Plant Physiol. (1974) 54, 886-891

Evidence for Active Phloem Loading in the Minor

Veins of Sugar Beett

Received for publication April 5, 1974 and in revised form August 15, 1974

SusAN A. SOVONICK,2 DONALD R. GEIGER, AND ROBERT J. FELLOWS3Department of Biology, University of Dayton, Dayton, Ohio 45469

ABSTRACT

Phloem loading in source leaves of sugar beet (Beta vulgaris,L.) was studied to determine the extent of dependence onenergy metabolism and the involvement of a carrier system.Dinitrophenol at a concentration of 4 mM uncoupled respira-tion, lowered source leaf ATP to approximately 40% of thelevel in the control leaf and inhibited translocation of exoge-nously supplied '4C-sucrose to approximately 20% of the con-trol. Dinitrophenol at a concentration of 8 mM inhibited ratherthan promoted C02 production, indicating a mechanism ofinhibition other than uncoupling of respiration. The 8 mMdinitrophenol also reduced ATP to approximately 40% of thelevel in the control source leaf and reduced translocation ofexogenous sucrose to approximately 10% of the control.Application of 4 mM ATP to an untreated source leaf promotedthe translocation rate by approximately 80% over the control,while in leaves treated with 4 mM dinitrophenol, 4 mM ATPrestored translocation to the control level. No recovery oftranslocation was observed when ATP was applied to leavestreated with 8mM dinitrophenol. The results indicate an energy-requiring process for both phloem loading and translocationin the source leaf.

Application of '4C-sucrose solutions in a series of concentra-tions through the upper surface of a source leaf produced abiphasic isotherm for translocation out of the fed region. Asimilar dual isotherm was obtained for phloem loading withleaf discs floated on 14C-sucrose solutions. The first and pos-sibly the second phases were attributed to active, carrier-mediated accumulation in the minor vein phloem. Autoradi-ography of the tissue confirmed that most of the sucrose waslocalized in the minor veins. Data from uptake through theabraded surface of intact leaves, the most reliable method, were

analyzed by the Hofstee method. Kinetic parameters, analogousto Km and Vmax of enzyme studies, were calculated to be: K, =

16 mM and Jmax = 70 ,g C/min dm2 or 490 nmoles sucrose/min-dm. Rates for phloem loading and translocation of ex-

ogenous sucrose are equal to or greater than those observedfor compounds derived from photosynthetically fixed C02. Thedata indicate that a free space sucrose concentration in theregion of the minor vein phloem of approximately 20 mMcan support translocation at the rates commonly observed forphotosynthetically produced sugars.

'This work was supported by National Science FoundationGrants GB-33803 (D.R.G.) and GZ-2460 (S.A.S.).

2 Present address: Harvard Forest, Petersham, Mass. 01366.3 Present address: Department of Botany, University of Illinois,

Urbana, Ill. 61801.

The source leaf, a sugar-exporting region of a plant, seemsto play a key role in providing the driving force for long dis-tance transport of organic solutes throughout the plant. Fewtranslocation studies have been focused on events in the sourceleaf, and some crucial questions remain unanswered. Is there,for example, an energy-requiring step in the transport of sugarfrom chloroplasts to sieve elements that is directly related tophloem loading and long distance transport. If an active stepis demonstrated, do the kinetics of uptake indicate that thisstep involves a membrane carrier?

In 1939, Curtis and Asai (3) suggested that the osmoticgradient from the mesophyll to the sink tissue was in the oppo-site direction required for a pressure flow mechanism. Roeckl(26) observed that the mesophyll cells, which produce mobilesugars, have a substantially lower osmotic potential than thephloem exudate. A gradient of increasing concentration towardthe sieve tubes would require energy to drive transport in thatdirection, suggesting the need for an active step between sugarproduction and translocation out of source leaves. Barrier andLoomis (1) termed the active step "loading," implying that therate-limiting step is transport into the veins, rather than asimple chemical transformation or synthesis of the translocate.There now seems to be general agreement that an active stepdoes occur in source tissue (10, 17, 22, 32). Several lines ofinvestigations have been pursued in order to substantiate thishypothesis, including the presence of a reverse gradient of su-crose in the leaf (11, 26), promotion of translocation by ATP(17, 18, 31), and inhibition by metabolic inhibitors (13, 16).

In addition to a need for metabolic energy, the participationof a carrier was further indicated in some translocation (16)and phloem loading (2, 30) studies by the presence of satura-tion kinetics. These studies, performed on a variety of ma-terials, support the operation of an active carrier. In this studythe data presented support the participation of at least one ac-tive, carrier-mediated step in phloem loading and translocationof exogenously supplied sucrose.

MATERIALS AND METHODS

Sugar beets (Beta vulgaris, L. cv. Klein E-type hybrid) weregrown for 5 to 6 weeks by solution culture as previously de-scribed (8). Translocation and phloem loading were measuredby monitoring the uptake or transport of "C-sucrose which wassupplied (a) to source leaf discs, (b) to the source leaf of anintact plant by reverse-flap feeding, or (c) through the abradedsurface of an intact leaf. Translocation was measured continu-ously throughout an experiment as described previously (9).The distribution of label within the tissue was visualized withautoradiography (5); invariably, the majority of label wasfound in the vascular tissues.

886

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021

ACTIVE PHLOEM LOADING IN SUGAR BEET

Reverse-Flap Feeding. Approximately 12 hr before an ex-periment, leaves were removed from the plant except for asingle source leaf approximately 0.5 dM2 in area and one sinkleaf approximately 0.1 dM2 in area. The plants were then placedin darkness for the normal night period. Immediately before anexperiment, plants were placed in dim light to reduce compet-ing effects from photosynthetically produced sugars and ATP.One end of a first-order vein of the source leaf was dissectedfree to produce a flap near its junction with the midrib, andthe flap was inserted into a small capillary tube filled with 100mM '4C-sucrose in 5 mm KH2PO4 buffer, pH 6.5. Uptake of thelabeled sucrose solution was measured periodically on a scaleplaced next to the capillary tube. A GM tube-ratemeter combi-nation positioned at the sink leaf monitored long distance trans-port of label into the sink leaf as described previously (8).Translocation rates were evaluated from the ratemeter output,sucrose specific radioactivity, and monitoring efficiency, usinga Fortran curve-fitting program on a Univac 70/7 computer.

Several experimental designs utilized this means of supplyinglabel, including ATP and dinitrophenol treatments and a su-crose concentration series. In the ATP and DNP4 experiments,the plants were supplied with a control solution of buffered4C-sucrose for 120 min. A steady state condition was attainedin which the rate of solution uptake and the arrival of labeledtranslocate in the sinks were relatively constant. A constant ar-rival rate in the sink implies isotopic saturation of the sucrosepools in the source and path regions as well as a steady rate ofphloem loading (9). In the ATP experiments, the control solu-tion in the capillary was withdrawn with a microliter syringeand replaced with a solution containing 4 mm ATP in additionto the labeled sucrose. The effect of ATP on the translocationrate was noted when a new steady rate was reached. In theDNP experiments, the control sucrose solution was changed toone containing sucrose and varying concentrations of DNP.This treatment was continued for about 2.5 hr. until the inhi-bition of the translocation rate was maximal. In most DNP ex-periments, the capillary solution was changed three times, DNPbeing finally replaced by 4 mm ATP in order to study recoveryfrom inhibition. The effect of increasing sucrose concentrationon the translocation rate was studied in two stages, from 10 to40 mm and from 40 to 160 mm. Within each stage, the sucroseconcentration was increased after a steady state rate of trans-port in the plant had been reached with the previous concentra-tion.A characteristic of this technique is widespread diffusion of

label from the point of application, a fact which makes the ex-porting area almost indefinable. Since the loading area wasdifficult to determine, the translocation rates with ATP andDNP were expressed as cpm/min, rather than as mass transferrates. However, an estimate of the loading area based on auto-radiographic evidence was made in the case of the sucrose con-centration series. Absolute rates of transport (,ug C/min dm2)were calculated as previously described (9), using the specificradioactivity of the '4C-sucrose.The reverse-flap capillary method was ultimately abandoned

in favor of an abraded-epidermis technique because the latterwas more reproducible. In the capillary method diffusioncaused the solutions supplied to the free space to be diluted toan unknown degree. This fact necessitated the use of relativelyhigh concentrations of DNP and ATP. In addition, the rateof solution uptake was reduced at high solute concentrations.A decrease in the translocation rate was correlated with this

'Abbreviation: DNP: dinitrophenol.

reduction. Finally, the source-leaf sucrose pools were difficultto saturate with isotope when the label supply rate was slow.ATP levels were assayed in the ATP and DNP experiments

using a modified luciferin-luciferase system (7). The respirationrate was measured as the rate of CO2 release, using the closedsystem developed by Geiger and Swanson (9). During therespiration measurements, experimental solutions were appliedto severed leaves via the petiole instead of the veins in order toproduce large enough changes in CO2 production to be detectedwith the apparatus used.

Abraded-Epidermis Feeding. The abraded-epidermis methodeliminated most objections to the reverse-flap method, since alarge volume of solution was supplied continuously to the freespace of the tissue, and the area of supply was clearly defined.

Plants were trimmed as described previously and wereplaced in the experimental hood overnight. Prior to the begin-ning of an experiment, the upper surface of a source leaf wasabraded over an area of 0.1 dM2 by rubbing gently with 600mesh Alundum paste, then rinsed with distilled water. Thesource leaf was then sealed with cord-type caulking compoundinto the supply apparatus which consisted of two chambers(Fig. 1). The lower section supplied a constant flow of air to thelower surface of the leaf, while the upper section supplied a"C-sucrose solution to the abraded portion of the upper leafsurface. The solution was introduced into the solution chambervia three inlet syringes. The sucrose solution was static andsimply bathed the leaf surface during an experiment. This ap-paratus was used to test the effect of increasing sucrose concen-tration on the translocation rate, using basically the same pro-cedure as in the capillary concentration series.Phloem Loading Isolated from Translocation: Sucrose Up-

take by Leaf Discs. Sucrose uptake by leaf discs was measuredto study more closely the process of phloem loading, as distinctfrom long distance transport. Discs were cut which excludedlarge veins and included primarily mesophyll and minor veintissue. Uptake times were kept short to avoid artifacts of over-loading the tissue, because the larger sinks had been removed inthis procedure.A standard source leaf was selected, and the upper epidermis

removed as described. One-centimeter square discs were cutfrom the interveinal areas with a razor blade and stored on 5mm potassium phosphate buffer, pH 6.5. Each experiment wasrun in triplicate with control and treatment sets of discs. Thediscs were floated on a series of "C-sucrose solutions, ranging

.1

FIG. 1. Chamber for applying a solution to the top of a sugarbeet leaf.

.887Plant Physiol. Vol. 54, 1974

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021

SOVONICK, GEIGER, AND FELLOWS

from 10 to 400 mm. Each disc was placed on 50 ,ul of solutionto take up labeled sucrose for 30 min; the uptake period wasfollowed by 30 min of exodiffusion, which removed most of thefree space sugars. Next, the discs were thoroughly rinsed withdistilled water and digested with H202 and perchloric acid at75 C for 1 hr. The samples were then counted on a NuclearChicago liquid scintillation system Model 6804. The countrates were converted to absolute amounts of radioactivity by aninternal standard method. The amount of sucrose absorbed wascalculated using the specific radioactivity of the sucrose sup-plied.Two additional experiments were performed. In the first, the

effect of a low temperature (O C) on the uptake process wastested by cooling the leaf discs with ice in a cold room. The sec-ond treatment investigated the effect of anaerobiosis on the up-take process. Before the experiment began, all solutions werepurged with nitrogen, and the entire apparatus, including leafdiscs, was kept in an atmosphere of nitrogen for 30 min. Theexperiments were performed in a glove bag, purged with nitro-gen both before and during the experimental period.

RESULTS

Effect of DNP Supplied to Source Leaf via Xylem on Respi-ration, ATP Level, and Translocation Rate. Although DNP isbest known as an uncoupler of oxidative phosphorylation, atrelatively high concentrations it can inhibit respiration andcause nonspecific tissue degradation. Consequently, we deter-mined at what concentrations DNP caused uncoupling insource leaves of sugar beet. CO2 production and ATP levelwere used as indicators of respiratory metabolism. Preliminaryexperiments, reported in Table I, indicated that concentrationsof DNP in the range of 1 mm were required to increase CO2production when the inhibitor was supplied via the xylem. Thisrelatively high level was probably necessary because of tissuedilution, a factor mentioned previously. Concentrations of 0.4and 0.8 mm DNP increased the rate of CO2 production onlyslightly, while 4 mm DNP increased the rate to approximately210% of the control. A concentration of 8 mm caused aninitial rapid rise in CO2 release of approximately the same mag-nitude as observed with 4 mm DNP, but this increase was fol-lowed by an inhibition of respiratory CO2 release. The finalrate after 2 hr was 40% of the stimulated rate and approxi-mately 90% of the control.

Table I. Effect of Various Concenitr-aionis of DNP Siupplied to

Soirce Leaf Blade by' Rever.se-Flap Capillary Method oln CO2Release, A TP Level, anld Tr-anislocationi of 100 m-i

14C SuctroseATP at 4 mm concentration was applied after approximately 2 hr

of DNP treatment. Translocation rates are averages with ranges in

parentheses and represent the maximum or minimum values aftertreatment.

DNPt'oncn

Mm.

Rate of C 2 |ATP Minimum

Praeofucton in Source TranslocationProduction ]ILeaf Blade' Rate +DNP

I0%

AlaximumTranslocation Rate-DNP, +ATP

0 100 100 100 183 (162-224)0.4 135 77 (75-78) 147 (110-183)0.8 1384.0 209 43 21 (2-42) 97 (79-123)8.0 (233) 239 36 9 (3-15) 11 (0-22)

After 3-hr treatment with ATP.2 Initial rate after 25 min.

Z Z 0806

o7- 20 04

02OL

100fz

2 75a-

zc 5cz0

, 25-J2

i00()u< n

DNP ATP

o I

DNP

ATP

OUT OF SOLUTION

- ---

-

5I.

200 400TIME (MIN)

600

FIG. 2. Effect of 4 mm DNP on the rate of translocation of"4C-sucrose supplied to the source leaf via reverse flap method. Atthe second arrow the sucrose solution containing DNP was re-placed with one containing 4 mm ATP. Typical graph depicting oneof 10 experiments of this type.

The 4 and 8 mm concentrations of DN P appeared to affectsignificantly the respiratory metabolism of the source leaf andthe ATP levels measured in that tissue. Both DNP concentra-tions lowered the ATP level to approximately 40% of the con-trol level after 3 hr of treatment (Table I). The significance ofthis degree of inhibition beyond the fact that energy metabo-lism is lowered is difficult to interpret because the ATP leveldepends on ATP utilization and production. The respirationand ATP data indicate that 4 mm DNP uncouples respiration,while 8 mm DNP inhibits the oxidation of respiratory sub-strates.The effect of DNP on the translocation of '4C-sucrose sup-

plied exogenously by the capillary method is also shown inTable I. The extent of inhibition again depended on the DNPconcentration employed. At 0.4 mm, DNP inhibited transloca-tion to a small extent, while a concentration of 4 mm decreasedthe translocation rate to approximately 20% of the controlrate. A typical time course for inhibition of sucrose transportby 4 mM DNP is shown in Figure 2. DNP at 8 mm inhibitedtranslocation from the source leaf almost completely. A highdegree of variability between plants was noted at all DNP con-centrations, which was probably due to individual differencesin sensitivity to the inhibitor and in the degree of its penetra-tion.

Effect of 4 mM ATP Supplied to Source Leaf via Xylem on

ATP Level and Translocation Rate. The effect of ATP on thetranslocation rate was studied using the capillary technique inboth DNP-inhibited and untreated plants. Figure 2 illustratesthe recovery of the translocation rate when ATP is added to

the source leaf previously treated with 4 mm DNP. The ca-

pacity of ATP to restore the rate of transport after inhibitiondepended on the degree of inhibition and on the concentrationof DNP applied (Table I). In control plants and those treatedwith 0.4 mm DNP, the added ATP increased translocation rates

to a level higher than the control. In the plants treated with 4

mm DNP, the ATP restored translocation to the control level,but ATP had little or no effect after 8 mm DNP treatment. Thestimulation generally lasted until the ATP solution was re-

moved. Promotion of the translocation rate in leaves treatedwith 4 mm ATP was accompanied by an increase in the ATP

concentration to 197% of that in the control leaves (Table I).The data from experimental treatment with DNP and ATPindicate the involvement of an active carrier in translocation.

::_

Plant Physiol. Vol. 54, 1974888

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021

ACTIVE PHLOEM LOADING IN SUGAR BEET

EFFECT OF SUPPLYING A SERIES OF SUCROSECONCENTRATIONS TO FREE SPACE

Kinetic Studies. To investigate the possible presence of asucrose carrier, saturation of phloem loading with increasingsucrose concentration was studied. The results obtained when aseries of "C-sucrose solutions was supplied to the free space ofsugar beet are shown in Figure 3. Both phloem loading in leafdiscs and translocation from intact leaves yielded the same typeof biphasic curve; apparently both segments show saturation.The data from Figure 3 for the abraded epidermis and disctechniques were analyzed according to the conventions of en-zyme kinetics by Hofstee plots (rate versus rate divided by con-centration). Data were separated into two groups, above andbelow the break in the curves for each technique. The bi-phasic nature of the curves was tested by analysis of covariancewhich in each case yielded a probability of less than 0.005 thatdata from the two segments were from the same system. Thedata for the capillary technique are provided for comparativepurposes but were not analyzed quantitatively because of thetechnical difficulties discussed previously. In addition, the in-tact plant technique did not strictly fulfill the usual require-ments for a Michaelis-Menten analysis. Steady state ratesrather than initial rates were used, and the internal sucrose wasremoved by long distance transport, once it had been accumu-lated. Since the analysis is not strictly analogous to those usedin enzyme kinetics, the flow terms, Kj and Jmax will be used inpreference to Km and V.,, (24). These calculations are pre-sented in Table II.

It can be seen that the Kj values for system I show muchmore variability than do the J1i.ax values for that system. TheKj for the disc data may be too high, due to a buildup of su-crose in the cells resulting from excision of the major sinks. Theresultant increase in leakage of label would cause the apparentuptake of sucrose to decrease at a given sucrose concentration.Both system II parameters (Kj and Jmax) display considerablevariability, to be discussed later. Whole tissue autoradiographyrevealed that most of the sucrose supplied accumulated in theminor veins by whichever method was used to supply the label.

Inhibitor Studies. Next, the role of energy in the phloemloading-step of the transport system was investigated with theleaf disc system. The uptake of sucrose by phloem loadingwithout translocation was determined under anaerobiosis andat 0 C. Inhibition was effective under both conditions over thewhole range of sucrose concentrations employed (Table II).

z 150

125-S~~

91 25 A7750

25

0 100 200 300 400SUCROSE CONCENTRATION (mM)

FIG. 3. Dependence of sucrose uptake on the concentration of"C-sucrose supplied by reverse flap capillary method (Q), throughthe abraded surface of source leaves (O), and through the surfaceof source leaf discs (A). Each point represents averages, reverseflap method of 5 replicates, intact leaf data of 3 replicates and discdata of 25 replicates.

Table II. Ki anzd Jma Values for Suigar Uptake anid TranislocatiolnRates for Both Sucrose Uptake Systems in Suigar Beet

Data from intact leaves and leaf discs under various treatments.

l__ System System II

Method of Sugar Treatmt Rate ParameterPresentation en Aleasured

Kj Jnmax Ki Jrmax

l C/min*2dii2 'C/n*n, din2Abraded epi- None Transloca- 16 70 620 330

dermis tion

Floating None Sugar uptake 88 66 2f0 140discs Cold Sugar uptake| 66 10 190 25

Anaero-I Sugar uptake! 94 23 190 40biosisI

The inhibition of phloem loading under anaerobic conditionsclosely resembled the inhibition of translocation in intact plantsunder the same conditions (unpublished data). This observationindicates that inhibition of long distance translocation primarilyreflects the process of loading and not a combination of pathand loading phenomena. A Q10 was calculated for the chillingdata at each sucrose concentration according to the van't Hoffequation. The average Q,, over the whole range of concentra-tions was 1.73, a value near the lower end of the range forthermobiological reactions. Since chilling also increases mem-brane viscosity, however, the Q., value does not unequivocallyindicate an active step.

DISCUSSION

The results obtained indicate that 4 mM DNP primarilylowers the energy status of the tissue, when supplied to thesource leaf of sugar beet. Data in support of this conclusion in-clude the stimulation of respiratory CO2 release as evidence ofuncoupling, the decrease in ATP level of the tissue, and thereversal of the effects of DNP on translocation by the additionof exogenous ATP. Thus, the decrease in translocation duringtreatment with 4 mm DNP was probably induced by a reduc-tion in ATP availability because its production was inhibitedby the uncoupling action of DNP. DNP at 8 mm, on the otherhand, inhibited oxidative metabolism and inhibited transloca-tion irreversibly despite the addition of ATP. Membrane dam-age or callose deposits may be responsible for these secondaryeffects through the production of toxic byproducts when res-piration is inhibited.

Previous studies have shown that DNP inhibits the uptakeof sucrose by castor bean cotyledons and its translocationwithin the seedling (16). Data on the inhibition of translocationby DNP as found by Qureshi and Spanner (25) and Harel andReinhold (13) also support the concept of active phloem load-ing as a necessary part of the translocation process, althoughthe latter work is somewhat difficult to interpret because ofthe methods by which sucrose and DNP were applied. It shouldbe noted that, although different conclusions were reached bythese workers concerning the role of energy in path tissue, aDNP inhibition of loading is evident in both cases. Thesestudies failed to demonstrate whether inhibition was caused bycytological damage or by a reduction in energy supplies be-cause the effect of the inhibitor on metabolism was not de-termined. The present study shows clearly that DNP appliedto the source leaf blade can both uncouple respiration and slowdown the translocation of exogenously supplied sucrose froma source leaf, an effect which is reversible.

889Plant Physiol. Vol. 54, 1974

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021

SOVONICK, GEIGER, AND FELLOWS Plant Physiol. Vol. 54, 1974

Not only does exogenous ATP effectively reverse DNP inhi-bition, but it also promotes translocation from uninhibitedsource tissue. Similar conclusions were reached previously byKursanov and Brovchenko (18) and Shiroya (28), using 14CO2-derived sucrose and "C-sucrose, and by Ullrich (31), usingfluorescein as tracer material. The nondestructive method ofmeasuring translocation used in the experiments described al-lows the time-course of the translocation rate to be observedthroughout the experiment. A continued supply of exogenous

ATP is required to promote translocation. Apparently, ATP is

necessary for short distance transport of sugar into the minor

vein phloem, but it is not known exactly how the moleculeplays this role. Possibly it promotes phloem loading by acting

as an anion (15), a source of energy for a sucrose carrier, a

phosphorylating agent, or even at the membrane level, by com-

bining with a membrane, causing it to change in conformation(29).

If ATP energizes a carrier directly, the proportion of theATP production of a leaf necessary to support a phloem-load-ing process in our sugar beet leaves can be calculated usingassumptions. We observed an average of 2.5 sieve element-companion cell complexes per minor vein and measured an

average combined perimeter of 126 Mm for the cells in the com-plexes of the minor veins sampled. Since there are 7 X 10° ,tmof minor veins/cm" of blade (6), we calculate 8.8 10' Mm2 ofcell surface/cm2 of blade, or 0.88 cm2 of surface/cm2 of blade.With a measured average translocation rate of 2.8 nmoles su-

crose/min cm' of blade, the calculated flux is 3.2 nmoles su-

crose/min cm2 of membrane. Based on a1 to 1 stoichiometry

for moles ATP utilized per sucrose transported, the flux wouldrequire 3.2 nmoles of ATP/min cm' of membrane or 2.8nmoles ATP/ min, cm2 of blade. Expressing the photosyntheticcarbon fixation rate in ATP equivalents, a photosynthetic rateof 160 Mg C/ min dM2 is equivalent to 22 nmoles of glucose/mincm2 of blade; at 38 nmoles of ATP per nmole of glucose,this fixation rate would potentially yield approximately 850nmoles of ATP/mincm2 of blade. Only 0.3% of this ATPwould be needed to supply ATP for loading. Based on the same

ATP yield per mole of hexose, approximately 1.4% of thetranslocated sucrose could supply the ATP required for phloemloading. Thus, it appears that metabolic energy is adequate fora short distance transport process with a membrane carrierdirectly powered by ATP. The membrane flux calculated aboveis several orders of magnitude above the reported values forpassive permeation (4) which itself indicates an active carrier.The high phloem-loading rates are particularly notable in viewof the high solute content of the minor vein phloem (11).The saturation curves (Fig. 3) provide further evidence that

an active carrier system operates in source leaf tissue. Manystudies, performed on ion uptake, have produced similar multi-phasic uptake curves and have generated a controversy over thephysical location of the two uptake systems (14). Some workershave proposed that both systems of ion uptake are in theplasmalemma, while others believe that the high affinity system(systemI) is in the plasmalemma, but the low affinity system(systemII) operates in the tonoplast. The present study indi-cates that both systems are located in the plasmalemma becauseboth produce sucrose loading into the translocation streamrather than transfer into the vacuole. In addition, it is mostprobable that the biphasic curve represents a single carrierpresent in the plasmalemma of the cells of the vascular system,rather than two carriers operating in series or in parallel. Thisconclusion is based on solute distributions in sugar beet leaves(11), as well as on the feasibility of the other possible distribu-tions (14, 21). The changes postulated in a single carrier en-zyme toproduce a biphasicuptake curve may involve isozyme-

like differences in the carrier enzyme, induced by the externalsubstrate concentration (19, 20, 23).The Kj for system I (Table II, abraded-epidermis uptake

data) is close to the Kin found by Cataldo (2) for sucrose up-take by isolated minor veins from tobacco leaves and fallswithin a range of Kj values reported by Linask and Laties (20)for 3-0-methyl glucose uptake by potato slices. However, sys-tem II data in the present study are not consistent with a num-ber of reports of a nonsaturating second phase (2, 16, 30).These studies stand in contrast to the work of Linask andLaties (20), who found multiple saturations of uptake. A non-saturating second phase could reflect diffusional uptake inducedby high external sugar concentrations, a possibility ruled outhere by the high sucrose concentration (approximately 0.8 M)observed in the minor vein phloem (11). However, the highconcentration of exogenous sucrose presented to the free spacemay have altered membrane permeability in some way. The Kjand J

.. , for system II show a wide spread (Table II), with theabraded-epidermis system yielding higher values. This factmay reflect an accumulation of sucrose within the isolated sys-tem or may mean that system II does not actually saturate, asdescribed by MacRobbie (21). No kinetic values have been re-ported elsewhere as high as those for SystemlI, and the systemmay not function under the usual physiological conditions.When the uptake by leaf discs is inhibited by either cold or

anaerobic conditions, reasonable changes occur in the calcu-lated Kj and J..a-, values (TableII). While the maximum uptakerate is much reduced when the tissue is placed in cold or undernitrogen, the Kj stays essentially the same. Thus, under condi-tions of limited ATP or lessened membrane flexibility, the rateof flux is reduced, but the carriers show the same saturation asunder control conditions. Much less variability is seen in sys-temI, and the figures for that segment are probably more ac-curate.

It is important to relate the data obtained with exogenouslysupplied sucrose to those obtained for similar leaves translocat-ing the products of photosynthesis. The rates of translocationof photosynthate obtained for young mature leaves by Servaitesand Geiger (27) are well within the range of system 1 rates. Thedata from the abraded-epidermis technique (Fig. 3) indicatethat a concentration of 20 mm in the free space is sufficient tosupport translocation at the rates observed for export of photo-synthate (27). SystemII is probably not of functional signifi-cance under ordinary circumstances.

Little work has been done to determine whether the rate oftranslocation of photosynthate saturates with increasing availa-bility of photosynthetic products. Using increasing light intensi-ties to raise the level of photosynthesis, Habeshaw (12) found asaturation of translocation at around 125Mug C/min dm'. Thisfigure is equivalent to approximately 87% of the carbon fixa-tion rate at the same time. Using younger leaves, Servaites andGeiger (27) found no comparable saturation, when photo-synthesis rates were manipulated by altering light intensityand CO2 andO2 concentrations. Even though the maximumphotosynthesis rates were high (approximately 250,ugC/mindm2), only around 20% of the carbon fixed was beingtranslocated in that study. It may be that younger leaves,though capable of higher net photosynthesis rates, do not trans-locate at limiting rates because much of the carbon fixed is di-verted to needs within the leaf. The absence of evidence forsaturation in the study of Servaites and Geiger (27) may alsobe the result of a feedback effect. As photosynthetic productionof sucrose increases, the level of sucrose in the free spacewould be expected to increase, increasing phloem loading andestablishing a steady state level of sucrose somewhat higherthan the previous level. Thus, the rate of production of sucrose

890

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021

or delivery to the loading site rather than the rate of phloemloading could limit translocation.The data presented in this paper support the existence of an

active, carrier-mediated transport system capable of loadingsucrose from the free space. Our data have been interpretedon the assumption that sucrose supplied exogenously to thesource leaf enters the vascular tissue in the same way as sucroseformed endogenously. If the free space is not a normal com-ponent of the short distance pathway in source leaves, the con-clusions reached above may be erroneous. A second paper fol-lows which concerns the role of the free space in short distancetransport in sugar beet source leaves and supports the interpre-tations presented here.

Ackniowledgmetnts-We thank Drs. John Milburn and Martin Zimmermann fortheir helpful (liscussions and their critical review of this work.

LITERATURE CITED

1. BAIRRIER, G. E. ANI) W. E. Looiss. 1957. Absorption an(d translocation of2,4-diclilorophenoxyacetic acid and 32P by leaves. Plant PhSysiol. 32: 225-231.

-2. CATALDO, D. A. 1974. Vein loading: the role of the symtplast in intercellulartransport of carbohydrate between the mesophyll and the miiinor veins oftobacco leaves. Plant Physiol. 53: 912-917.

3. CURTIs, 0. F. AND G. N. ASAI. 1939. Evidence relative to the supposed per-meability of sieve tube cytoplasm. Amer. J. Bot. 26: 16s-17s.

4. EDELMAN, J., A. I. SCHOOLAR, AND W. B. BON-N-ER. 1971. Permeability ofsugar-cane chloroplasts to sucrose. J. Exp. Bot. 22: 534-545.

5. FELLOWS, R. J. AND D. R. GEIGER. 1974. Structural and physiological changesin sugar beet leaves during sink to source conversion. Plant Physiol. 54:877-885.

6. GEIGER, D. R. AND D. A. CATALDO. 1969. Leaf structure and translocation insugar beet. Plant Physiol. 44: 45-54.

7. GEIGER, D. R. AND A. L. CHRISTY. 1971. Effects of sink region anoxia on

translocation rate. Plant Physiol. 47: 172-174.8. GEIGER, D. R. AND C. A. SWANSON. 1965. Sucrose translocation in the sugar

beet. Plant Physiol. 40: 685-690.9. GEIGER, D. R. AND C. A. SwANsoN. 1965. Evaluation of selected parameters

in a sugar beet translocation system. Plant Physiol. 40: 942-947.10. GEIGER, D. R., J. MALONE, AND D. A. CATALDO. 1971. Structural evidence for

a theory of vein loading of translocate. Amer. J. Bot. 58: 672-675.11. GEIGER, D. R., R. T. GIAQUINTA, S. A. SOVONICK, AND R. J. FELLOWS. 1973.

Solute distribution in sugar beet leaves in relation to phloem loading andtranslocation. Plant Physiol. 52: 585-589.

12. HABESHAW, D. 1969. The effect of light on the translocation froni sugar-beetleaves. J. Exp. Bot. 20: 64-71.

891

13. HAREL, S. AND L. REINHOLD. 1966. The effect of 2,4-dinitrophenol on trans-location in the phloem. Physiol. Plant. 19: 634-643.

14. HIGINBOTHAM, N. 1973. The mineral absorption process in plants. Bot. Rev.39: 15-69.

15. HuMPHREYS, T. R. AND L. A. GARRARD. 1971. Stucrose leakage from themaize scutellum: evidence for the participation of the phloem. Phyto-chemistry 10: 981-995.

16. KRIEDEMANIN, P. AND H. BEEVERS. 1967. Sugar uptake an(d translocationin the castor bean seedling. I. Characteristics of transfer in intact andexcised seedlings. Plant Physiol. 42: 161-173.

17. KURSA'NOv, A. L. 1963. Metabolism and the transport of organic substancesin the phloem. Adv. Bot. Res. 1: 209-278.

18. KURSAN-Ov, A. L. A.ND M. I. BROVCHENKO. 1961. Effect of ATP on the entryof assimilates into the conducting system of sugar beets. Soy. Plant Physiol.8: 211-217.

19. LEON-ARD, R. T. AND T. K. HODGES. 1973. Characterization of plasma mem-

brane-associated adenosine triphosphatase activity of oat roots. PlantPliysiol. 52: 6-12.

20. LiNASK, J. AND G. G. LATIES. 1973. Multiplhasic absorption of glucose and3-0-methyl glucose by aged potato slices. Plant Physiol. 51: 289-294.

21. MAcRORRIE, E. A. C. 1971. Fluxes and compartmentation in plant cells.Annu. Rev. Plant Physiol. 22: 75-96.

22. 'MILTHORPE, F. L. AND J. MIOORBY. 1969. Vascular transport and its signifi-cance in plant growth. Annu. Rev. Plant Physiol. 20: 117-138.

23. NISSEN, P. 1971. Uptake of sulfate by roots and leaf slices of barley: nmediatedby single, multiphasic mechanisms. Physiol. Plant. 24: 315-324.

24. NOBEL, P. S. 1974. Introduction to Biophysical Plant Physiology. W. H.Freeman and Co. San Francisco. pp. 133-138.

25. QURESHI, F. A. AND D. C. SPANNER. 1973. The influence of dinitrophenolon phloem transport along the stolon of Saxifraga sarmentosa. Planta 111:

1-12.26. ROECKL, B. 1949. Nachvweis eines Konzentrationshubs zwischen Palisadenzellen

und Siebrihren. Planta 36: 530-550.27. SERVAITES, J. C. AND D. R. GEIGER. 1974. Effects of light intensity and oxygen

on photosyntlhesis and translocation in sugar beet. Plant Physiol. 54: 575-578.

28. SHIROYA, 'M. 1968. Comparison of upward and downward translocation of 14Cfrom a single leaf of sunflower. Plant Physiol. 43: 1605-1610.

29. STUART, D. M. 1973. Reduction of water permeability in potato tuber slicesby cyanide, ammonia, 2, 4-dinitrophenol and oligomycin and its reversalby adenosine 5'-triphosphate and cytidine 5'-triphosphate. Plant Physiol.51: 485-588.

30. TURKINA, M. V. AND S. V. SOKOLOVA. 1972. 'Membrane transport of sucrose

in plant tissue. Soy. Plant Physiol. 19: 912-919.31. ULLRICH, WV. 1962. Zur Wirkung von Adenosintriphosphat auf den Fluorescein-

Transport in den Siebrohren. Planta 57: 713-717.32. ZIMMERMANN, 'M. H. 1969. Translocation of nutrients. Ini: 'M. B. Wilkins,

ed., The Physiology of Plant Growth and Development. McGraw-HillPublishing Co., New York. pp. 383-417.

Plant Physiol. Vol. 54, 1974 ACTIVE PHLOEM LOADING IN SUGAR BEET

Dow

nloaded from https://academ

ic.oup.com/plphys/article/54/6/886/6074149 by guest on 17 Septem

ber 2021