4C-Photosynthate in Morning Glory - Plant Physiology · Kinetic studies using '4C frequently require destructive sampling of at least several plants, an ap-proach which introduces

Plant Physiol. (1978) 61, 283-290

Kinetics of _4C-Photosynthate Translocation in Morning GloryVines"' 2

Received for publication April 1, 1977 and in revised form September 8, 1977

A. LAWRENCE CHRISTY3 AND DONALD B. FISHER4Department ofBotany, University of Georgia, Athens, Georgia 30602

ABSTRACT

The movement of '4C-photosynthate in morning glory (Ipomea nilRoth, cu. Scadet O'Hara) vines 2 to 5 meters long was followed bylabeling a lone mature leaf with 14CO2 and monitoring the arrival rateof tracer at expanding sink leaves on branches along the stem. To a firstapproximation, the kinetic behavior of the translocation profiles resem-bled that which would be expected from movement at a single velocity("plug flow") without tracer loss from the translocation stream. Therewas no consistent indication of a velocity gradient along the vine length.The profile moved along the vine as a distinct asymmetrical peak whichchanges shape only slowly. The spatial distribution of tracer along thevine reasonably matched that predicted on the basis of the arrivalkinetics at a sink, assuming plug flow with no tracer loss. Theseobservations are in marked contrast to the kinetic behavior of anymechanism describable by diffusion equations.

However, a progressive change in profile shape (a symmetricalwidening) was observed, indicating a range of translocation velocities.A minimum of at least two factors must have contributed to theobserved velocity gradient: the exchange of 14C between sieve elementsand companion cells (demonstrated by microautoradiography) and therange of velocities in the severd hundred sieve tubes which carried thetranslocation stream. Possible effects of these two factors on profilespreading were investigated by means of numerical models. The modelsare necessarily incomplete, due principally to uncertainties about theexchange rate between sieve elements and companion cells and thedegree of functional connectivity between sieve tubes of differentconductivities. However, most of the observed profile spreading may bereasonably attributed to the combined effects of those two factors.The mass average velocity of translocation (calculated from the mean

times of 14C arrival at successive sink leaves) was about 75% of themaximum velocity (calculated from the times of initial detection at thesame sink leaves), which was usually between 0.6 and 1 cm min-'.Owing to tracer exchange between sieve elements and companion cells,the mass average velocity of tracer in the sieve tubes was probablycloser to 86% of the maximum velocity, a figure which agreed with apredicted velocity distribution based on calculated sieve tube conductiv-ities and the size distribution of functional sieve tubes.

Most experiments on phloem translocation have employedrelatively short pathways, usually less than 50 cm in length.

' This work was supported by National Science Foundation GrantsGB 33905 and PCM 75-09537 to D. B. F.

2 Portions of this work were presented at the NATO AdvancedStudies Institute on Phloem Transport, held in Banff, Alberta, Canadain 1974.

3Present address: Monsanto Co., Agricultural Research Dept. T1G,800 N. Lindbergh Blvd., St. Louis, Missouri 63166.

4 To whom reprint requests should be addressed.

This is particularly true where the movement of radioactivesugars has been followed. Kinetic studies using '4C frequentlyrequire destructive sampling of at least several plants, an ap-proach which introduces substantial variability and becomesincreasingly tedious with larger plants. Labeling with "IC allowsrepeated in situ measurements of activity in the same plant butrequires elaborate equipment and its short half-life precludesexperiments lasting longer than about 3 hr. As a result, evencrude estimates of a translocation velocity have rarely beenmade for pathways longer than a meter in length.

In spite of some increased technical difficulties, longer path-ways offer some important advantages. Experiments requiringdifference measurements (e.g. of time, pressure, or concentra-tion) are carried out more readily with long pathways. Therelationship between the translocation profile and the rate oftracer efflux from a source leaf would be clearer if the entireprofile, from the front to the source, could be visualized after 2to 3 hr, since this is usually the interval over which changes inthe specific radioactivity of the translocated sugar take place.Where the question of mechanism is concerned, translocationover long distances poses a more difficult problem of explana-tion.The purpose of the present experiments was to investigate

the kinetics of '4C-photosynthate translocation through moder-ately long pathways (2-5 m). Of particular interest were thevelocity of translocation, the localization of the sugar beingtranslocated, the kinetic behavior of the translocation profile,and the relationship between source kinetics and the transloca-tion profile as they relate to the mechanism of translocation.The experimental plants were morning glory vines, which canreadily be grown to lengths of several meters.

MATERIALS AND METHODS

Growth of Plants. Japanese morning glory plants (Ipomea nilRoth, cv. Scarlet O'Hara) were grown in a growth chamber in2-liter containers of aerated, full strength Hoagland solution for5 to 10 weeks. The nutrient solution was renewed when theplants were about 3 weeks old and at weekly intervals thereafter.A mixture of fluorescent and incandescent lights supplied aphotosynthetically active radiation (PAR) intensity of about100 ,tE m-2 sec-1 (about 5,000 lux). The plants were grown ona 14-hr photoperiod at 25 C and a night temperature of 21 C.The lowermost mature leaves were continually removed fromthe vines during their growth, leaving about five to sevenmature leaves on the uppermost part of the vine, plus theexpanding leaves and shoot above the mature leaves. Underthese conditions, lateral shoots developed from serial buds inthe leaf axils along the length of the vine. Most of thesebranches were continually removed as the plant grew.

Labeling Experiments. In most experiments, the movementof "4C-photosynthate along the vine was followed by pulse-labeling a mature leaf with "CO2 and monitoring the appearance

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Plant Physiol. Vol. 61, 1978

of radioactivity in expanding leaves on the side shoots which

had developed at the older nodes along the vine. This approach

is basically similar to that introduced by Geiger and Swanson

(10) for following the kinetics of 14C arrival in expanding sugar

beet leaves. One day prior to an experiment, a plant was

selected with two to four (usually three) side shoots at roughly

equal intervals along the vine. Each of the side shoots had an

expanding sink leaf blade 2 to 3 cm in length, so that most of its

area could be monitored by a thin end-window Geiger tube. All

other side shoots were excised, as were all mature leaves on the

experimental side shoots and the shoot just above the expanding

leaves. A single mature leaf near the apex on the main vine was

selected for labeling and all others were excised, as well as the

stem above the node of this leaf. The experimental plant, then,

consisted of the roots, a length of mature stem with a single

mature leaf at its apex, and the short side shoots (usually

three), each of which had an expanding sink leaf at its terminus.

The vines were usually 2 to 5 m long with side shoots ranging

from 2 to 10 cm in length.The mature leaf was sealed into a Plexiglas labeling chamber

(14 cm square by 1.5 cm deep), with the chamber top removed.

A light intensity of 500 ,uE m-2 sec-1 was supplied by two 150-

w flood lamps, filtered through 5 cm of water. After an

overnight dark period of 8 hr, the lights were turned on about 4

hr before the labeling period. The expanding leaf blades on the

side shoots were flattened against the windows of thin end-

window Geiger tubes connected either to a scaler or to one

channel of a dual channel analytical rate meter. Data from the

latter were recorded on a strip chart recorder.

To initiate labeling, the top of the labeling chamber was

sealed in place and 300 ,uCi of '4CO2 was generated from

Na'4CO3 (about 60 mCi/mmol) with 40% HC104. The labeling

system consisted of the 14CO2 generator, the leaf chamber, a

rotary peristaltic action pump, and interconnecting lengths of

plastic tubing. Five min after generation of14CO2, the labeling

period was terminated by opening the chamber in the draft of

the fume hood. Photosynthesis during the remainder of the

experiment took place in ambient '2CO2.The arrival rate of 14C in each of the sink leaves (the "kinetic

translocation profile" [7]) was calculated as the first derivative

of a fifth degree polynomial which was fitted to the data curves

for total activity versus time. To facilitate comparisons between

curve shapes, the arrival rate was divided by the final counts

present in the leaf and expressed as the fraction, in per cent, of

the14C which arrived per min. This plot is essentially a probabil-

ity density distribution representing the chances of14C-photo-

synthate arriving at each of the sink leaves during the course of

the experiment. The mass average velocity of translocation was

calculated from the difference between the mean times of

arrival of tracer at successive sink leaves divided by the distance

between the leaves. Since the arrival of tracer did not decline to

zero during the measurement period, the data used for calcula-

tion of the mean time of arrival were truncated at '/io of the

peak height. In some cases this necessitated a brief extrapolation

of the arrival curve.

In several experiments, the distribution of tracer along the

vine (the "spatial translocation profile" [7] was determined by

extraction of stem segments. The procedure was similar to that

described above except that when tracer was first detected in

the lowermost sink leaf, the experiment was terminated by

cutting the stem into 2-cm segments, extracting alternate seg-

ments in boiling 80% aqueous ethanol, and counting aliquots of

the extract. In one experiment, additional aliquots were sub-

jected to one-dimensional paper chromatography in propionic

acid-butanol-water (23:12:15, v/v), and the insoluble com-

pounds remaining in the extracted stem segments were digestedin Protosol (New England Nuclear Corp.) and counted in a

liquid scintillation spectrometer. The identity of sucrose in

ethanolic extracts was established by its RF and by hydrolysis toequal amounts (as cpm) of glucose and fructose on treatmentwith yeast acid invertase (Sigma Chemical Co.).

Anatomical Observations. Microautoradiographs were pre-pared of stem cross-sections taken from four internodal regions10, 60, 110, and 160 cm below the labeled leaf of a vine 180cm long. The leaf was labeled for 6 hr with constant specificradioactivity '4CO2 with a steady-state labeling apparatus similarto that described by Coulson et al. (4). Each sample was quickfrozen in situ by isopentane-methylcyclohexane (9:1, v/v) cooledto about -175 C with liquid N2. Pieces of the internodes werethen freeze-substituted in acetone, embedded in Spurr's epoxyembedding medium, sectioned (complete cross-sections), andautoradiographed as described by Fisher and Housley (8). Thesections were stained after autoradiography with 1% (w/v)aqueous methyl violet.

Sieve element dimensions were measured in hand-cut freshsections stained with aniline blue for callose detection by fluo-rescence microscopy (11). Areas of sieve plates and of sievetube cross-sections (from microautoradiographs) were calculatedfrom the formula for an ellipse after measuring the longest andshortest distances across the plate or lumen.

Mathematical Models for Interpretation of Changes in ProfileShape. Two factors were viewed as necessarily contributing tochanges in the shape of the translocation profile as it movedalong the pathway: exchange of sucrose between sieve elementsand companion cells, and the probability that velocities weredifferent in different sieve tubes, particularly in different sizedsieve tubes. The effect of the latter was approached by assumingthat there were 40 entirely separate translocation streams, eachwith its own translocation velocity. The kinetics at any particularpoint along the pathway would be the sum of the contributionsby all 40 streams at that point of the pathway. The effects ofvarious velocity distributions and sieve tube cross-sectional areadistributions could be varied by changing the relative masstransfer rates and velocities of the 40 separate streams. Thenecessary arithmetical computations for this model were carriedout by FORTRAN programming of an IBM System 360 com-puter.The partial differential equations describing tracer exchange

between sieve elements and companion cells are identical tothose presented by Cataldo et al. (2) for water exchange betweenthe translocation pathway and surrounding tissues. However,their solutions were for steady-state labeling of the incomingstream. A simple FORTRAN program was written to provide anumberical solution for an arbitrary input function.The combined effects of tracer exchange and a velocity

gradient on profile spreading were modeled by a linear combi-nation of the above two models for 30 separate streams whichmoved at different velocities and made variable contributions tothe total mass transfer rate.

RESULTS

Kinetics of Arrival Rate at Successive Sink Leaves ("KineticProffles"). Figure1 illustrates some typical kinetic data fromthese experiments for the arrival rates of'4C-photosynthate insuccessive sink leaves along morning glory vines. Translocationvelocities (calculated from the time of 14C detection at a sinkleaf) were usually between 0.6 and 1 cm min-'. The velocity tosink leaves in side shoots at the node of the labeled leaf or atthe first node below it (a distance of about 10-20 cm) wereconsistently less than to other nodes along the vine. All datareported are taken from more distant sinks. There was noconsistent indication of a velocity gradient along the vine length,and the differences which were observed were in most cases notvery large. In some experiments, the velocity in the upper partof the stem was somewhat greater than in lower parts, while in

284 CHRISTY AND FISHER

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TRANSLOCATION IN MORNING GLORY VINES

MINUTES

FIG. 1. Kinetics for the arrival rate of '4C-photosynthate at expand-ing sink leaves along morning glory vines after pulse-labeling a matureleaf with '4C02. All other mature leaves had been excised, as well asthat portion of the vine above the labeled leaf. w: measure of peakwidth; p-d: time from detection to peak import rate. Three representa-tive experiments.

others the reverse was true, or there was no apparent difference.In 18 pairs of profiles from 10 experiments, the ratio of themass average velocity between successive sinks to the velocitycalculated from the difference in times of detection at the sinkswas 0.75 + 0.19.Although the kinetic curves for all sink leaves on a vine were

quite similar, there was a progressive and fairly symmetricalspreading of the profiles as they moved along the vine. This wasobserved in all experiments. Some quantitative aspects of thisspreading are summarized for 15 experiments in Figures 2, 3,and 4. Figure 2 shows the increase in profile width, measuredat one-half the peak height (w in Fig. 1 A), as a function of thetime at which the peak occurred. The increase in profile widthproceeded at an average rate of 0.41 min/min. That the spread-ing of the profile was fairly symmetrical is reflected in Figures 3and 4. Figure 3 shows the relative constancy of ratio of the timebetween tracer detection and the maximum import rate to theprofile width (i.e. p-d/w in Fig. 1A). The ratio starts at about0.57 and increases only slightly, with a slope of 0.0003 min-'.The apparent constancy of this ratio suggested that a fairlyuniform curve shape might be obtained for all times by express-ing the arrival rate as a function of a suitably chosen dimension-less time. That this was the case is illustrated in Figure 4, where, the dimensionless time, is defined as T = (t-tM)/(td-tM) (t =

time, t;, = mean time of arrival, and td = time to detection).The data are taken from 10 experiments, and include 26profiles. The points were taken from each of the profiles atequal time intervals and least squares fitted by an eighth degreepolynomial. Individual profiles, when plotted as a function of T,showed no evident trend in profile shape as a function of theirdistance from the source.

TINE TO PEAK (MIN)

FIG. 2. Widths of kinetic profiles (i.e. of 14C arrival rates), measuredat half the peak height (w in Fig. IA), as a function of the time atwhich the peak occurred. Numbers indicate individual experiments;circled numbers are spatial profiles. Profile width increased at a rate ofabout 0.41 min/min.

TIME TO PEAK (WON)

FIG. 3. Behavior of profile shape, expressed as ratio of time betweendetection and peak import rate to the profile width, (i.e. p-d/w in Fig.1A), as a function of the time at which the peak occurred. Numbersindicate individual experiments. Ratio increased at a rate of about0.0003 min-'.

100-

~50

0.5 1.0 1.5 2.0 2.5T (DIMENSIONLESS TIME)

FIG. 4. Arrival rate as a function of dimensionless time, defined as T= (t-tm)/(td-t), where t = time, tX = mean time of arrival, and td =time to detection. Points were taken at equal time intervals from 26profiles and are least squares fitted by an eighth degree polynomial.

Plant Physiol. Vol. 61, 1978 285




Distribution of Labeled Photosynthate Along the Pathway("Spatial Profiles"). Figure 5 shows the distribution of 14Calong a morning glory vine 330 min after pulse-labeling themature leaf with '4CO2. A distinct peak of radioactivity appearedalong the stem. Very little of the activity was in an insolubleform. Even in segments close to the labeled leaf, insolubleactivity accounted for only about 25% of the total and in thepeak region only about 1%. Most of the soluble activity was inthe form of sucrose; it accounted for about 98 to 90% of thesoluble activity in the front and peak regions, respectively,dropping to about 75% in segments close to the labeled leaf.The locations along the path of three sink leaves are also

indicated in Figure 5. The rates of 14C arrival (i.e. kineticprofiles) were obtained for the first two sink leaves; the experi-ment was terminated as soon as a detectable level of activity(twice background) was observed in the third sink leaf. Thecurves for the arrival rates at the first two sink leaves werequite similar and the velocity to all sink leaves was 0.73 cmmin-'. If all of the translocated 14C moved with that velocityand there was no loss of activity from the translocation stream,the kinetic profiles can be used to predict the expected distribu-tion of 14C along the pathway (see under "Discussion"; also ref.7). The predicted position of the peak with respect to sinks 2and 3 is particularly easy to calculate. Since the kinetic profile(measured from the 14C arrival rate) reached a peak 75 minafter detection and the translocation velocity was 0.73 cmmin-', the predicted peak for the spatial profile should havebeen about 55 cm (0.73 cm min-' x 75 min) upstream fromthe third sink. Since the experiment was terminated 90 minafter the peak had apparently passed the second sink, the peakshould have been about 66 cm downstream from there, whichagrees with the prediction based on the third sink. The completecurve for the predicted distribution of 14C along the pathway isshown by the solid line in Figure 5. Although the shape of thepredicted curve strongly resembled, in most respects, the actualcurve, it was about 30 cm more advanced and the experimentallydetermined distribution had a "tail" of low levels of activityprojecting ahead of the main front. Three experiments of thiskind were run; all gave similar results.Anatomical Observations. Figure 6 illustrates microautoradi-

ographs of cross-sections from the internode immediately below(10 cm) a mature leaf blade which had been labeled with '4CO2for 6 hr. In comparison to other cell types, all sieve elementsand their companion cells were heavily labeled. Autoradi-ographs from other internodes 60, 110, and 160 cm below thefed leaf were qualitatively similar to Figure 6. However, there

7 SIlNK SIM 2 +(1.0) OW 3

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FIG. 5. Distribution of 14C along a morning glory vine 330 min afterpulse-labeling a source leaf with "4CO2. The experiment was terminatedas soon as activity was detected at third sink leaf. Curve without pointsindicates predicted position of the profile, based on kinetics of 14Carrival at first two sink leaves. Note that the scale for insoluble activitydiffers from that for soluble activity by 10-fold.

A ~ ~ ~ ~ g --A

/,.,

Y<2-~:.7

A A1

FIG. 6. Microautoradiographs of 2-,um Epon sections from a morn-ing glory vine, taken from internodal tissue 10 cm below labeled leaf.All mature leaves except one had been removed from vine. Theremaining leaf was steady-state-labeled for 6 hr with 14CO2. The sectionswere stained with methyl violet. A: low magnification; bar indicates200 ,um. B: high magnification; bar indicates 20 ,um.

seemied to be a regular decrease in labeling intensity withincreasing distance. Although the difference in autoradiographsfrom successive positions was not necessarily persuasive (noattempt was made at utilizing quantitative procedures), therewas an obvious difference between the 10-cm and 160-cmpositions.The distributions of sieve element sizes in each of the autora-

diographed internodes are shown in Figure 7. A fairly widerange of sieve element sizes were represented in all internodes,with each size range contributing substantially, in most cases, tothe total cross-sectional area of the translocation stream. In theinternal phloem, there was a consistent decrease, toward theroots, of the cross-sectional area of sieve elements.Measurements of sieve element lengths, cross-sectional areas,

and sieve plate dimensions provided an estimate of sieve tubeconductivity as a function of sieve element size (i.e. their cross-sectional area or sieve plate area). The major contributingfactor to differences in conductivity was the variation in sieveplate pore diameter. Only minor differences, if any, were notedin length (300 ,um), a plate thickness (1.4 gm), or in thefraction of the sieve plate occupied by pores (60%). The cross-sectional area of sieve elements was about 7/10 of their sieveplate area, due in part to their slightly bulbous ends and in partto inclination of some sieve plates. Figure 8A illustrates therelationship between sieve element area, sieve plate area, andsieve plate pore diameter. Sieve element conductivities, calcu-lated from the Hagen-Poiseuille equation and assuming unob-





EXTERNAL

287TRANSLOCATION IN MORNING GLORY VINES

INTERNAL

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38430 02152 ST

160 cm 32050 jj220 152 ST

OI0r nre zKI

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zLu Duo 750 z250 50 750SIEVE TUBE AREA (Y2)

FIG. 7. Size distribution of sieve elements in internal and externalphloem of a morning glory vine at four distances from a labeled leaf.Measurements were taken from microautoradiographs (same experi-

ment as in Fig. 6). Total sieve tube cross-sectional area and number ofsieve tubes are indicated in upper right. Each bar represents the per

cent of total sieve tube area contained in a class of sieve tube areas

extending over an interval of 50 ,um2.

structed pores and sieve element lumen (e.g. see ref. 3), are

plotted as a function of sieve plate area and sieve element area(7/Io of the sieve plate area) in Figure 8B. Sieve plate porediameter increased with increasing size of the sieve element upto a sieve plate area of about 500 tLm2, but all sieve elementswith plates larger than 500 gm2 had similar pore diameters.Sieve element conductivity increased linearly with size up to a

sieve plate area of about 500 gm2 and was fairly constant abovethis, reflecting the strong dependence of conductivity on sieveplate pore diameter. Since there is appreciable uncertainty inthe calculation of sieve tube conductivities, particularly wherepore dimensions are concerned, they have been calculated for a

range of pore diameters.The expected velocity distribution in a morning glory stem

may be calculated from the conductivities in Figure 8B and thedistribution of sieve element sizes in Figure 7, assuming thatthe sieve elements function separately, have uniform conductiv-ities along their lengths, and are subjected to the same pressuregradient (Fig. 9). The mass average velocity for the predictedvelocity distribution is 87% of the maximum velocity.

Mathematical Models of Profile Spreading. Figure 10 illus-trates the effects on profile spreading of three hypotheticalvelocity gradients. The source kinetic data used in the calcula-tions were chosen to approximate reasonably the observedkinetics for morning glory. In Figure 10A, where the mass

transfer rate is evenly distributed between streams moving at

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A 15 a r% cr%^ V C ^ SIEVE PLATE AREA (pt)FIG. 8. Relationship between sieve plate area, sieve element area

(= 0.7 x sieve plate area), and sieve plate pore diameter (8A), andcalculated conductivities of sieve tubes (8B), based on a sieve elementlength of 300 tLm and a sieve plate thickness of 1.4 tLm, 60% of theplate occupied by pores and a viscosity of 1.4 x 10-2 poise. Conductivi-ties in 8B are calculated for pore diameters of measured values (8A)and for diameters of 0.9, 0.8, 0.7 and 0.6 times the measured porediameters.

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RELATIVE VELOCITYFIG. 9. Expected translocation velocity distribution in a moming

glory vine, calculated from conductivities in Figure 8B and distributionof sieve element sizes in Figure 7.

velocit-les ranging from 0.5 to 0.8 cm min-' (inset), there tendsto be a broadening and flattening of the kinetic profile. With a

marked preponderance of tracer moving at higher velocities,there is a greater tendency for the profile to retain its originalshape, although with substantial spreading (Fig. I OB). Whenslower moving streams account for most of the tracer movement,the effect on profile shape is most noticeable, with a pronouncedshift toward later arrival times for most of the tracer (Fig.

I I I

II I I




MINUTES

FIG. 10. Theoretical effects of three hypothetical velocity distribu-tions (upper right of each figure) on profile shape observed at 100,200, and 300 cm from source. In graphs of velocity distribution,velocity is in cm min-', and w is the relative amount of translocationstream moving at a particular velocity. The calculation assumes thatthere are 40 independent streams, each moving with its own velocity.Velocities range from 0.5 to 0.8 cm min-'.

OC). When expressed on the same basis as for Figure 2 (peakwidth at half-maximum versus peak time), the spreading rate isabout 0.26 min/min. However, it is not constant, but decreasessomewhat with time.The effect on profile spreading of the expected distribution of

mass transfer rates in morning glory vines (Fig. 9) is shown inFigure 11, assuming a maximum velocity of 0.8 cm min-'. Theeffect is fairly symmetrical except for the latter part of theprofile, which is progressively skewed toward longer times. Thespreading rate is about 0.13 min/min.Exchange of tracer between sieve elements and their compan-

ion cells also represents a potentially significant cause of profilespreading, although it is appreciable only when the exchangerate between the two is fairly slow. Figure 12 illustrates theeffect on profile shape for the case where the translocationvelocity is 0.8 cm min-' and the companion cell volume is 1/7 ofthe sieve element volume. Sucrose concentration is assumed tobe equal in the two cell types. The approximate spreading ratesfor turnover times in the companion cells of 10, 20, and 40 minare 0.07, 0.11 and 0.17 min/min, respectively. Again, theprofile does not, in fact, spread linearly with time. In thismodel, the mass average velocity, as calculated from the differ-ence in mean times of tracer arrival at successive distances, is0.7 cm min-' rather than 0.8 cm min-', the mass averagevelocity in the sieve tube. The mean times of tracer arrival atsuccessive distances (hence the maximum velocities) are thesame for all three exchange rates between sieve tubes andcompanion cells.

400MINUTES

FIG. 11. Theoretical effect of velocity distribution shown in Figure 9on profile shape observed at 100, 200, and 300 cm from source,assuming a maximum velocity of 0.8 cm min-'. Other assumptions as inFigure 10.

SOURCE 10 min

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FIG. 12. Theoretical effect on profile shape of sucrose exchangebetween sieve elements and companion cells. Velocity of translocationstream is 0.8 cm min-1, and companion cells are assumed to contain '/7the amount of sucrose in the sieve elements. Calculations are presentedfor sucrose turnover times in companion cells of 10, 20, and 40 min.

An example of the combined effects of tracer exchange and avelocity gradient is shown in Figure 13. Turnover time in thecompanion cells was assumed to be 20 min, and the assumedvelocity distribution was that expected for a morning glory vine(Fig. 9), with a maximum velocity of 0.8 cm min-'. The profile




TRANSLOCATION IN MORNING GLORY VINES

z8 <200 cm

Z :1300 cm

4

° 200 400 SooMiNUTE S

FIG. 13. Combined effect of tracer exchange and velocity gradienton profile shape. Assumed velocity distribution is as in Figure 9(maximum velocity = 0.8 cm min-'), and turnover time for sucrose incompanion cells is 20 min. Mean times of tracer arrival for all companioncell turnover times: 0 cm: 65.7 min; 100 cm: 207.8 min; 200 cm: 350.1min; 300 cm: 492.7 min.

spreads in a fairly symmetrical manner at a rate of about 0.22min/min.

DISCUSSION

The simplest model for translocation kinetics would be one inwhich all of the translocation stream moved with the samevelocity ("plug flow") with no loss of tracer from the stream.This would result in a linear displacement of the (spatial)translocation profile along the pathway with time, with thesame kinetic profile being observed, after a delay proportionalto the distance, at each point along the pathway (7). As a firstapproximation, the kinetics in morning glory vines may bereasonably characterized by that description. There was a strongresemblance between the spatial and kinetic profiles, the shapeof which altered only slowly with time. The preponderance oflabel in the translocated sugar (sucrose), its localization in sievetubes even after several hr of labeling, and the small amount oftracer remaining in the stem after passage of the peak all attestto the low rate of loss from the translocation stream. Thisinterpretation requires that the profile shape must be generatedprincipally by the rate of 14C export from the labeled leaf, a

condition which has strong experimental support (9). The obser-vation that the translocation velocity to sinks at the first twonodes was consistently less than to subsequent nodes suggests a

more indirect route of transport to those sinks, presumably dueto the necessity for lateral movement from the leaf traces toother vascular tissue. However, this effect was secondary in

comparison to the effect on profile shape of the rate of 14Cexport from the source leaf (9).The observations contrast sharply with the kinetic behavior

predicted by any mechanism describable by diffusion equations(1). In addition to the resemblance between the kinetic and

spatial profiles, and the relative constancy of the profile shape,neither of which can be accounted for by diffusion equations,the formation of a peak of tracer which is propagated along the

translocation is a diffusion-like process. Since diffusion occurs

down a concentration gradient, a peak of tracer could not even

be generated unless the labeled leaf, after exporting tracer for

an hr or so, then began to import it at a similar rate. This kind

of behavior has never been observed in any plant; once a

mature leaf is labeled with radioactive CO2 there is a continual

net loss of tracer which eventually approaches zero but never

reverses. Furthermore, a symmetrical peak, once formed, would

spread with equal rapidity in both directions. This predictioncontrasts sharply with the observed profile behavior in morning

glory vines. (For a more detailed quantitative analysis of thisproblem, see ref. 5.)However, the characterization of the translocation process in

morning glory vines as one of plug flow with negligible loss isobviously useful only as a first approximation. The progressivespreading of the profile with time and the consistent lag of thespatial profile behind its predicted position both preclude thecharacterization of the transport process by a single velocity.The discrepancy between the predicted and actual profile posi-tions appeared to be caused by an acceleration of part of thetranslocation stream as it nears a sink. The reason for this isunclear, although it is reasonable to assume that the explanationmay lie in the functional interconnections between sieve ele-ments and their resistance to flow, both of which are verydifficult to investigate experimentally. Although the discrepancyintroduces an element of error into velocity measurements andcharacterization of the profile shape, it does not seem suffi-ciently great to affect our basic interpretations of the kinetics.The peak of the spatial profile had always moved 75 to 80% ofits predicted distance from the source and showed a strongresemblance to its predicted shape.Owing to the exchange of tracer between sieve tubes and

companion cells, the mass average velocity of translocation, ascalculated from the difference between mean times of arrival atsuccessive distances, will be less than the mass average velocityin the sieve tubes (i.e. on the average, a sucrose moleculewould spend only a fraction of its time in the sieve tubes). Atany given instant, the mass average velocity will be representedby the mean velocity of sugar in the sieve tubes and companioncells, hence it will not be affected by the rate of exchangebetween the two (Fig. 12). Since the sucrose concentration isthe same in sieve tubes and companion cells, the fraction of thetotal stream (sieve tubes and companion cells) which is movingis represented by the fraction of the total cross-sectional area

(sieve tubes plus companion cells) occupied by the sieve tubes.In the models with exchange (i.e. Fig. 11), this fraction is 7/8;the velocity calculated from mean times of arrival is 0.7 cm

min-', while that in the sieve elements is 0.8 cm min-'. Notethat the latter value (i.e. the maximum velocity) would be thatobtained by dividing the time to detection by the distance alongthe path. In the case of the experimental values, the averageratio of the mass average velocity to the maximum velocity was

0.75. Correcting this for the effect of exchange, the ratio of themass average velocity in the sieve tubes themselves to themaximum velocity should be closer to 0.86 (0.75 x 8/7). This isin good agreement with the ratio predicted in Figure 9 (0.87)from the sieve element dimensions and size distribution in a

morning glory vine.The progressive spreading of the profile shape indicates that

translocation proceeded with a range of velocities rather thanwith just a single velocity (7). The fairly symmetrical spreadingof the profile suggest that, whatever the precise features of thevelocity distribution, there was a skewing of the velocity profiletoward lower velocities (i.e. most of the tracer moved at

relatively higher velocities). The basis for this conclusion lies inthe fact that a given velocity distribution will tend to shift a

kinetic profile toward a shape which is the mirror image of thevelocity distribution (Fig. 10). This is perhaps more easilyimagined by considering the fate of a hypothetical very narrow

pulse in the translocation stream. In time, the spatial profile ofthe pulse, which is similar to the mirror image of the kineticprofile, would resemble the velocity distribution. Since thekinetic profile near the source was skewed toward later timesand spread symmetrically, the velocity distribution must havebeen skewed toward lower values. The velocity distribution(Fig. 9) suggested by the range of sieve tube sizes and conductiv-ities (Figs. 7 and 8B) is, in fact, skewed toward lower velocities.With respect to the mechanism of translocation, the signifi-

289Plant Physiol. Vol. 61, 1978



290 CHRISTY AND FISHER Plant Physiol. Vol. 61, 1978

cance of an observable velocity range lies in whether it occurswithin individual sieve elements. The movement of sucrosemolecules with differing velocities would be detectable only iftheir movement were substantially restricted in comparison tofree diffusion, since the effect of the parabolic velocity gradientaccompanying simple pressure flow would be undetectable (13).Although morning glory sieve elements have not been investi-gated specifically, recent work on sieve element ultrastructurein angiosperms does not suggest any cytological basis for com-partmentation (6, 12). The most reasonable explanation for thevelocity range seems to lie in the demonstrated exchange oftracer between sieve elements and companion cells and a rangeof velocities in the more than 200 sieve tubes which carried thetranslocation stream. Both factors must be taken into consider-ation when attempting to account for profile behavior and,taken together, it seems likely that they provide an adequatebasis for an explanation of spreading. None of the profiles inthe models actually showed a spreading rate equivalent to theaverage experimental values (0.41 min/min). (Note that thevalues for spreading rates have been introduced only as aroughly quantitative but convenient means of comparing profilebehavior under different circumstances.) When the effects oftracer exchange and velocity distribution were combined (Fig.13), the spreading rate was 0.22 min/min, less than observed inmost experiments. Nevertheless, it is apparent that any numberof not unreasonable combinations of velocity gradients andturnover times could satisfactorily mimic observed profile be-havior. A more exact model of profile spreading would be

desirable, but would require more complete knowledge of theexchange rates between sieve elements and companion cells, ofthe degree of functional interconnectedness between sieve tubesof various conductivities (ignored in the present model), and ofthe pressure differentials between source and sink regions(assumed here to be the same for all sieve tubes).

LITERATURE CITED

1. CANNY MJ 1973 Phloem Translocation. Cambridge Univ Press, London2. CATALDO DA, AL CHRISTY, CL COULSON, JM FERRIER 1972 Solution-flow in the' phloem.

I. Theoretical considerations. Plant Physiol 49: 685-6893. CHRISTY AL, JM FERRIER 1974 A mathematical treatment of Munch's pressure-flow

hypothesis of phloem translocation. Plant Physiol 52: 531-5384. COULSON CL, AL CHRISTY, DA CATALDO, CA SWANSON 1971 Carbohydrate translocation

in sugar beet petioles in relation to petiolar respiration and adenosine-5'-triphosphate.Plant Physiol 49: 919-923

5. FERRIER JM, MT TYREE 1976 Further analysis of the moving strand model of translocation,using a numerical calculation. Can J Bot 54: 1271-1282

6. FISHER DB 1975 Structure of functional soybean sieve elements. Plant Physiol 56: 555-5697. FISHER DB 1975 Translocation kinetics of photosynthates. In S Aronoff et al, eds, Phloem

Transport. Plenum Press, New York, pp 327-3588. FISHER DB, TL HOUSLEY 1972 The retention of water-soluble compounds during freeze-

substitution and microautoradiography. Plant Physiol 49: 166-1719. FISHER DB, TL HOUSLEY, AL CHRISTY 1978 Source pool kinetics for '4C-photosynthate

translocation in morning glory and soybean. Plant Physiol 61: 291-29510. GEIGER DR, CA SWANSON 1965 Sucrose translocation in the sugar beet. Plant Physiol 40:

685-69011. JENSEN WA 1962 Botanical Histochemistry. WH Freeman & Co, San Francisco12. PARTHASARATHY MV 1975 Sieve element structure. In MH Zimmermann, JA Milburn,

eds, Transport in Plants. 1. Phloem Transport. Springer-Verlag, Berlin, pp. 3-3813. TAYLOR G 1953 Dispersion of soluble matter in solvent flowing slowly through a tube.

Proc R Soc Lond A 219: 186-203



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4C-Photosynthate in Morning Glory - Plant Physiology · Kinetic studies using '4C frequently require destructive sampling of at least several plants, an ap-proach which introduces