Measurementof PhloemTransportRatesby an ... - Plant physiology · Plant Physiol. Vol. 94, 1990 this is the mostconvenient for phloemtransport studies. For amorecompletediscussion

Plant Physiol. (1990) 94, 455-4620032-0889/90/94/0455/08/$01 .00/0

Received for publication March 19, 1990Accepted May 22, 1990

Measurement of Phloem Transport Rates by anIndicator-Dilution Technique1

Donald B. FisherDepartment of Botany, Washington State University, Pullman, Washington 99164-4238

ABSTRACT

An indicator-dilution technique for the measurement of flowrates, commonly used by animal physiologists for circulationmeasurements, was adapted to the measurement of phloemtranslocation rates in the wheat (Triticum aestivum L.) peduncle.The approach is based on the observation that, during the trans-port of a given amount of solute, its mean concentration will beinversely proportional to flow rate. For phloem transport in thewheat peduncle, the necessary measurements are (a) the timecourse of tracer kinetics in the peduncle phloem, (b) the volumeof sieve tubes and companion cells in the monitored segment ofthe peduncle, and (c) the amount of tracer transported past thatpoint. The method was evaluated by in situ monitoring of 32PO4transport in pulse-labeling experiments. Specific activities (i.e.32p concentrations) of phloem exudate were in good agreementwith those calculated from in situ count rates and measuredphloem areas. Mass transport rates, calculated from volume flowrates and phloem exudate dry matter content, also agreed wellwith expected mass transport rates based on measurements ofgrain growth rate and net CO2 exchange by the ear. The indicator-dilution technique appears to offer good precision and accuracyfor short-term measurements of phloem transport rates in thewheast peduncle and should be useful for other systems as well.In contrast to velocities based on time-delay measurements, it ismore precise, more accurate, and produces an estimate of mean,rather than maximum, velocity. Also, since only a single detectoris required, it can be applied to very short transport paths.

Radiotracers are often used to measure the velocity ofphloem transport, usually by labeling a source and determin-ing the time to reach a detector, or by determining the timedelay between tracer appearance at two points along thetranslocation pathway. However, these approaches do notprovide a value for the mean velocity of movement along thephloem. The first method is subject to several uncertainties,including time delays associated with phloem loading, whilethe second applies only to the fastest-moving tracer. Theseinaccuracies are an important practical consideration in meas-uring phloem transport rates, since the volume rate of flow isthe product of the mean velocity and cross-sectional area offlow. Provided the sieve tube sap concentration is known, the

Supported by a National Science Foundation grant, PCM 83-04693, and by a U.S. Department of Agriculture Competitive Re-search Grant, 88-37262-3426.

2 Except in one instance (the rate of grain growth), the error limitsare ±SD.

455

volume rate of flow can be used to calculate the rate of masstransport, the most fundamental value for phloem transportrates.

Several factors contribute to the range of times taken fordifferent molecules to traverse the same distance along thepathway and, therefore, to the range of velocities observed.(The mean velocity is this distance divided by the mean transittime.) Solute exchange between sieve tubes and companioncells and the fact that many vascular bundles and hundredsof sieve tubes are involved are probably the main contributors(1). That there is a dispersion of velocities involved in phloemtransport has been recognized for some time; unfortunately,it is quite difficult to account for, as must be done to obtaina mean transport velocity. Probably the most promising ex-perimental and theoretical approach to the problem has em-ployed input-output analysis of kinetic data obtained by invivo monitoring of "C movement (10). However, aside fromcomplications arising from the short half-life of "C (whichmay be circumvented by the use of alternative tracers, at leastunder some conditions), the method requires monitoring attwo well-separated points along the phloem, so cannot beapplied to a short path.A promising alternative approach to measuring flow rates,

not yet employed with plants, is the use of 'indicator-dilution'techniques. This is an established methodology for the meas-urement of flow rates in the circulatory systems of animals(8). As the name suggests, it relies on following the concentra-tion oftracer ('indicator') in the flowing stream. For any givenamount of tracer transported, its mean concentration (dilu-tion) will be directly dependent on the volume rate of flow.Doubling the flow rate, for example, would halve the meantracer concentration in the stream. I have adapted this ap-proach to the measurement of phloem transport rates in thewheat peduncle by in vivo monitoring of 32P translocation.The method appears to provide accurate values of volumeflow rates along the phloem and, in conjunction with phloemexudate concentrations, of mass transport rates as well. Thus,the results allow assessment ofshort-term responses ofphloemtransport (here, solute movement into developing wheatgrains) to changing conditions of solute concentrations, tem-perature, partial degraining, etc.

MATERIALS AND METHODS

Theoretical

Several variants of the indicator-dilution method exist; thefollowing brief exposition will apply to pulse-labeling, since

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Plant Physiol. Vol. 94, 1990

this is the most convenient for phloem transport studies. Fora more complete discussion of the indicator-dilution methodand its application in animal physiology, see Larsen et al. (8).The topic is also covered in other treatments concerned withmathematical techniques in animal physiology (1 1, 12).The amount of solute transported by a flowing stream in a

time interval, At, is simply the product of concentration andflow rate. That is,

AI = CQAt

the product of phloem area (sieve tubes plus companion cells,= Aj; the requirement for including the companion cell areais summarized in the next section) and mean velocity, Vj,

= T

I. = V.A. Ci(t) dt.i (6)

Since Cj(t) = Rj(t)/Aj, where Rj(t) is activity per unit lengthof the jth vascular bundle, substituting for Cj(t) gives:

(1)where AI is the amount of solute transported during At, C isconcentration, and Q is the volumetric flow rate. Both C andQ may be time-dependent. During any period of time, T,there will be T/At = m intervals, and the total solute trans-ported, I, will be

m mI= 2AlJ,= 2C,QiAt. (2)

i=I i=I

Taking the limit as At approaches zero,

rT*= fo C(t)Q(t) dt. (3)

If the flow rate is constant,'T

I = C(t) dt. (4)

Solving for Q yields

Q T * (5)JC(t) dt

This is the Stewart-Hamilton equation, which provides arelationship between volume flow rate (not directly measur-able) and values which are experimentally accessible, so canbe used to calculate Q. (As used by animal physiologists, I isthe amount of tracer injected, hence the upper limit of theintegral in Equation 5 is set to infinity. Here, however, I refersto the amount of tracer that has been carried past the obser-vation point at time T.)An important assumption in deriving Equation 5 (actually

introduced in Eq. 1) is that tracer is evenly mixed across thestream, so the mean flow rate of tracer molecules is the sameas that of the stream. This condition is not met for phloemtransport in the wheat peduncle. Although diffusion providesessentially instantaneous mixing across individual sieve tubes,and microautoradiography likewise indicates uniform tracerdistribution across individual vascular bundles (6), tracerconcentrations are demonstrably different between vascularbundles generally and between those of the wheat pedunclespecifically. Thus, Equation 5 may be assumed with reason-able confidence to apply to transport within a particularvascular bundle, but not necessarily to the phloem as a whole.Furthermore, in monitoring the activity in a length of thepeduncle, one obtains activity per unit length rather thanconcentration (activity per unit volume). For the system athand, some further modification of Equation 5 is required.

If the volume flow rate in a vascular bundle is expressed as

rT.j= J'J Rj(t) dt.

Summing for n = all 40 or so vascular bundles,

n n rTI= zij= I J Rj(t) dt.

j=l j=l 0

(7)

(8)

If the mean velocity, V, is the same for all vascular bundles(this condition will be discussed in the context of the results),then solving for Vresults in an analog ofthe Stewart-Hamiltonequation which can be applied to phloem transport:

V= I

JR(t) dt(9)

where I is the total tracer transported (obtained by assayingtracer in the ear) and R(t) is the total count rate from allvascular bundles in a unit length of peduncle (corrected forrelative counting efficiency). The volume rate of flow may becalculated as the product of Vand total sieve tube-companioncell area, and the mass rate of flow by multiplying by sievetube sap concentration.A somewhat different approach may be taken if sieve tube

sap is collected from exuding aphid stylets, which provides aconvenient determination of specific activity on a weightbasis. Simply by changing the units in Equation 5, one maywrite

W= TIj'S(t) dt

(10)

where W is the mass flow rate and S(t) is activity per unitmass. Substituting S(t) = a(t)/g(t), where a(t) is the rate atwhich tracer is exuded and g(t) is the mass rate of exudation,

W= I

I t(t)d

and, if the mass exudation rate is constant,

fW= T i

a(t) dt

(1 1)

(12)

For an experiment of duration, T, the denominator is simplythe total radioactivity exuded from the stylet, and g is the dryweight of exudate divided by T. Note that Equation 12 mayperhaps be viewed more conveniently as stating that the mean

456 FISHER



PHLOEM TRANSPORT RATES BY AN INDICATOR-DILUTION TECHNIQUE

specific activity of tracer transported to the ear is the same asthat collected in the stylet exudate, i.e.

rT

, Jofa(t) gt (13)WT gT

Again, possible differences between vascular bundles must beconsidered in the use of Equations 12 and 13 to calculatetransport rates.

Phloem Area Measurements

In calculating volume flow rate as the product of meanvelocity and area (Eq. 6 et seq.), the relevant area is that ofthe sieve tubes plus companion cells, rather than of the sievetubes alone (1). The reason for including the companion cellarea lies in the fact that, although flow occurs only in thesieve tubes, the mean velocity of solutes will be decreased byany time spent in a nonmoving pool. Microautoradiographyand negative staining (2) demonstrate that solute exchangetakes place readily between the sieve tubes and companioncells and, further, that concentrations in the two are quitesimilar. In a sieve tube-companion cell pair, then, the proba-bility that a solute molecule will be moving or stationary isgiven by the fraction of area occuppied by each of the twocell types. The mean velocity would be the weighted averageof moving solutes (sieve tube) and stationary solutes (com-panion cell).The cross-sectional area of phloem (sieve tubes and com-

panion cells) in the peduncle was measured in fresh sectionsstained with I2-Kl to enhance the visibility of phloem, asdescribed previously (6). However, a more accurate correctionto this figure for the fraction of area occupied by cell wallswas obtained from electron micrographs ofrepresentative veincross-sections. Sieve tube and companion cell protoplast areaswere measured by digital planimetry and the wall areas wascalculated as the difference between the phloem area andprotoplast areas.

Plant Material

Plants of Triticum aestivum L. (cv. Sun 9E) were grown ina growth chamber on a 16 h photoperiod at a light intensityof 450 uE m-2 s-'. Temperature was 22°C during the day and1 6°C at night. The plants were grown in pots containing equalvolumes of perlite, vermiculite, and potting soil, and wereirrigated automatically with water at 16 h intervals and witha complete nutrient solution once a week. Ears were taggedat anthesis and used for experiments 15 to 25 d later. Grainfilling was complete at about 30 d postanthesis.The time course ofgrain dry matter accumulation for these

conditions of plant growth was determined by harvesting earsat various times from 1 to 40 d postanthesis, drying themovernight at 80C, and weighing the grains.

Indicator-Dilution Experiments

Labeling experiments were run in the laboratory at 22 to25°C under a water-filtered metal halide lamp providing illu-

mination of about 800 ,uE m-2 s-'. Plants were brought to thelaboratory and placed under the light at least 2 h beforelabeling. For 32P, about 0.5 cm2 of the flag leaf, about 3 cmfrom the tip, was abraded with moist diatomaceous earth and100 ,uCi Of 32P04 in 10 to 15 ML of 10 mM K-PO4 buffer [pH7], was applied to the abraded area. After most of the labelingsolution had been absorbed into the leaf, the abraded areawas covered with Parafilm. After about 30 min, the distalthird of the leaf was excised. This labeling protocol produceda well-defined pulse of radioactivity without affecting sievetube sap concentration (monitored by following aphid styletexudate osmolarity (3)).

Radioactivity in the peduncle was monitored by a dualchannel ratemeter equipped with thin end-window Geiger-Muller detectors shielded with lead to admit radiation onlyfrom a 1 cm segment of the peduncle. The detectors werepositioned either on opposite sides of the same length of thepeduncle or, to obtain a conventional velocity measurement,as far apart as possible along the peduncle (8-12 cm). Inseveral experiments, one detector monitored total 32p in thedistal two spikelets of the ear, while the other monitoredactivity in the peduncle.

After the pulse had mostly passed the detectors, the pedun-cle and ear were dried overnight at 80°C. A scaler with a thinend-window detector was used to assay radioactivity in the 1cm segment of monitored peduncle, the peduncle above thispoint, the rachis, the glumes, and the grains. The latter werefirst ground to a fine powder with a mortar and pestle, andweighed subsamples were counted. The integration requiredfor determining the translocation velocity (see Eq. 9) wasperformed by measuring the area under the recorder trace forthe peduncle kinetics with a digital planimeter. The result wascorrected for the relative counting efficiency (usually about40-60%) between the ratemeter (in vivo count rate) and scaler(dried tissue) and Equation 9 was used to calculate the meantranslocation velocity in the peduncle. When two positions ofthe peduncle were monitored, a translocation velocity wasalso calculated from the mean of the time differences between(a) the times to first appearance of tracer at the two positionsand (b) the times to reach one-half the peak value at eachposition (6). In all experiments, the volume flow rate was alsocalculated as the product of velocity and phloem area (seebelow for measurement) and, when desired, the mass flowrate was calculated from pedicel phloem exudate osmolality(see below).

In experiments where stylet exudate specific activity wasused to calculate mass transport rates (from Eq. 12), 15 to 20exuding stylets were established on the peduncle by severingthe stylets of Rhopalosiphum padi (L.) by radiofrequencymicroautery (3, 4). For 32P experiments, labeling was accom-plished as described above. For 14C experiments, the distalthird of the flag leaf was allowed to photosynthesize for 10min in 50 ,Ci of '4C02 and the labeled area was excised 30min later. Previously accumulated exudate was washed offjust before labeling; during the experiment (5 h), exudate wasallowed to dry on the peduncle surface. At the end of theexperiment, the exudate from each stylet was dissolved in asmall amount of water and placed on squares of aluminumfoil preweighed to ±1 Mg on a Cahn electrobalance. After

457



Plant Physiol. Vol. 94,1990

drying overnight at 80°C, the foil squares were reweighed. Thescaler and thin end-window detector were used to assay 32pdirectly in the dried exudate, while 14C was assayed in a liquidscintillation counter after dissolving the exudate in 0.5 mL ofwater. To determine the total 14C translocated, tissue sampleswere extracted with 80% aqueous ethanol followed by hotwater, and an aliquot of the combined soluble activity wasassayed for 14C in a liquid scintillation counter. Starch wasthen hydrolyzed with amyloglucosidase for assay of "1C.

Collection of Phloem Exudate from Broken GrainPedicels

Phloem exudate concentration and specific activity wasdetermined in several experiments by collecting exudate fromthe surface of broken grain pedicels (5). Evaporation wasprevented during collection by flooding the pedicel surfacewith mineral oil. Exudate volume was determined by trans-ferring the sample to mineral oil in a Petri dish and measuringthe droplet diameter with an ocular micrometer.

Relationship of Phloem Exudate Osmolality to Dry MatterContent

Due to the small volumes usually obtained, the dry mattercontent of phloem exudate was estimated from cryoscopicmeasurements. Previously, this relationship was based onanalytical measurements which accounted for most of theosmotic content of the sieve tube sap (3). The relationshipwas established more directly for the present experiments bycollecting approximately 50 mg (dry weight) of pedunclephloem exudate from severed stylets and measuring the os-molality-dry weight relationship for successive dilutions.Starting with the highest concentration, osmolality was meas-ured with a nanoliter osmometer (Clifton Technical Physics)and a 5 kiL aliquot was removed and dried, with washings, ona preweighed square of aluminum foil. After repeating atsuccessive dilutions, exudate dry weights were determinedafter 3 h in an oven at 80°C. Similar measurements weremade for dilutions of 'synthetic' phloem exudate (0.6 Msucrose, 0.3 M glutamine, and 0.025 M KCl; compositionbased on earlier measurements (3)).

CO2 Loss/Gain by the Wheat Ear

The contribution to the carbon balance from CO2 loss orgain by wheat ears under these experimental conditions wasdetermined from changes in the CO2 concentration in a closedsystem, as measured with an infrared gas analyzer. A wheatear was sealed into a chamber under the experimental lightconditions and, after admitting enough CO2 to raise theconcentration to 400 to 420 ,uL L', the system was closeduntil CO2 had dropped to about 300 ,L L' (about 8 min),whereupon the chamber was darkened until the CO2 increasedto about 450 ,uL L' (about 5 min). The system volume (0.42L) and the slopes of the CO2 traces were used to calculaterates of repiration and photosynthesis. Respiration rates wereindependent of CO2 concentration; however, photosyntheticrates often slowed at lower CO2 concentrations. Photosyn-thetic rate was calculated from the slope at 330 ,uL CO2 L'.

RESULTS AND DISCUSSION

Typical Kinetics and Sample Calculation

A representative recorder trace for 32P kinetics in the pe-duncle is shown in Figure 1. Typically, activity was initiallydetected about 1 h after labeling, and reached a maximum 30to 45 min later. It then declined more slowly to about 15 to30% of the maximum count rate, whereupon it remainedfairly constant, though still decreasing. At this point, usually4 to 5 h from initial labeling, the experiment was terminated.In some (infrequent) cases, probably caused by greater thanusual basipetal 32P movement in the leaf xylem, decline afterthe peak count rate was not so pronounced. Since this in-creased the possible error in subsequent calculations (seebelow), experiments were usually discarded if the rate did notdecline by at least 60% from the peak.

Since the integration required by Equation 9 applies onlyto tracer in the phloem, some correction must be applied toaccount for activity which has been lost to surrounding tissues.An experimental approach to this question will be presentedbelow, where it will be shown that tracer in the phloemaccounted for about 20 to 50% of the count rate at the endof an experiment. However, even without that figure, it canbe noted that it will not greately influence the value obtainedby integration. It may reasonably be assumed that, relative tothe several-hour duration of the experiment, tracer loss willbe permanent and, further, that the rate of loss would beproportional to the amount present in the phloem. Giventhese assumptions, the amount of tracer outside the phloemat any time would be proportional to the integrated value ofthe tracer in the phloem until that time, i.e. the amount oftracer outside the phloem would show a sigmoid-shaped timecourse reaching a maximum at the experiment's end. This isshown by the lower curve in Figure 1, which was calculatedon the assumption that one-third of the final count rate

, I I

l0

7 8 - ~~~TotalIE

o4

2 Non-phloem (calculoted)-,,

50 100 150 200Time (min)

Figure 1. Tracer kinetics in a 1 cm segment of the peduncle afterpulse-labeling the flag leaf with 32P04. Time zero is the time at whichtracer was first detected; the trace is a time-smoothed reproductionof the recording. The kinetics for nonphloem tracer were calculatedby assuming irreversible loss from the phloem, with one-third of theactivity still in the phloem at the end of the experiment. Total amountof tracer transported was 4.72 x 1 05 cpm. An illustrative calculationof velocity using these data is provided in the text.

458 FISHER




represented tracer still in the phloem. The correction requiredto account for activity outside the phloem, represented by theshaded area, will not be greatly influenced by uncertaintyconcerning the proportion of counts outside the phloem aslong as (a) there is a substantial decrease in counts from themaximum, demonstrating that most counts were moving inthe phloem in any event, and (b) the experiment does nottake overly long, unduly increasing the uncertainty of thecorrection. Furthermore, the justifiable degree of accuracy inthe integration required to account for the correction can beperformed visually. That is, tracer outside the phloem willreach half the final value when equal areas under the pedunclecurve lie to the left and right; several similarly determinedpoints provide an adequate basis for completing the plot.Another ambiguity arises concerning the value of I, the

total amount of tracer transported, in Equation 9. Although32PO4 is an excellent tracer for photosynthate movement inthis system (5, 9), it clearly is not a perfect one (e.g. CO2production and refixation) and, in addition, at least somemovement may occur in the xylem. Again, however, thepotential error is not large, since the grains, which importonly via the phloem (14), accounted for by far the largestamount of activity (about 85-90%). Most of the remaindercan also be reasonably presumed to have been phloem trans-ported. Nevertheless, the glumes represent the major transpir-ational surface in the ear, and consistently contained appre-ciable amounts of the total tracer. This proportion was some-what higher in illuminated than in darkened ears (9.2 + 4.0%,n = 15 versus 6.5 ±3.1%, n - 37)2. The latter figure iscomparable to the proportion of tracer in the glumes ofdarkened ears when 14C was used (4.5 ± 0.2%, n = 4), so mayrepresent mostly phloem import; the source of 32P reachingilluminated glumes is less clear. Given these ambiguities andthe relatively small amount of tracer involved, activity in theglumes was ignored in calculating the flow rate from Equation9, i.e. it was assumed that I was the sum of activity in therachis and grains.

Velocity is calculated from the data of Fig. 1 as follows:

Area under the curve for 'Total' cpm

= 1.51 x 104 cpm h cm-'

Correction for non-phloem cpm (shaded area)

= -0.30 x 104 cpm h cm-'

Corrected integral (non-shaded area)

= 1.21 x 104cpmhcm-'

From Equation 5, V

Table I. Calculated versus Measured Values of Phloem ExudateSpecific Activities

Calculated values are based on peduncle count rates and phloemareas. Measured values were obtained from pedicel phloem exudatescollected (a) just after tracer in the peduncle reached its peak or (b)after most of the decline was complete.

Sampling No. of Relative Specific ActivityTime Experiments (Measured/Calculated)

% SD

At peak 15 98 19After decline 11 34 17

large ones around the peduncle, so account for about half thenumber of bundles but only about 25% of the phloem area.To determine whether there was any systematic differencebetween large versus small bundles, a flag leaf was pulse-labeled with "4CO2 and the specific activities of accumulatedstylet exudates were determined after 5 h. The vascular bundleinto which each stylet was inserted was determined by ex-amination of unstained fresh sections. Considerable differ-ences were noted in the specific activities of exudate collectedfrom individual stylets (see the final section); however, therewas clearly no systematic difference between large versus smallbundles (data not shown). Furthermore, there was never anyindication in monitoring the peduncle during 32P experimentsthat there might be streams with markedly different velocities(i.e. there was no double-peaking, and only slight spreadingof the profile occurred between detectors). Finally, there wasgood agreement between measured exudate specific activitiesand those calculated from experimental count rates and totalphloem area (next section).The fraction of the phloem cross-sectional area occupied

by walls was 20% in a large vascular bundle and 26% in asmall one. Thus, the total phloem area was multiplied by 0.78

0to

'20E

0C0

V

(D10

E

(4.72 x I05 cpm)(1.21 x 104 cpm h cm')

= 39.0 cm h-'.

Phloem Area

Observations from earlier experiments, based partly onmicroautoradiographs (6), raised the question of whether thesmaller vascular bundles in the peduncle were active inphloem transport. These small bundles tend to alternate with

0 100Time (min)

Figure 2. Time course of accumulated tracer in the terminal 4 grainsof a wheat ear after pulse-labeling the flag leaf with 32P04. Time zerois the time at which tracer was first detected; the trace is a time-smoothed reproduction of the recording. The rate of accumulation(slope) is assumed to reflect specific activity in the translocationstream at that time; thus, the ratio of final to maximum rate shouldreflect relative phloem specific activities at those times. This ratio iscompared with phloem exudate specific activities in Figure 3.

459




1_1~~ I I 0I0E.0.2

0~~~~~~~~_ ,'

0~~~~0 /

~~0.l /~U 0.1 /,'_

'I ,' 0

o/w 0

i / I I I0 0.1 0.2 0.3

Import Rate Ratio (final/max.)

Figure 3. Comparison of two estimates of relative phloem specificactivities during the course of 32P experiments. Ratios on the abscissawere estimated from import rates (see Fig. 2); ordinate values arefrom phloem exudate collected after the experiment and the maxi-mum specific activity calculated from phloem area and peduncle countrate. The strong correlation between the two (r = 0.82) indicates thatphloem exudate specific activity at the end of the experiment was agood indicator of the proportion of tracer in the phloem then.

to give the actual area involved in transport (i.e. sieve tubeplus companion cell protoplast area).

Specific Activity of Tracer in the Translocation Stream:Calculated Versus Experimental Values

Two approaches were taken to compare calculated to ex-perimental values of specific activities during the course of32P experiments. One was based on phloem exudate samplingand the other on the rate of tracer import by grains in the ear.Phloem exudation offered the most direct means of vali-

dating the specific activity values calculated from pedunclecount rates and phloem area. Exudate from grain pedicelswas used for this purpose since, compared to stylet exudate,it has the advantage of mixing the contents of many sievetubes and, to an extent, of different vascular bundles, thusproviding more representative values of overall concentra-tions. Comparisons of calculated and measured specific ac-tivities were made for two conditions: (a) shortly after thepeduncle count rate reached its peak, when all of the tracershould still be in the translocation stream, and (b) after it haddeclined to its ending value, when the proportion of activityoutside the stream would be greatest. In the first instance,sampling of pedicel exudate was delayed until about 10 minafter the count rate at the detector had reached a maximum,to allow the peak of activity to move into the rachis. (Giventhe time course of a typical 32P experiment, the phloemspecific activity would remain within 5% of its maximumvalue for about 15 min, and within 10% for about 35 min.)During the next 10 to 15 min, exudate samples were obtainedfrom four to six spikelets in the central part ofthe ear. Resultsof these experiments are summarized in Table I. Substantialvariation was encountered in replicate exudate samples. Forsamples taken when the peduncle count rate reached its peak,

the mean coefficient of variation was 17%, with a range of 10

to 37%. (Samples taken after the count rate had declined werepooled, rather than quantified separately.) Calculated andmeasured specific activities at the peak were nevertheless ingood agreement. As expected, exudate specific activities afterthe peduncle count rate had declined were substantially lessthan the calculated values, indicating that most of the tracerremaining in the peduncle at that time was outside thephloem. On average, radioactivity in the peduncle declinedby 75% (±10%, n = 20) from its maximum value in experi-ments where final exudate specific activity was measured.Although it might be reasonably anticipated that a greaterdecline might indicate a lower proportion of radioactivity inthe phloem at the experiment's end, there was no evidentcorrelation between the two.

Observations on the rate of tracer import by grains in theear confirmed the general validity, at least, of the estimates ofthe proportion of tracer still moving in the translocationstream at the end of the experiment. As expected from thetracer kinetics in the peduncle, total tracer in the grainsfollowed a sigmoidal time course (Fig. 2). An estimate of theproportion of tracer moving in the phloem at the end of theexperiment, relative to the peak rate, can be obtained bycomparing the maximum rate of accumulation with that atthe end of the experiment. These are the slopes indicated inFigure 2, and their ratio (final slope/maximum slope) iscompared in Figure 3 to the ratio of final phloem exudatespecific activity (measured) to the calculated maximum spe-

cific activity. Although the ratios are not equal, this may beascribed partially, at least, to the time lag between the pedun-cle and ear, and to some distortion of count rate in the grainsby the greater importance of self-absorption there. Signifi-cantly, however, there was a strong correlation between theratios (r = 0.82), indicating that the measured values for

60

_ 40

0

4.

qJ 20c:

0

._

0 20 40Indicator-Dilution Flow Rate

(jjl h-')

60

Figure 4. Comparison of two methods for calculating translocationflow rates in the wheat peduncle. Velocity was estimated in one casefrom the time delay between kinetic profiles at two points along thepeduncle, 8 to 12 cm apart. In the other, velocity was calculated fromthe indicator-dilution equation (Eq. 9); both values were multiplied byphloem area to give volume rates of flow. The lowest flow rates wereachieved by partially degraining the ear.

I I

0

°o'00,

08 //cPa

°,00/0 0

L o' I I

460 FISHER




100 200Concentration (;jg jjV1)

A series of experiments compared the two approaches formeasuring flow rates in the peduncle phloem; the results areshown in Figure 4. Again, the earlier method consistentlyoverestimated the translocation rate. In these experiments,the overestimate was not so pronounced (about 20-25%). Thecloser agreement is at least partly due to an undercorrectionfor walls in the phloem area (previously estimated to be only7%, versus the 22% measured here). There may also havebeen some improvement in accuracy from the more distincttracer pulse achieved in the present experiments.

Accuracy and Reproducibility of Transport Rates Basedon Indicator-Dilution Measurements

300

Figure 5. Relationship between osmolality and dry matter content ofphloem exudate, and comparison to similarly-obtained values for'synthetic exudate' based on earlier analytical data. Dry matter valuesfor dilutions of the synthetic exudate agreed with expected values to+1%.

exudate specific activities did, in fact, provide meaningfulestimates of the proportion of tracer still moving in thephloem.

Comparison of Methods for Estimating Volume FlowRates in the Phloem

In earlier experiments (6) translocation rates in the wheatpeduncle were estimated by following 32P kinetics at twopoints along the peduncle. While they provided useful resultson relative rates under two different conditions (two sets of32P measurements were obtained from the same plant) theabsolute rates of translocation were obviously high by 30 to100%, and expected light/dark differences were not detected.At least part of this systematic overestimate arose from thevelocity measurement (described in "Materials and Meth-ods"), which unavoidably emphasizes faster-moving tracer.

In one series of experiments, as complete a set of measure-ments as possible was obtained to evaluate the accuracy andreproducibility of the indicator-dilution method. This in-cluded monitoring the peduncle at the same point with twodetectors, measurements ofear photosynthesis and respirationrates, and sampling of pedicel phloem exudate at the end ofthe experiment for specific activity (% oftracer in the phloem)and osmolality (for conversion to dry weight, using the exper-

imentally determined relationship shown in Fig. 5). Measure-ments were made for darkened ears, illuminated ears, and on

peduncles in which about half the vascular bundles were

severed before labeling (shown by Wardlaw and Moncur notto affect transport rate into the ear [ 13]).

Expected transport rates, to which the measured rates werecompared, were calculated on a per grain basis from thegrowth rate per grain (1.94 ± 0.12 mg d-' grain-' [5% confi-dence interval, n = 74] =81 gg h-' grain-') plus the rate ofCO2 gain (loss) by photosynthesis (respiration) as glucoseequivalents. On average, net photosynthesis was 18 ± 6 sg

h-' grain-' (n = 15) while dark respiration averaged 21 ± 6,gg h-' grain-' (n = 15). (These values are in good agreementwith those assumed in earlier calculations [6].) The expectedtransport rate in the light should have been about 63 ug h-'grain-' and, in the dark, 102 usg h-' grain-'. (Darkening hasno evident effect on grain filling rate [7; also, see 6].) However,

Table II. Calculated versus Expected Mass Transport Rates in the Wheat PeduncleCalculated rates are based on indicator-dilution measurements. Expected rates are based on grain

growth rate and ear gas exchange measurements.

Treatment No. of Exudate Calculated ExpectedExperiments Osmolality Rate" Rate

mOsm Agh-' grain-1A. Using specific activity on a volume basis (Eq. 9; 32P04 labeling)

Ear darkened 4 941 ± 86 112.0 ± 19.8 101.2 ± 6.7Ear illuminated

Peduncle intact 6 980 ± 33 62.1 ± 4.9 61.8 ± 3.8Approximately half of vascular 4 993 ± 38 59.2 ± 5.2 60.1 ± 4.0

bundles severedbB. Using specific activity on a weight basis (Eq. 12; 14C02 labeling)

Ear darkened 4 165 ± 50 1 02Ca Integrations of duplicate peduncle tracer kinetics (32P experiments) rarely differed by more than 5%,

so were not included in the error estimate. b Calculations were based on the phloem area of intactbundles. c Average value; gas exchange measurements were not made in these experiments.

1200

800 .

E0E2-

0

E0

I I I I I

O Phloem exudate

* Synthetic exudate 20.6M sucrose0.3M glutamine *0.025M KCI

I I

400 L

-LI I

1,n

461




calculated ear demand was based in each experiment on thegas exchange measurements for that ear.

Results from these experiments are shown in Table IIA.The measurements clearly showed the expected differencesbetween net ear demand in illuminated versus darkened ears,

and their absolute values were in good agreement with ex-

pected rates. Severing more than half the vascular bundles inthe peduncle had no detectable effect on the rate of transportinto the ear or on sieve tube exudate concentration.

Several "4C02 labeling experiments (Table IIB) were run toassess the feasibility of using exudate specific activity on a dryweight basis for the calculation of transport rates, as suggestedby Equation 12. Considerable variation was encountered inthis approach. In general, the values were higher than expectedrates, but not significantly so. The lack of precision in themethod clearly lay in the wide range of exudate specificactivities from different stylets; the coefficient of variation forstylet specific activity in the experiments ranged from 40 to65% of the mean specific activity. Only two experiments were

run using 32p, since it was anticipated that unevenness in32P04 uptake into the abraded leaf would be even more likelyto result in variability. This did not seem to be the case, andthe calculated rates were more comparable to expected valuesthan in the 14C experiments. However, the variability was stillsimilar to that found in the 14C experiments (data not shown).

CONCLUSIONS

At least in the case of the system used in these experiments,the indicator-dilution method provided a convenient andaccurate approach to the short-term measurement of phloemtransport rates. In contrast to conventional velocity measure-

ments, the method not only yields a mean value for thevelocity, which is more significant physiologically, but alsohas the potential for use with very short pathways (i.e. thepath only has to be long enough to allow placement of a singledetector). Its usefulness in other systems remains to be seen.

In general, its main limitation would appear to arise from thepossibility of preferentially labeling streams of a particularvelocity class (cf Eqs. 8 and 9; this was a concern for the largeversus small vascular bundles in the wheat peduncle). Anotherobvious example would be a case in which bidirectional

transport was occurring in the same axis, unless it was clearwhich phloem area was involved and there was no recircula-tion. However, the possibility of error arising from systematiclabeling differences remains even when transport would beoccurring only in one direction (e.g. petioles, fruits, etc.).Each system would have to be evaluated for this.

LITERATURE CITED

1. Christy AL, Fisher DB (1978) Kinetics of '4C-photosynthatetranslocation in morning glory vines. Plant Physiol 61: 285-290

2. Fisher DB (1978) The estimation of sugar concentration inindividual sieve-tube elements by negative staining. Planta 139:19-24

3. Fisher DB (1987) Changes in the concentration and compositionof peduncle sieve tube sap during grain filling in normal andphosphate deficient wheat plants. Aust J Plant Physiol 14: 147-156

4. Fisher DB, Frame JM (1984) A guide to the use of the exuding-stylet technique in phloem physiology. Planta 161: 385-393

5. Fisher DB, Gifford RM (1986) Accumulation and conversion ofsugars by developing wheat grains. VI. Gradients along thetransport pathway from the peduncle to the endosperm cavityduring grain filling. Plant Physiol 82: 1024-1030

6. Fisher DB, Gifford RM (1987) Accumulation and conversion ofsugars by developing wheat grains. VII. Effects of changes insieve tube and endosperm cavity sap concentrations on thegrain filling rate. Plant Physiol 84: 341-347

7. Jenner CF (1968) Synthesis of starch in detached ears of wheat.Aust J Biol Sci 21: 597-608

8. Larsen NA, Henriksen 0, Sejrsen P (1983) Indicator methodsfor measurement of organ and tissue blood flow. In JT Shep-herd, FM Abbond, eds, Handbook of Physiology. AmericanPhysiological Society, Bethesda, MD, pp 21-64

9. Marshall C, Wardlaw IF (1973) A comparative study of thedistribution and speed of movement of 14C assimilates andfoliar-applied 32P-labelled phosphate in wheat. Aust J Biol 26:1-13

10. Minchin PEH, Troughton JH (1980) Quantitative interpretationof phloem translocation data. Annu Rev Plant Physiol 31:191-215

11. Sheppard CS (1962) Basic Principles of the Tracer Method. JohnWiley & Sons, New York

12. Simon W (1972) Mathematical Techniques for Biology andMedicine. MIT Press, Cambridge

13. Wardlaw IF, Moncur L (1976) Source, sink and hormonal con-trol of translocation in wheat. Planta 128: 93- 100

14. Zee SY, O'Brien TP (1970) A special type of tracheary elementassociated with "xylem discontinuity" in the floral axis ofwheat. Aust J Biol Sci 23: 783-789

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Measurementof PhloemTransportRatesby an ... - Plant physiology · Plant Physiol. Vol. 94, 1990 this is the mostconvenient for phloemtransport studies. For amorecompletediscussion