of ionmovements between ithe 2 systems. T'hus, one findsthat excised roots placed in a KCI solution lose Caand accumulate K and Cl (fig 1). These valuesrepresent net tuptake of K and Cl and the Ca con-tent at the indicated times. It is readily apparentthat one is observing a dynam'ic system and hencemust proceed cautiously with finterpretations foundedon observations of a particullar ion. The hi'gh rateand extent of Ca loss was tunexpected. Loss ofCa by soybean roots was not peculiar to this KCIconcentration. However, as ito be expected, theCa 'loss decreased with decreasinig KCG concentra-*tions with the 'loss to 10-4 N KCI being 0.5 of thatin 10-2 N KCI. Excised roots in water for aneqtiuivalent time period as in salt solutions did notlose Ca. Thus, one must conclude a negligibleassociation of a Ca-salt in t'he apparent free spaceof the root utnder these experimental conditions.

The initial time couirse of Ca loss fro'm soybeanroots placed in a M'gCl2 solution was fouind to be

a

A Ca('i M g

10 N MgCI,

30 60 90 120M inutes

FIG. 2. Influence of 10-2 NANgCl1 on the grosscontelnt of Mg and loss of Ca by soybean roots as afunction of time. Line a: Theoretical loss of Ca bydiffusioni where D - 3 X 10-8 cm2 sec-1 and r =0.3 mml.

simillar to the loss in KC1. NevertheIless, Ca losswas greater for roots placed in MgCl, than a KCIsolution (figs 2, 3, 4). The data in figures 2, 3,and 4 are for the amount of the indicated elementpresent *in the root at the time of mea,surement.Entry of Mg is always equal or slightly greaterthan the Ca loss at all times of measurement. Ingeneral, uiptake of Cl from a MgC'l2 solution doesnot occur ito any measurable extent. Although theentry of MVig can approach that of K, the Cl uptakedoes not appear to be associated nor mediated byMg.

The loss of Ca from excised roots to a solutioncontain,ing both Mg and K is similar to that ob-served in the absence of K. Da'ta of figure 3were obtained on roots placed in a 5 X 10-3 NMIgCI., solution for various btime periods. Oneobserves an immediate loss of Ca and an accumu-lation of K and Cl. Potassium 'loss was apparent

8

4

; O

; 603c * --s0 50 I*

0--, 40 .

cv 3030

20

l0

* ci4 KA CoMg -A1

2 4 6 8 10' 12 14 16 18 20 2 2 2 4H o u r s

FIG. 3. Change in the gross content of K and Caand net uptake of Mg and Cl of soybean roots in ai X 10-3 N KCl + 5 X 10-3 N MgCl, solution as afuncltioni of time.

--

1659

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PLANT PIIYSIOLOGY

ait tiLmeCS CXceed(inig 2 houirs and(i conitininiig to thelast observatjion at 24 houirs.

Soybean pl)lalts wer-e grow,n in a S X 10-; NIKCI plis 2 X 10 M CaSO4 SOlUtiOI for 24 houirsp)rior :to root excisioin in order to exam-Tine Ca lossfrom high-salt roots. 'I'hese roots were place(d in1 X 10 2 N MgCl.. and at variouis times u1p to 24houirs examinecd for Ca, K, Cl, and M\g content(fig 4). The loss of Ca began imnmediately, anohservation in agreemeint with the previouis (lata.As was the case in figuire 3, the loss of K becamexvery evidelnt near the 2-hour periodl xwhere the Caloss was asyml)totically approaching zero. Thechloride colntent of these roots decreased nearly tozero after l)eing in MgC,l2 for 24 hours. Exalminal-tioIn of data in figuire 3 and 4 also indicates thatthe rates of K loss and M,g entry were greater fromthe MgCl. solution than in the MgCl. pltus KCIsolution without a noticeable influience on the Caloss.

No attempt is made to evade the fact thatsoybean roots may lose K because they are damagedin some manlner as a restult of Mg uptake. Thislamage may be explained on the basis of a Ca losswhich is (irectly associated with the structulralchalinges of membranes as observed by Marschneranlld(1lGunther (21). However, damage as expressedby the K loss was not evident as assayed by res-piration rates. The rate of 02 consumption andCO., production was ofbserved to be approximately4 and 3 tumole hr-' g-', respectively, under all ex-perinilental conditions. Respiration of the Mg-treated roots was slightly higher than for those illCa, thereby eliminatiing a possilbility of a Mg im-pairnment of the respiratory enzymes or loss ofsuibstrates to the external so,lilbion through a leakymembrane.

The fact that root damage (loes occur undercertaini of these experimental conditions may bebeneficial with respect to attempts at relative local-

6# [ * K

A Ca4ik Mg

C I~~~~~~~~~~~C

80 LI00

80o

6 0 Jt

40 OF

t~~~~~~~~~ t-

2 4 G a3 10 12 14 16 18 20 22 24H c u rs

FIC. 4. Influiencee of 10-2 xN MgCl, on tlh g-rosscontent of AI'g. Ca, K, anid Cl of soybean roots Uls Uftunictioni of timie. Root pretreated for 24 lhours priorto excisioni in a 5 X 10 2 N KCI + 2 X10X Cr CaSO4'0lliltion.

ilation of ionls within a root. 'I'huis, we observedthe rates of loss of particuilar ions as well as ther-atio of their loss as an indicationi of their tlistribui-tion in the root. 'I'he shift in ion contenit of theKCl-loa(led roots (luring Mg uptake in the 0- to2-houir time period was an accumuilation of Mg anda loss of Ca, K, and Cl of 13.9, 5.2, 7.0, andl 1.2,aeq g 1, respectively (fig 4). Expressed as a per-cent of the initial content, the Ca decreased by 87 %and the K by 9 %. These resutlts present ev.idenceof a heterogenic (listribtition of a major portion ofthe Ca wx-ith the K of the root. Ho,wever, theremaininig Ca, K, and Cl would seem to be dis-tribu1ted homogeneouisly x-ithin the root volumebecause each decreased at the rate of 9 % every 2hoturs after the second houir of the experiment.

. K

-0 ACa6 0 Co l0- N KC I

. 3IT N CaCl_550

4 0

Io '"I

2 4 6 8 10 12 14 16 18 20 22 24

H o u r s

F1G. 5. Iinfluieice of 10 2 N KCI + 10 3 N CaCI.,oni the net uptake of K and(l Cl anid the gross contentof Ca of soybean roots as a function of time.

Both potassiumIll aIld chloride uptake were in-creased by the presence of Ca in the external solil-tioil (see figs and 5). Values reported for Kand Cl represent net uiptake, whereas those for Carepresent total content at indicated times. The in-crease for botlh K and( Cl utptake in the presence ofCa wxas approximately 1.6-fold at the end of 24houirs. Calciulm increased in some manner both therate and amoulnt of Cl tuptake beginning at zerotime. As for K, an increase in rate and amountof uptake wNas more pronotiince(d between the 4- and24-hotur time period. 'rhat the in,fluence of Ca onthe uiptake of K an(l Cl mtust be at an interfacebathed by the external soluttion is conclutdedl fronithese observations becauise (1) the effect of CaNwas immediately observed, and( (2) the Ca contentof the root did not increase duiring anI tuptake periodof 24 hoturs (fig 5).

The influence of Ca on the preference of uptakebetween 2 cations mav also indicate the localizationof the interaction. Soyblean roots accuimtulate rela-tively more MgI, than K from a soluttion containiingequlivalent concenltrationis of Ag and K (table I).However, addition of Ca to suich a solution reversesthe ulpt)ake ratio, i.e. the r-oots accuimullate more Kthan AIg. Ftirther increases ini Ca concentrationresulted in a slight inierease in the preference for

166,f0




Talble I. Effect of Ca oit K, Mg, and Cl Absorptioiby Excised Soybean Roots

Albsorption solution: KCl = MgCl2 = 5 X 10-3 N.Ab)sorption period: 24 hours. Initial content: K = 46;Ca = 6: Mg = 3; Cl = 0.2 /eq/g.

Solution Tissue contentconcCa K Mg Cl Ca

miecq/l ,ueq/g fr wt0 350.1 60()5 1011.0 1135.0o 10310.0 106

694017161612

173459666162

0.31.01.32.04.35.8

K as well as an increase 'in the level of Ca. Thisrelationship was surprising because the addition of0.5 me/liter of Ca induced an infinite net changerelative to absence of Ca in the K-Mg rajtio, whilethe root content of Ca had decreased 4.7 ueq/gbelow that of the initiail value. Because Ca in theexternal solution induced such a change without amajor change in the root Ca conitent suggests adrastic modification of the interface between theexternal .solution and the root.

The rate of removal of Ca and K from rootswas determined for roots placed in 0.1 N HCI tofturther elutcidate the proximity of root Ca to theexternal soltution. It was observed that in 10seconds the roots lost 40% and 3 % of the initialconitent of Ca and K, respectively (fig 6). Theloss of Ca was initially fast followed by a slower

rate al)proaching that observed for K. Potassiumcontent of the root iiiitially decreased as a linearfunction of time as if it had a homogenous dis-tribution in the root. 'l'he distribtution of K rela-tive to ithat of Ca is heterogenous and the externalsolution must be readily accessible to the Ca becauseof its initial rapid loss.

Calcium Loss-Actdal and T'heoretictl DiffusionRaites. Investigations of ion uptake by excisedroots usually leads to consideration of separationof accuimulaited ions from those associated in theapparent free space (AFS). This free space coIn-tains the cations and anions in the same concentra-tion as the external solution as well as someexchangeable cations (22). The salt in the freespace is readily lost to water or solution of adifferent salit. Half-time of this reaction was re-ported to be only a few seconds (1). The ex-changeable cation wilil be removed at a rate coni-trolled by those limitations imposed upon a diffusionexchange reaction.

The data 'presented for Ca loss probably doesnot contain a significant fraction of the diffusibleCa-sa1t in the free space of the soybean roots.This conclusion was drawn from the observationthat excised roots did not lose a measurable amountof Ca nor K to distilled water in 24 hours followingexcision and 3 water rinses.A major portion of the Ca in a root is considered

to be held in an exchangeable form (9) and is notaccumulated to any extent by the individual cells(18). The exchangeable fraction must thereforeexist either near the root surface, in the cell walls,or distrilbuted in some ratio between these 2 loca-tions. The possibility exists that one can use theequation describing diffusion from a cylinder ofinfinite length as an aid in the localizati-on of Cain a soybean root. It is necessary at this point toagree (1) that diffusion rather than exchange willbe the rate-limiting step, (2) that the total volumeof the root be considered, and (3) that althoughCa probably is confined to the cell wall region, theoverall di,stribution can be treated as being uniformthroughout the cylinder.

Compar.ison of the time course of Ca loss withthe theoretical loss by diffusion from a cylinderwhere the average concentration C, in the cylinderof radius, ro, whose initial concentration had beenCi, while its final concentration is Cf, is given bythe series ( 12):C - Cf I

= 4Ci - Cf Z12

e -DtZ12/r20 +

M n ut e s

FIG. 6. Influence of 0.1 N HCi on the gross contentof Ca and K of soybean roots as a function of time.Line a: Theoretical loss of Ca by diffusion whereL) = 5 X 10J- cm2 sec-1 and r = 0.3 mm.

e -DtZ.2/r20 +Z 2

Z1 and Z. are series coefficients. Defining the ioncontent of root in terms of concentration is at bestan approximation, hence the left side of the equa-tion is taken to be F, the fraction of the initial ioncontent still remaining in the root at time, t.

1661



1662

TIhe above equation was uised to calculate line a

of figures 2 and 6. A value for the diffusioncoefficient, D, was determined for a 0.06 cm diam-eter root and the valuie for F seleicted at 120minutes and 3 minutes from figures 2 and 6,respectively. The calculaited values for D were3 X 10-8 cm2 sec-' for figures 2, and 5 X 10-cm2 sec-1 for the data in figure 6. Tihese valuesare to be compared to 5 X 10 cm2 sec' fortritiated water in a maize root (27) and 3 to4 X 10- cm' sec-' as diffusivit- o,f K and Na inbarle- root (22). On the basis of these reportedXvalues, use of ouir calculated apparent diffusioncoefficients appears to be a realistic assumption toobtain a theoretical curve.

It has been assumed that Ca was in the AFSof a pilant root and that the loss of exchangeableCa was rate limited by diffusion. However, we

find that the ini,tial loss of Ca in both cases (figs2 and 6) was greater than the theoretical value as

obtained by use of the equation. If one attemptsto fit the equation to the initial portioil of thecurve, then the latter portion of the curve is noteven close to the experimental data. The valuteso f D selected to describe a tangent to the initialtime course experimental curve was 5 X 10- cm2

sec-1 aind 1 X 10-5 Cm2 sec-1 for data in figures 2and 6, respectively. Using these values for D, thetheoretical loss would be 90 % complete in 10minutes for data of figure 2, and 29 seconds fordata of figure 6. If one sele,cts smailler values ofD, the curve will be above line a with the initialreaction being much less prominent.

The in;itial loss of Ca is not adequately describedas a diffusion-limited process, although the loss atlonger time periods appears to be more nearlytypical of diffusion ouit of a cylinder. Inspectionof the theoretical and experimental curves indicatesthat 'the initial loss of Ca was more rapid thanpredicted by the equation. Thus, this indicates a

shorter path length for Ca (liffusion than the 1assuimed and that ithis amount of Ca imust be asso-

ciated near the root surface relative to the totalCa. The Ca loss at the longer time periods appearsto be described by the diffusion equation, with theimplication that this fractional Ca content is uni-formly distributed in the root.

Bernstein and Nieman (1) placed the free spaceregion exterior to ithe endodermis of a root, whilePitman (22) and Woolley (27) did not find any

evidence of a diffusion barrier at the endodermis.The uptake data on intact and separated cortex andstele of corn roots by Yui and Kramer (28) alsocontain,s evidence that the endodermis doesn't re-

strict movement of ions. Since these conclusionswere from data on the movement of both cationsand an anion, we assumed that they are also validfor Ca movement throughout the root.

The evidence presented indicates that the Caloss measuired in these experiments probably resuiltsfrom 2 regions of the root. A portion of the Ca

PHYSIOLOGY

comes from the eintire radial sectioin anid anotherportion from near the root surface. This is seenifrom the calculated valuie of the apparent (liffusioncoefficient for the initial loss being larger thain forlonger time periods showing a negligible torttiosity.This being the case, the Ca mtust be localized nlearthe epidermal layer in order to minimiize movementthrough the tortuious pathway of the cell walls.One must keep in mind that the produict Dt wviliremaiin essentially constainit with chaniges in theinternal concentration, whereas one expects varia-tion in the D anid t values. Thus, it is the relativeshape of the curves which is important and not theabsolutte values of the constants.

Discussion

The identificatioin of ions in a tissue by micro-autoradiographic studies ha,s met only limited suec-ces,s (2). These investigations must be concernedwith possible movement of the isotope duiring samplepreparation and the resolution may not l)ermitidentification of an isotope with a particullar cellcomponent or in some cases even a single layer- ofcells. However, if we accept the results, wi thoiitthe possible modifving influence of sample prepara-tion, they- indtlicate that after 45Ca ulptake by beaniroot, the isotope concentratioin is greater in thestele and epidermis than in the cortical cells. Thework of Luttge and Weigl f(17) was similaar to,although not (lirectly comparable to, Biddulph's (2)because the former primarily observed the unildiffer-entiated region while the latter the more matuireregions, i.e. greater than 8 mm from the root apex.

Lauchli (14) has made use of anl N-ray micro-analyzer to detect localization of ions in thill sec-tions of plant material. These results inidicate(d agreater concentration of Sr and presumably Ca inthe epidermis and stele of the differentiate(d partof the root. Strontium was only in the dermatogenof the root tip. The findings are similar to thoseobtained by use of microautoradiographic techni(queBidduJph (1), and(l Luittge anid WAiegl (17). How-ever, determinationi of amounts in a given regionof the root is not obtainable by either technique.Nevertheless, on a relative basis, the data suiggestthat a large portion of the divalent cation in theroot is located in the epidermis.

The epidermis was considered by Sandstrom(25) to be (lireetly involved in the selective absorp-tion of iolls. This is in agreement w-ith the )ro-posal that a large portion of the Ca is located inthe epidermis of the soybean root. That the Ca islocated in Or niear the epidermis is also suggestedfrom the rapid exchange of Ca (18) alnd Sr (9)and(I the interaction of Ca wxith 32P uptake (15) bN-plant roots. The time of this Ca exchange isuisually 30 minutes, but should not be eq(utate(d todepth of location since a period of 2 to 3 hours may'be required for the exchange reaction in baker syeast (16). The path length of ionl mio-vement in




this single cell organismn is less than 3 /A, comparedto a thicktiess of 30 to 50 p, for a soybean epidermalcell.

Further identification o4f the Ca reactioni withthe root surface area was ideduced from the re-versal of K-Mg utptake ratio upon addition of Ca.The effect was evident immediately and occurredwithout a large change in Ca content. Any ob-served increase in Ca 'was relatively fast, while therates and uptake levels of K increased andl those ofMg decreased. I f Ca or Mg penetrated the root,o,ne would expect an inhibition of K retention atsome level of internal divalent cation. Since entryof Mg was shown to induce a loss of K (figs 3and 4) and was reversed by Ca (table I), one mustdeduce an interaction 'blocking Mg uptake at theroot-solution 'interface 'or more specifically at theepidermal cells.

The movement of an ion inito a root may beconsidered as a series of interactions occurringstepwise at succeedingly snaller and smaller cvlin-drical interface's unti'l reaching a conducting tissuesuch as a xylem element. Furthermore, the entryof an i'on either results in the exit of another ionof similar charge in order to maintain electrostaticneutralitN or entry of an ion of opposite charge(K accompanied by Cl). It is al,so possible thatuptake o'f 1 ion 'may increase the ileakage of otherions (see fig 4). The Mg content of the roots isconsidered to be uptake without stipul1ation as toinvolvement of an energy step. Magnesium entryis considered in this case to be a radial movementacnd induces leakiness in each cell layer as it pro-cee(ls toward the stele. In which case a givenamouint of Mg would be required to inidtuce total Kand C1 leakage. T'hus, it atppears possible that theentry of Mg would be related to K an(d Cl effluxby the effected root volume on the assumption ofuniform distribution. That the rate of change ofK and Cl is similar to the Mg doesn't implystoichiometry, bu't instead indicates equivalency involuime distribution. From the above deductions,one theni places a large part of the total Ca in theepiderinal layer rather than as uniformly distributtedin *the root. If Ca has been equally distributed,theni all cell membranes would have been immedi-ately effected and an initial surge in K loss shouldliave been observed as was for the Ca loss.

Additional information on the localization ofthe Ca and K in the soybean root with respect tothe external solution was obtained by placing theroots in 0.1 N HCO for short-time periods. TheHCI is considered to enter the root and disruptcellular membranes in its radial movement to thecenter of the root. T,he loss of Ca and K, underthese conditions, would be in sequential orderbeginning with the epidermal layer and approachingthe stele. Uniform distribution of K and Ca woutldbe indicated by a constant K/Ca in their loss.Because the ini!tial loss of Ca was greater than Ksuggests that relatively more Ca is in the cell layer

adjacent to the external solution-the epidermis.If the major part of the Ca as an exchangeableion or salt associated with the soybean root isuniformly distributed in or external to the l)las-malemma in the free space, then the loss of Ca toHCl solution should near completion in 30 seconds(1). Since this was not observed is evidence that:(1) 'the total Ca in a soybean root is not equallyavailable to the external solution, (2) penetrationof the root free space by HCI as assayed by Calo,ss is not as rapid as one might assulme, all(n (3)the rapid initial Ca loss is indicative of loss froma region proximal to the external solution.

The observed loss of Ca and K as a percentageof the initial content should be useful in evaluatinigthe root volume containing a large fraction of theCa. Examination o'f figture 4 sh'ows that in the first2 houirs in MgCl2 'solution, the roots lost - 90%of their Ca and only 9 % of the K. After 2 houirs.the amount of Cl and K decreased by 9 % every2 hours, indicating homogeneity of the K and Cldistribution. It appears that 10 % of the Ca con-tent is distributed in the same -space as 91 % ofthe K 'because the rate of loss of thi's remaining Caapproaches 9 % per 2 hours as found for K and Cl.Accordingly, 90 % of the root Ca was located inthe root volulme occupied by 9 % of the total K.Since K appears to be uniformlly distribtutedthroughout the root cross section, it follows that90 % off the root Ca was localized in the 9 % ofthe root voltume nearest the external solution.

The calculated thickness of an outer cylindricalvolume containing 9 % of the root volume is 14 u

ba'sed upon a uniform rooit diameter of 0.6 mm.This calculated thickness would place 90 % o,f theCa in association with the epidermal layer of cellIswhich have an average thiekness of 30 to 50 ,u.On the basis of the calculation and the assumptionthat Ca associated in this volume is uniformlydistributed, the minimum Ca concentration is5 X 10-2 N. Hence, it becomes more plausiblethat this concentration o-f Ca can react with thecell componenits at this interface to modi'fy the ionuptake process.

Literature Cited

1. BERNSTEIN, L. AND R. H. N IEMAN. 1960. Ap-parent free space of plant roots. Plant Physiol.35: 589-98.

2. BIDDULPH, S. F. 1967. A microautoradiographicstudy of 45Ca and 35S distribution in the intactbean root. Planta 74: 350-67.

3. BOZLER, E. AND D. LAVINE. 1958. Permeabilityof smooth muscle. Am. J. Physiol. 195: 45.

4. BURNSTOCK, G. AND R. W. STRAUB. 1958. Amethod for studying the effects of ions and drugson the resting and action potential in smoothmuscle with external electrodes. J. Physiol. 140:156.

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PLANT PHYSIOLOGY

5. CHANCE, B. AND1 l. L-ENNA. 1966. A hydrogenion concenitrationi glradient in a mnitochondrialmembranie. Nature 212: 369-72.

6. COTLOVE, E., H. V. TRANTHAM, AND R. L. Bow-MAN. 1958. An instrument and method for au-tomatic, rapid, accurate, and sensitive titrationof chloride in biological samples. J. Lab. Clin.Med. 51: 461.

7. DIAVID, D. J. 1960. The determination of ex-changeable sodiumn, potassium, calcium, and mag-nlesium in soils by atomlic-absorptioni spectopho-tometrv. Analyst 85: 495-503.

8. EPSTEIN, F. 1961. The essenitial role of calciunin selectiv e catioIn tranlsport by plalIlt cells. PlantPhvsiol. 36: 437 45.

9. EPSTEIN, AND J. E. LEGGETT. 1954. The ab-sorptioni of alkaline earth cations by barley roots:kinetics anid mechanismii. Amii. T. Botany 41:785-91.

10. FOOTE. B. 1). AND J. B. HANSON. 1964. Ioni up-take bv soybeani root tissue depleted of calcium byetlhvlenediaminetetraacetic acid. Plant Physiol.39: 450-60.

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