saturable of indol-3yl-acetic roots · hilary v. martin and paul-emile pilet* institute...

Plant Physiol. (1986) 81, 889-8950032-0889/86/81 /0889/07/$0 1.00/0

Saturable Uptake of Indol-3yl-Acetic Acid by Maize RootsReceived for publication November 8, 1985 and in revised form March 11, 1986

HILARY V. MARTIN AND PAUL-EMILE PILET*Institute ofPlant Biology and Physiology, Biology Building, University ofLausanne, 1015Lausanne, Switzerland

ABSTRACT

The uptake of 5-[3Hlindol-3yl-acetic acid (IAA*) by segments of Zeamays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found tohave a nonsaturable component and a saturable part with (at pH 5.0) anapparent K, of 0.285 micromolar and apparent V,,,, 55.0 picomoles pergram fresh mass per minute. These results are consistent with thosewhich might be expected for a saturable carrier capable of regulatingIAA levels. High performance liquid chromatography analyses showedthat very little metabolism of IAA* took place during 4 minute uptakeexperiments. Whereas nonsaturable uptake was similar for all 2 milli-meter long segments prepared within the 2 to 10 millimeter region,saturable uptake was greatest for the 2 to 4 millimeter region. Highlevels of uptake by stelar (as compared with cortical) segments are partlyattributable to the saturable carrier, and also to a high level of uptake bynonsaturable processes. The carrier may play an essential role in con-trolling IAA levels in maize roots, especially the accumulation of IAA inthe apical region. The increase in saturable uptake toward the root tipmay also contribute to the acropetal polarity of auxin transport.

The natural occurrence of IAA in maize roots was unequivo-cally established by GC-MS (3, 5, 26). There is evidence thatIAA plays a part in the regulation of root growth and gravireac-tion, though it is not clear how this control is exercised. Thegrowth rate of maize roots was found to be inversely correlatedwith the level of endogenous IAA in the elongating zone (24).Applied IAA has been shown to inhibit root growth (2) butstimulates the growth of auxin-depleted roots (23).When roots are treated with inhibitors of auxin transport,

gravireaction is strongly retarded and growth is inhibited (6, 12).When triodobenzoic acid was applied only to the caps of pearoots (11) gravireaction was inhibited but growth appeared to beunaffected. The effect of auxin on growth and gravireaction maytherefore depend on the level of IAA in the elongating zone or,more specifically, on the action of the carrier for IAA efflux.

Transport between tissues and subcellular compartments isone ofthe factors influencing the levels ofIAA in different tissuesand in various parts of the cell. The transport of auxin in theroot tip is acropetally polarized (20, 21, 30, 31) and involvesmovement from cell to cell. The transfer of IAA across cellmembranes comprises uptake and efflux, both of which may bepartly by diffusion and partly via carriers.According to the chemiosmotic polar diffusion theory (25, 28)

energy is expended creating a gradient of pH and of electricalpotential across the plasmalemma and IAA, which is a weak acid(pK 4.7), accumulates as the anion inside the cytoplasm. Polarityof auxin transport is conferred by a carrier for the efflux of IAAacross the plasmalemma, located preferentially at the apical end

of the cell (basal end in the case of shoots). This carrier has beenshown to be sensitive to auxin transport inhibitors such as NPA'(32) and other phytotropins (10). An indirect immunofluores-cence method (9) has shown that a NPA binding site is indeedlocated at the basal end of particular cells in pea stems.IAA uptake across the plasmalemma has also been studied

(27) and found to have a saturable component. However, thiscarrier has received less attention than the efflux carrier.The purpose of this study was to find whether, in maize roots,

IAA uptake does have a saturable component. Then, by com-paring published data for levels of IAA in the root with kineticproperties related to the saturation and capacity of the carrier(apparent Km and Vm,x), we wished to gauge whether such acarrier has the potential for regulating IAA movement to anextent which could alter IAA levels and influence physiologicalprocesses. To gain some information concerning the physiologi-cal role of a saturable carrier for IAA uptake, it was interestingto determine its activity in different parts of the root. Compari-sons were made between stele and cortex, and different zonesalong the root.

MATERIALS AND METHODS

Plant Material. Caryopses of Zea mays L. cv LG 11 (Ass.Suisse des Selectionneurs, Lausanne) were germinated for 48 hin the dark at 19°C (22), and plants with roots 15 ± 2 mm longwere selected. Segments were prepared in three different ways(Fig. 1): (a) successive 2 mm long segments corresponding to theregions 2 to 4, 4 to 6, 6 to 8, and 8 to 10 mm counting from thetip (A); (b) 5 mm segments cut at 2 mm from the tip (B); and(c) cortex and stele located 5 to 10 mm from the tip separatedby withdrawing the stele through the side of the cortex (C).

Radiochemicals. 5-3H IAA (29 Ci/mmol) was purchased fromCEA, Gif-sur-Yvette, France. Purity was checked at intervals(about 3 months) by TLC (16) or HPLC using a Perkin ElmerSeries 4 Liquid Chromatograph fitted with a 250 x 3 mmWhatman Partisil 5 ODS 3 column. HPLC elution was at 2 mlper min using the gradients mentioned in the results, and wasfollowed by LS counting of 0.5 min fractions using a PackardTri-Carb LS Spectrometer.

Incubation. Groups of 20 segments were incubated at 20.0 +0.2°C in tubes containing 1.0 ml of medium and shaken at 150rpm. The medium contained 2.0% w/v glucose, IAA* at 5 nMor 10 nm, and IAA° at different concentrations, and was bufferedat pH 5.00 ± 0.02 (Na2HPO4 6.6 mm plus 3.4 mm citric acid).Experiments were all carried out in daylight, as preliminary testshad shown that IAA* uptake from 10-7M solution was identicalin daylight and dim green light.

Radioactivity Determination. After incubation, segments were

'Abbreviations: NPA, N-l-naphthylphthalamic acid; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; 1AA°, 'H-IAA; IAA*, 5-3H-IAA; Ki, Michaelis constant of IAA°; Km. Michaelis constant of IAA*;LS, liquid scintillation.

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Plant Physiol. Vol. 81, 1986

FIG. 1. Diagram showing the preparation of maize root segments. A,Successive 2 mm segments corresponding to the regions 2 to 4, 4 to 6, 6to 8, and 8 to 10 mm counted from the tip; B, 5 mm segments cut at 2mm; C, cortex (1) and stele (2) separated in the zone 5 to 10 mm.

rapidly (less than 5 s) surface rinsed with distilled H20 andtransferred to scintillation vials (4 segments per sample). Scintil-lation fluid was added, and radioactivity was measured as de-scribed above. Experiments were always performed twice, eachexperiment comprising 5 scintillation vials per treatment. Nosignificant difference between duplicate experiments was ob-served.Metabolism of IAA*. Two groups of 20 segments (2-7 mm

region) were incubated in medium containing 10 nM IAA*.Twenty segments were frozen in liquid nitrogen and stored at-80°C until required (less than 1 week). The remaining segmentswere used for LS counting as already described, and the quantity(moles) of IAA* taken up was calculated. From this the amountof carrier IAA° to be used was determined (I03 times the amountof IAA* present). Extractions were therefore made by grinding10 segments in 1.0 ml ethanol with carrier IAA, using a pestleand mortar. The homogenate was transferred to an Eppendorftube and centrifuged for 5 min. The supernatant was kept, andthe pellet reextracted as before but using 80% ethanol. The twosupernatants were combined, and reduced using a stream ofnitrogen. Portions ofextract were analyzed by TLC (16) or HPLCas described above.

Calculation of Kinetic Parameters. If IAA* is at concentrations, and IAA° at concentration i, and the Michaelis constants arerespectively Km and Kj; and if v is the initial rate of saturableuptake of IAA* and Vmd the maximum rate (when substrate isin excess) then according to Dixon (4)

V = (SVmax/ (Km(l + i/Ki) + s)In our case Km = Ki (since both correspond to IAA). Therefore

I/v = (Km + i + s)/ (SVmax)

or

l/v = (i/SVmax) + (Km + s)/ (SVmax).

Plotting (l/v), i.e. (saturable part of IAA* uptake rate)-' in gfresh massmin/pmol-' as ordinate, and i (concentration ofIAA° in AM) as abscissa, Km and V,. can be determined, sincethe intercept on the i axis will be -(Km + s) and the gradient(SVmax}'

RESULTS AND DISCUSSION

First, using 2 to 7 mm segments, the time course of IAA*uptake was followed (Figs. 2 and 3) in order to select a period

80

60

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FIG. 2. Time course of [3H]IAA uptake (expressed in Bq ± SE ofmean per segment) from media containing 10-8 M [3H]IAA plus ['H]-IAA at different concentrations to give total IAA concentrations (radio-active plus nonradioactive) of 10-8 M to 10' M. All solutions were

buffered at pH 5.0.

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FIG. 3. Time course of saturable component of [3H]IAA uptake (ex-pressed in Bq per segment) for 10-, 10-', and I0O M total IAA concen-

tration minus the corresponding value for 10-l M total IAA (Fig. 2).Since these are difference curves from Figure 2, SE are not given.

during which uptake was linear with time, for the determinationof initial uptake rates. Second, tests were conducted to check theextent of IAA* metabolism during uptake (Fig. 4).To investigate the saturability ofIAA uptake, the rate ofuptake

of 5 nM IAA* was determined in the presence of IAA° at (0-20Mm) (Fig. 5). These data were further analyzed to give the appar-ent Km and V,,,. for the saturable component of IAA uptake(Fig. 6). Finally, to obtain information concerning the distribu-tion ofthe IAA uptake carrier in different tissues, uptake ofLAA*(10 nM) was measured in the presence of IAA° giving a total IAAconcentration (IAA* + IAA°) of 10 nm to 10 AM using isolatedcortical and stelar segments (Fig. 7). Similarly, the distribution

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IAA UPTAKE BY MAIZE ROOTS

0 5 10 15 20 25TIME IN MINUTES

FIG. 4. Radioactivity (Bq per fraction) in 0.5 min fractions obtainedby HPLC of (A) 5-[3H]IAA solution and (B) extract from maize rootsegments (2-7 mm region) which had been incubated for 4 min inmedium (pH 5.0) containing 10-8 M 5-[3H]IAA. Elution was at a rate of2 ml/min using a linear gradient of methanol and 1% (w/v) acetic acid(0-75% methanol over 25 min).

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0 5 10 15 20CONCENTRATION OF 'H-IAA IN liM

FIG. 5. Uptake of radioactivity (Bq per segment) by maize root seg-ments (2-7 mm region) after 4-min incubation in media (pH 5.0)containing 5-[3H]IAA (5 x 10-9 M) with the addition of ['H]IAA atdifferent concentrations (0-2 x 10-0 M). SE are shown where larger thanthe points. Nonsaturable component of uptake (NS) = 10.0434 Bq persegment.

of the uptake carrier along the root axis was determined, underthe same conditions, by comparing IAA* uptake in 2 mmsegments cut from different zones along the root (Fig. 8).

Effect of pH on IAA Uptake. Using 2 to 7 mm segments (Fig.1B), the uptake of 10-7M auxin (10 nM IAA* plus 90 nM IAA°)was tested over 30 min using media at different pH values withinthe range 4.0 to 7.0 (buffer components as described for pH 5.0,with the sum ofthe concentrations ofthe two components always10 mM). Net uptake was found to decrease with increasing pHof the medium (results not shown). This is in agreement withpublished results (27). pH 5.0 was chosen for subsequent exper-iments, as it is a compromise between high uptake of IAA and a

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FIG. 6. Dixon (1953) representation of the saturable part of IAAuptake by 2 to 7 mm maize root segments. Data are taken from thesubmicromolar range of Figure 5, after subtraction of nonsaturableuptake (also from Fig. 5) and expressed in (g fresh mass x min) (pmol)-'.

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10 8 10-7 10-6 10o-TOIAL IAA CONCENTRATION IN M

FIG. 7. Uptake of 5-['H]IAA (Bq per segment of stele or cortex), after4-min incubation in solutions (pH 5.0) containing 10-8 M ['H]IAA plus'H-IAA at different concentrations, to give total IAA concentrations of10-8 to I0-' M. SE of the mean are shown by vertical bars. Numbersadjacent to points indicate uptake for a given concentration of total IAA(Bq per segment) expressed as a percentage ofthe uptake (Bq per segment)from 10-8 M solution.

pH which is sufficiently near the wall pH for equilibrationbetween medium and wall to occur rapidly and with minimalinterference with physiological processes.Time Course of IAA Uptake. When testing whether the uptake

of IAA has a saturable component, some of the experimental

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FIG. 8. Uptake of 5-[3H]IAA (Bq per 2 mm segment) after 4-minincubation in solutions (pH 5.0) containing 10-8 M 5-[3H]IAA plus ['H]IAA at different concentrations to give total IAA concentrations of 1O-8to 10-5 M. SE of the mean are shown by dotted lines.

conditions must be chosen with great care. The most criticalparameter is probably the incubation time. If the data are to beanalyzed using a model based on reaction kinetics, it is necessaryto find the initial rate of uptake. In this experiment, 2 to 7 mmsegments were incubated in medium at pH 5.0 for periods of 30s to 5 min. All results are corrected for a zero time incubation(segments plunged into medium and immediately rinsed).

First, when the curves in Figure 2 are compared it can be seen

that the uptake of IAA* declined for increasing total IAA con-centrations from 10-7M to 10-5 M. This indicates that at least acomponent of uptake was becoming saturated. Second, when theinitial uptake rates are considered (tangent to each curve at timezero), it can be noticed that for all concentrations of IAA testedthe uptake rate declined with time. This effect was most pro-nounced for the highest concentration tested (10 Mm), suggestingthat nonlinearity was mainly in the nonsaturable component. Itwas important to check this point since, to determine the kineticsof saturable uptake, it is essential to measure the rate of saturableuptake while it is linear. In addition, if the rate of uptake wasdetermined using longer incubations, the nonlinearity of theuptake curves obtained for higher concentrations with timewould appear to suggest that saturation of hormone uptake withconcentration took place.To separate the saturable and nonsaturable parts of uptake,

the following calculation was therefore made for each point alongthe curve: the nonsaturable component of IAA* uptake wassubtracted from the total uptake, and three difference curves

were obtained. The value obtained for 10- M IAA concentrationwas considered to show nonsaturable uptake, so the differencecurves comprise (10-8 M minus 10-5 M values), (10-7 M minus10-5 M values), and (10-6 M minus 10-5 M values). This choiceof 10-5 M as a saturating concentration will later be shown (Fig.5) to be valid. These curves (Fig. 3) therefore show the uptake ofIAA* by saturable process(es) as a function oftime. The saturableuptake shows a high degree of linearity (better than Fig. 2),indicating that the nonlinearity of Figure 2 is largely due tononlinearity of nonsaturable uptake. This can be explained by

an increase in the efflux of IAA* as the concentration inside thetissue increases (with time). From Figure 3 it can be seen thatthe values obtained by calculating uptake rate after 4 min incu-bation are very similar to those which would be obtained bydrawing a tangent at time zero. Thus, it is justifiable to use 4min incubation to study the kinetics of saturable uptake, andthis was the period chosen for further experiments.

Metabolism of IAA* during Uptake Experiments. The advan-tages of using short incubation periods are that IAA metabolismis reduced. In the present study, no metabolites could be detected(results not shown), by TLC of extracts prepared from rootsegments incubated 4 min in medium containing IAA*.HPLC analyses of a solution of IAA* and an extract prepared

from root segments which had been incubated in medium withIAA* indicate that the solution of IAA* (Fig. 4A) contained fewimpurities, with most of the radioactivity in the major peak(retention time 18.5 min). HPLC analysis of the radioactivity inthe root extract (Fig. 4B) indicted a major peak exactly co-chromatographing with the IAA standard, and minor peaks atretention times of 14, 15, and 16 min. When the extract shownin Figure 4B was prepared, 99.5% of the total radioactivity inthe tissue was extracted showing that metabolism of ethanol-insoluble conjugates was negligible. Preliminary experimentsshowed that if the carrier IAA° was omitted only 82.9% of theradioactivity was extracted (mean of 83.8% and 82.0%). Theextract contained few detectable metabolites, and they contrib-uted only about 5% to the total radioactivity (8% of totalradioactivity in minor peaks for root extract, as compared with3% for the original solution).

Similar results were obtained when the gradient used com-prised methanol 0 to 75% with pH 5.0 buffer (ammoniumacetate/acetic acid, containing 10 mM total acetate). The reten-tion time for IAA with this gradient was 15 min.

It has been shown that, when maize root segments are incu-bated for longer periods in radioactive IAA, several metabolitesare formed (18) especially in the stele (19). However in this study,after the 4-min incubation, detectable metabolites were few andin small quantities. It is, therefore, justifiable to use a measure-ment of radioactivity in the tissue after a 4-min incubation toassess the amount of IAA* present.A further advantage of short experiments is that aerenchyma

formation, which takes place after prolonged incubation of rootsin liquid media (13, 14), is avoided. Short incubation periodsgive lower levels of radioactivity in the tissue, and very shortexperiments lead to higher percentage errors in the incubationtime.The second major parameter which must be carefully selected

is the concentration of radioactive IAA. This must be low inorder to avoid saturation of low capacity, high affinity sites bythe radioactive hormone.Uptake of IAA* in the Presence of IAA° (0-20 gM).The uptake

of IAA* (5 nM) was determined using 2 to 7 mm segmentsincubated for 4 min with different (0-20 ,M) concentrations ofIAA°. A zero time incubation was performed for each concentra-tion (value approximately 0.2 Bq per segment) and these valueswere subtracted from the results of the 4-min incubations toallow for immediate adsorption to the surface of the tissue. Theradioactivity taken up per segment declined with increasingconcentrations of IAA° (Fig. 5) from 37 Bq per segment (noIAA°) to 10 Bq per segment for concentrations above 4 gM IAA.The decrease was particularly marked within the submicromolarconcentration range. These data indicate the presence of a satu-rable component of IAA* uptake, and a nonsaturable part.To calculate the apparent Km and VmD ofthe saturable process,

the nonsaturable component must first be removed. To deter-mine the radioactivity taken up by nonsaturable processes, themean value for all concentrations of IAAM from 4 AmM upwards





was calculated, and gave 10.043 Bq per segment. This impliesthat for 5 nM 3H-IAA alone 27.5% of the uptake is by diffusionand other nonsaturable mechanisms, and 72.5% enters via asaturable process or processes. Nonsaturable uptake probablycomprises diffusion into the cell wall, diffusion of IAA into thecytoplasm, and nonspecific, low affinity binding to different partsof the cell.

Before discussing the values of apparent Km and Vmax for IAAuptake obtained, the meaning of these terms in the context ofhormone transport should be mentioned. We must assume thatthere is a rate determining step in the transport process. Thisstep must be second order overall, that is first order with respectto IAA concentration and first order with respect to carriermolecules. No other molecular species should participate in therate determining step, which is a reasonable assumption if weare considering a uniport, but could be questionable for anantiport, or particularly in the case of a symport. It has beensuggested (8) that the IAA anion is transported across the plas-malemma into the hyaloplasm with two protons. If the protonsare involved in a rate-determining link between hormone andcarrier, this would influence the reaction kinetics.The uptake of IAA by maize roots could involve many com-

ponents: one or more saturable carriers and several factors con-tributing to nonsaturable uptake. The following analysis seeks todistinguish the saturable part of IAA uptake. This appears torepresent saturable influx at the plasmalemma and not diffusionfollowed by saturable binding of IAA at an intracellular sitebecause there is no lag in the time course of saturable uptake(Fig. 3). Our conclusion is confirmed by the effect of FCCP onIAA uptake (HV Martin, PE Pilet, unpublished data). Pretreat-ment of maize root segments with FCCP (10-4 M) had no effecton the uptake of IAA at I0-' M, whereas the uptake of IAA at 5nM was inhibited. Therefore, the saturable component of IAAuptake depends on the proton gradient across the plasmalemma.The nonsaturable part of IAA* uptake is subtracted from the

total uptake of radioactivity and the data for 0 to 1 AiM IAA areplotted according to Dixon (4), (Fig. 6). The equation of this lineis found to be (least squares regression; correlation coefficient =0.985):

l/v = 3.6375 i + 1.0556

This gives Km = 0.285 gM and Vm = 54.98 pmol/(g freshmass- min). This value of Km is lower than those previouslyreported: 0.65 gM for suspension cultures of crown gall cells atpH 5.0 (27), and approximately 0.6 Mm for maize roots at pH4.0 (1). The present Vm- is much lower than that found forcrown gall cells (1.0 nmol/(g. min)-') (27). The difference in Vmax

can be explained by the fact that the surface area in direct contactwith the medium per unit mass of tissue is smaller for rootsegments than for cell suspension cultures.A comparison beween the observed values ofKm and Vm_ and

the level of endogenous IAA in the tissue should give an indica-tion as to whether such a carrier could be of physiologicalsignificance. Twenty-eight ng IAA/g fresh mass of tissue has beenfound in elongation zones (2.5-5 mm region) of 15 mm longroots of LG 11 maize (24). The segments used in the presentexperiment were prepared from identical plants but using the 2to 7 mm region. Taking tissue density as 1 g cm-3, if IAA wasdistributed evenly through the tissue, this would correspond to aconcentration of 0. 16 uM. When this value is compared with thevalue for half saturation (Kin) of 0.285 Mm, it can be seen thatthe carrier has spare capacity which, however, is not excessive.This gives credence to the idea that such a carrier could have a

regulatory role.When the rate at which the carrier could operate is taken into

consideration, we obtain Vmdl, = 5.5 x 10-" mol (g fresh massmin)-'. If this is compared with the level of endogenous IAA in

1 g fresh mass of tissue (elongation zone) which is 1.6 x 10-10mol, the carrier would be capable (if substrate was in excess, andunder the experimental conditions described) of accounting foran uptake equivalent to 34.4% of tissue content per min. Asshown above, the carrier would normally be considerably lessthan 50% saturated and therefore might actually operate (underthe conditions of pH and temperature given above) at 10% orless of tissue content per min. Again this seems adequate forregulating IAA levels which might normally exist.

In the above analysis, a certain number of approximationswere necessary. The assumption was made that the concentrationof IAA applied is the concentration presented to the carrier.However, the IAA naturally present in the wall also plays a partand will compete with IAA* and applied IAA for uptake. Anotherproblem is that if the carrier transports auxin anions (27) thecritical concentration is not that of total IAA, but the concentra-tion of auxin anions. However, it is difficult to assess the latterbecause the wall may not attain the pH of the bathing medium(pH 5.0) within the 4-min incubation period.

Distribution of Saturable Uptake Carrier in Stele and Cortex.The uptake of IAA* (10-8 M) in the presence of IAA° at differentconcentrations was determined using 5 mm segments of isolatedstele or cortex cut 5 to 10 mm from the apical end of the root(Fig. IC). For both steles and cortices (Fig. 7) the radioactivitytaken up per segment declined with increasing total IAA concen-tration, indicating that, in both tissues, IAA uptake has a satu-rable component. These results are in contrast to previous results(1) where no saturability of IAA uptake was found for stelar orcortical tissue ofPhaseolus coccineus roots. This discrepancy canprobably be explained by the species difference and the fact thattheir experiment was performed at pH 4.0, whereas pH 5.0 wasused in the present study. Furthermore, their tissue was preparedfrom more basal regions of the root.A comparison of the results for steles and cortices in Figure 7

shows that the uptake per stele is about half that per cortex, andthis holds for all concentrations of IAA tested. Taking intoaccount the fresh mass of the segments (Table I) and expressinguptake as radioactivity per unit fresh mass, the value is abouttwice as high for steles as for cortices. A similar result is obtainedbased on tissue dry mass (from Table I). It is interesting toconsider whether this difference in total uptake reflects a differ-ence in saturable or nonsaturable uptake. The numbers next tothe points in Figure 7 indicate the percentage uptake of radio-activity compared with uptake from a 10-8 M solution (100%).These values were similar for stele and cortex, indicating that theratio of saturable to nonsaturable uptake is very similar in bothcases. This suggests that the Km values are similar for stelar andcortical segments, but that Vmav is greater for stelar tissues.However, the nonsaturable component of uptake is also greaterfor stelar tissue, so the difference in VmKa can perhaps be explainedby the geometry of the two types of segment.

Table I. Fresh and Dry Weights of5 mm Segments Citt 2 to 7 mmfrom the Apical End ofMaize Roots and ofCortical and

Stelar Segments Preparedfrom the Region 5 to 10 mm Couintedfrom Root Tip

Results are the means of 8 determinations. See Figure 1 for zones.

Five mm segments

Weight 2-7 5-10 5-10

B (cortex) (stele)C C2

mg ± SE ofmeans per 20 segmentsFresh 112 ± 2 89 ± 2 22.0 ± 0.4Dry 12.0 ± 0.3 7.3 ± 0.2 2.33 ± 0.06

893




The high level of IAA* uptake by stelar as compared withcortical segments per unit mass of tissue is attributable to twofactors: a high degree of uptake by nonsaturable processes and ahigh level of saturable uptake. Both of these factors may contrib-ute to the high level of IAA found in stelar as compared withcortical tissues (3, 7, 29).When the results in Figure 7 are compared with those obtained

for 2 to 7 mm segments (Fig. 5) and 10-5 M is considered to bea saturating concentration, uptake from 10-8 M solution is 64%saturable for cortices, 61% saturable for steles, and 68% saturablefor 2 to 7 mm segments. Therefore, at 10-8 M, the contributionof the saturable component to total uptake of IAA* is similar forthese different tissues. However, when considering tissue freshmasses (Table I) and calculating total uptake from a 10-8 M

solution from Figures 6 and 7 the following values are obtained(in pmol [g fresh mass 4 min]-'): 22.8 for steles, 12.4 for cortices,and 10.6 for 2 to 7 mm segments. This means that total uptakefrom 10-8 M solution expressed per unit fresh mass of tissue iscomparable for 2 to 7 mm segments and isolated cortical seg-ments, but is about twice as great in the case of stelar segments.

Distribution of Saturable Uptake Carrier Along the Root Axis.The uptake of IAA* was determined in the presence of IAA atdifferent total concentrations using 2 mm segments cut at differ-ent points along the roots (Fig. IA). The radioactivity taken up(Fig. 8) was greatest when the total IAA concentration was 10-8M (no IAA° supplied) and declined with increasing concentrationsof IAA°. This is in agreement with the results obtained usingother parts of the root, and indicates uptake by both saturableand nonsaturable processes.When 2 mm long segments were incubated in 10-8 M IAA,

uptake was greatest for the 2 to 4 mm region and uptake wasprogressively less for more basal segments. On the other hand,for segments incubated in 10-5 M IAA (total concentration), theuptake was similar throughout the 2 to 10 mm region whenexpressed per 2 mm segment (Fig. 8). However, the fresh massis greatest for basal segments (Table II) and if this is taken intoaccount, uptake per gram fresh mass was least for the basalsegments. If IAA uptake from 10-5 M solution is expressed pergram dry mass of tissue (from Table II) the uptake is greatest forthe 4 to 6 mm region.

Considering 10-5 M as a saturating concentration, it is againpossible to estimate the contribution of saturable and nonsatu-rable processes to IAA* uptake from a 10-8 M solution. Thecalculation is made as described for 2 to 7 mm segments, steles,and cortices. Along the root, going from tip to base, uptake wasfound to be 73, 62, 61, and 55% saturable for the differentsegments. These data for 2 to 4, 4 to 6, and 6 to 8 mm segmentsare in agreement with the value of 68% already calculated for 2to 7 mm segments. In addition, the percentages which werecalculated for cortical (64%) and stelar (61%) segments aresimilar to those for the corresponding 2 mm regions.Comparing the distribution of saturable and nonsaturable

uptake for 2 mm segments within the 2 to 10 mm region,nonsaturable uptake was similar throughout, whereas saturableuptake increased towards the root tip. This can be comparedwith the natural occurrence of IAA in the root: high levels of

Table II. Fresh and Dry Weights of2 mm Segments Cut at DifferentPositions Along the Maize Root Axis

Results are the means of 1O determinations. See Figure 1A for zones.

WeightTwo mm Segments


IAA were found in the cap and apex of maize roots (26).Similarly, when radioactive IAA was applied to maize shootapices or caryopses, it became concentrated in the apical 2 mmof the primary root ( 15, 16). This carrier could therefore play animportant part in controlling the accumulation in the elongationzone of IAA transported in the stele from the aerial parts of theplant.

It has recently been noted that if the activity of an uptakecarrier gradually increases along the root, then transport willoccur preferentially towards the region where the number ofcarrier sites is the greatest (17). The distribution of the IAAuptake carrier along the root reported here will in this waycontribute to the acropetal polarity ofauxin transport. Transportwould be polar even ifthe carrier was at the same density at bothends of the cell, but acropetal polarity would be greater if theuptake carrier was located preferentially at the basal end of thecells.Although the results reported here give evidence that a carrier

for IAA uptake plays an essential part in controlling IAA move-ment and IAA levels in the root tip, the location and activitiesof both uptake and efflux carriers may be important. Moreover,if both carriers are present in the same or neighboring cells, theiractions may be interdependent, and could be synergistic. Theuptake carrier will supply IAA to the efflux carrier if both arepresent in the same cell, thereby stimulating IAA effiux. Simi-larly, efflux of IAA into the cell wall will increase the activity ofthe uptake carrier in the same and nearby cells. These processeswould permit the precise regulation ofIAA exchanges and levelsin the root tip, and could play an essential role in the control ofroot growth and gravireaction.

LITERATURE CITED

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Growth rate distribution during response and the effects of applied auxins.J Exp Bot 8: 105-124

3. BRIDGES IG, JR HILLMAN, MB WILKINS 1973 Identification and localisationof auxin in primary roots of Zea mays by mass spectrometry. Planta 115:189-192

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5. ELLIOTr MC, MS GREENWOOD 1974 Indol-3yl-acetic acid in roots of Zeamays. Phytochemistry 13: 239-241

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7. GREENWOOD MS, JR HILLMAN, S SHAW, MB WILKINS 1973 Localization andidentification of auxin in roots of Zea mays. Planta 109: 369-374

8. HERTEL R 1983 The mechanism of auxin transport as a model for auxinaction. Z Pflanzenphysiol 112: 53-67

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13. KoNINGs H 1982 Ethylene-promoted formation of aerenchyma in seedlingroots of Zea mays L. under aerated and non-aerated conditions. PhysiolPlant 54: 119-124

14. KONINGs H, G VERSCHUREN 1980 Formation of aerenchyma in roots of Zeamays in aerated solutions, and its relation to nutrient supply. Physiol Plant49: 265-270

15. MARTIN HV, MC ELLIOTT 1984 Ontogenetic changes in the transport ofindol-3yl-acetic acid into maize roots from the shoot and caryopsis. Plant Physiol74: 97 1-974

16. MARTIN HV, MC ELLIOTT, E WANGERMANN, PE PILET 1978 Auxin gradientalong the root of the maize seedling. Planta 141: 179-181

17. MILBORROW BV, PH RUBERY 1985 The specificity of the carrier-mediateduptake of ABA by root segments of Phaseolus coccineus L. J Exp Bot 36:807-822

18. NONHEBEL HM, A CROZIER, JR HILLMAN 1983 Analysis of ['4C0indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using

2-4 4-6 6-8 8-10

mg ± SE ofmeans per 20 segmentsFresh 43.9 ± 0.4 46.7 ± 0.3 51.1 ± 0.5 55.7 ± 0.9Dry 5.4 ± 0.2 3.99 ± 0.09 4.1 ± 0.1 4.46 ± 0.08




reverse-phase high-performance liquid chromatography. Physiol Plant 57:129-134

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In PE Pilet, ed, Plant Growth Regulation. Springer-Verlag, Heidelberg, pp115-128

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25. RAVEN JA 1975 Transport of indoleacetic acid in plant cells in relation to pHand electrical potential graident, and its significance for polar IAA transport.

New Phytol 74: 163-17226. RIVIER L, PE PILET 1974 Indolyl-3-acetic acid in cap and apex of maize roots:

identification and quantification by mass fragmentography. Planta 120: 107-112

27. RUBERY PH 1978 Hydrogen ion dependence of carrier-mediated auxin uptakeby suspension-cultured crown gall cells. Planta 142: 203-206

28. RUBERY PH, AR SHELDRAKE 1974 Carrier-mediated auxin transport. Planta118: 101-121

29. SAUGY M, PE PILET 1984 Endogenous indol-3yl-acetic acid in stele and cortexof gravistimulated maize roots. Plant Sci Lett 37: 93-99

30. ScoTT TK, MB WILKINS 1968 Auxin transport in roots. II. Polar flux of IAAin Zea roots. Planta 83: 323-334

31. Scorr TK, MB WILKINS 1969 Auxin transport in roots. IV. Effects of light onIAA movement and geotropic responsiveness in Zea roots. Planta 87: 249-258

32. SUSSMAN MR, MHM GOLDSMITH 1981 Auxin uptake and action of N-1-naphthylphthalamic acid in corn coleoptiles. Planta 151: 15-25

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