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Plant Physiol. (1 992) 100, 1878-18840032-0889/92/100/1 878/07/$01 .00/0

Received for publication March 25, 1992Accepted August 10, 1992

Carbon Fluxes in Mature Peach Leaves

Annick Moing*, Francis Carbonne, Mohamed H. Rashad, and Jean-Pierre GaudillereInstitut National de la Recherche Agronomique, Station de Recherches Fruitieres (A.M.) and Station de Physiologie

Vegetale (F.C., M.H.R., I.-P.G.), Centre de Recherches de Bordeaux, BP81 33883 Villenave d'Ornon, France

ABSTRACT

The turnover and transport of sugars are described in peach(Prunus persica L. Batsch), a species exporting both sucrose andsorbitol. Apparent export rate was slower in peach leaves than inleaves of herbaceous species. Sorbitol was the major soluble endproduct of photosynthesis and the major soluble carbohydrate inthe leaf (higher than sucrose). Carbon fluxes were described using14C labeling, radioactivity loss curves, and compartmental analysisduring the second half of the photoperiod when chemical steadystate was reached for soluble carbohydrates. The measured specificradioactivity of sucrose was typical of a primary product. Thedelayed decrease in specific radioactivity of sorbitol indicated thatpart of it was secondarily synthesized. Sucrose is proposed to bethe carbon source for the delayed synthesis of sorbitol in the light.The sorbitol to sucrose ratio was higher in the petiole than in theleaf tissues. In phloem sap, obtained using stylectomy of aphidsand collected from the main stem between source leaves and apex,this ratio was lower than in the petiole, suggesting a preferentialsorbitol demand by sinks.

In most higher plants, the primary photosynthetic productsare sucrose and starch. Both may be stored in the leaf bladeduring the day. In some species, alditols are additionallysynthesized (1). Sorbitol was reported as the major photoas-similate in the woody Rosaceae, e.g. Prunus, Pyrus, and Malusspecies (27), where it is also translocated via phloem (30).

In comparison with species where mannitol is a primaryphotoassimilate, e.g. celery (5, 13), less is known about carbonpartitioning in sorbitol-synthesizing species. Since the label-ing study of Redgwell and Bieleski (20) and the purificationof aldose-6-P reductase (EC 1.1.1.200) from loquat (10) andapple leaves (18), the precursor for sorbitol biosynthesis inmature leaves is known to be glucose-6-P. The catabolicpathways of sorbitol involve sorbitol dehydrogenase (NAD-dependent, EC 1.1.1.14 [17] or NADP-dependent [29]) or

sorbitol oxidase (28) in sink tissues. These enzymes are notexpressed in mature leaves (2, 12, 15). Therefore, the fate ofsorbitol in mature leaves is either translocation or storage.The primary partitioning of photoassimilated carbon be-

tween sucrose and starch and its regulation are now wellknown (11, 24). However, the involvement of sorbitol is lessclear. The fate of glucose-6-P, one of the regulatory substratesof the sucrose pathway (25), may be altered by sorbitolsynthesis. In addition, sucrose and sorbitol are both export-able carbohydrates via phloem, and the control of theirtranslocation remains unclear.The present work investigates the tumover and transport

1878

of nonstructural carbohydrates in a leaf exporting both su-crose and sorbitol. The relative carbon fluxes through starch,sorbitol, and sucrose pools, three major end products ofphotosynthesis, for storage and/or export are studied in amature peach (Prunus persica L.) leaf. Pulse-chase experi-ments, recording of leaf radioactivity loss curves, compart-mental analysis, and examination of total changes in solublesugars and starch contents are used to describe carbon fluxesthrough the leaf at chemical steady state. This description iscomplemented by an analysis of the composition of sievetube sap collected using stylectomy of aphids.

MATERIALS AND METHODS

Plant Material

Seedlings of the autogamous and near homozygous peach(Prunus persica L. Batsch cv GF 305) were used at the 2-month-old stage. The seedlings were potted and trained to asingle shoot. They were cultivated in a growth chambermaintained at 250C day/200C night, 75% RH, and a 15-hphotoperiod with Na-vapor lamps (Philips SON-T) giving200 ,mol photons m-2 s-' in the 400- to 700-nm range atplant height. The CO2 concentration was approximately 400,uL L1. For fertilization, Osmocote (15% N, 10% P, 12% K,from Sierra Chemical Co.) and Fe-ethylenediamine-N,N-di(2-hydroxy-5-methylphenyl)acetic acid (150 mg/pot) wereadded to the substrate (peat:sand:soil, 1:1:1 by volume) oncea month and once a week respectively.Mature leaves were studied during the second half of the

photoperiod. Leaf samples were collected 7, 9, 12, and 15 hafter the start of the photoperiod.

Photosynthesis

Photosynthesis and transpiration were measured on at-tached leaves using an IRGA, a dew point hygrometer, anda mass-flow meter (8) under the same temperature, lightconditions, and CO2 concentration used during plant growth.Photosynthesis was monitored during 14CO2 labeling to allowcalculation of total fixed radioactivity.

Pulse-Chase Labeling

A portion (75 mm long) of an attached mature leaf (thesecond youngest mature leaf) was inserted into a small cham-ber and fed with 1110 MBq mol' 14CO2 at a concentrationof 411 ,uL L` for 4 min. Following a chase of 2 min with thesame concentration of "2CO2, the leaf was removed from thesmall chamber and discs (1 cm2) were rapidly cut from the

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CARBON FLUXES IN MATURE PEACH LEAVES

leaves and fixed in hot ethanol:water (80:20, v/v at 800C).The chase period was prolonged until the end of the photo-period, under the usual conditions in the growth room, andother samples were harvested on the same half of the leaf,at 2, 5, and 8 h after labeling for labeling applied 7 h afterthe beginning of the photoperiod, and at 3 and 6 h afterlabeling for labeling applied 9 h after the beginning of thephotoperiod. Labeling was also applied 12 and 15 h after thebeginning of the photoperiod to evaluate carbon transferthrough the petiole.Continuous monitoring of 14C in the other half of the fed

leaf was carried out until the end of the photoperiod using aGeiger-MIller tube and a data logger (Delta-T). Owing to thehigh sensitivity of the Geiger-Muller counting yield to leafposition, the coordinates of leaf position on the Geiger-Mullertube were precisely noted and maintained throughout theexperiment. At the end of the experiment, other leaf discs (2cm2) and petioles were sampled and frozen rapidly in liquidnitrogen. They were freeze-dried at -200C for the determi-nation of dry weights. Lyophilized petioles were then ex-tracted for further carbohydrate analysis, using the procedureadopted for fresh tissues. Therefore, for the petiole study,labeling was applied either 7, 9, 12, or 15 h into the photo-period, and petiole samples were always collected 15 h intothe photoperiod.

Compartmental Analysis

Quantitative information on photosynthate compartmen-tation and export was obtained using the method of com-partmental analysis described by Moorby and Jarman (16).When chemical steady-state conditions were fulfilled, theloss in radioactivity could be analyzed as a sum of exponentialfunctions. Compartmental analysis was performed using totalradioactivity loss curves. The precision of mathematical curvefitting did not allow identification of more than two expo-nential functions. The model used to estimate the partitioningof exportable carbohydrates in illuminated leaf blades usedthe sum of two exponential functions and a constant. Theequation was

Hie-9gt + H2e 82t + H3 (1)

where t was the time in hours, gi and g2 were exponentialcoefficients (h-1), and H1, H2, and H3 were the fractions ofinitial, i.e. after a 2-min chase, labeling for each pool. Thetwo exponentials could be attributed to an exportable pooland a transient storage pool of soluble sugars. The constantH3 was attributed to a nonexportable pool. Equation 1 wasfitted to the data, which were expressed as a percentage ofthe initial 14C content (Fig. 1).Double exponential plus residual curves were fitted to the

data with Fig.P software (Fig.P Corp., Durham, NC) using aleast-square iterative method to calculate H1, H2, H3, gi, andg2. This method of curve fitting was chosen because it is moreaccurate than graphical methods. Curve fitting was per-formed for three experiments for each of two labeling times(7 and 9 h). For the exportable pool and the transient storagepool of soluble sugars, rate constants were calculated accord-ing to Shipley and Clark (23) after subtracting the calculatedvalue for H3 from the experimental data. Because the transient

100

.E4- 80

00604-

3 40I._ 404-

0

X~ 20

EU

0 82 4 6

Time (h after labeling)

Figure 1. Decrease in leaf blade radioactivity, measured with a

Geiger-Muller tube, after 14CO2 labeling of a mature peach leaf.Pulse labeling was performed 7 h after the beginning of the pho-toperiod with a chase of 8 h. Symbols represent the experimentaldata for one leaf. The lines illustrate curve fitting, following thedouble exponential plus residual model, performed with Fig.Psoftware for this leaf.

storage compartment was supposed to be in chemical steadystate, carbon flux out of this compartment was equal tocarbon flux entering this compartment. Therefore, the ratioof the content of the exportable pool to the transient storagepool could be calculated as the inverse of the ratio of theirrespective rate constants. The sum of transient storage andexportable pools was estimated as the sum of soluble sugars.

The contents of transient storage and exportable pools were

calculated from their ratio and sum.

Carbohydrate Determinations

Soluble sugars were extracted with hot ethanol:water(80:20, v/v, and 50:50, v/v at 800C) and purified on tandemion-exchange resins. For 1 cm2 of leaf blade, 0.4 milliequiv-alent of Bio-Rad AG 1-X8 resin in the carbonate form and0.5 milliequivalent of Dowex 5OW resin in the H' form were

used. The purified soluble carbohydrates were analyzed byHPLC (Aminex HPX-87C column from Bio-Rad with wateras eluent at 10-8 m3 s-' and 750C). Starch was determined inthe wet pellets after rinsing with water and heating at 1350Cfor 1 h to gelatinize starch, followed by hydrolysis withamyloglucosidase (EC 3.2.1.3., hydrolyzing 1,4- and 1,6-a-Dglycosidic linkages, from Merck) in 0.2 M acetate buffer (pH4.6). Buffer salts were removed using the anion- and cation-exchange resins described above and glucose levels were

analyzed by HPLC. For comparative purposes, soluble car-

bohydrate and starch contents, as well as sorbitol to sucrose

ratio, were expressed as moles of carbon and moles of carbonper moles of carbon, respectively.

Radioactivity in Carbohydrates

Soluble carbohydrates or glucose obtained from starchhydrolysis were separated by HPLC and collected using a

1 6.5e-O.S5t

~-- - - .~constant-3.2.................i... I I I... , .; . .

1 879



Plant Physiol. Vol. 100, 1992

volume drive fraction collector (Gilson 201). The radioactivityof the fraction was counted on a Packard Tri-carb 2000 CAliquid scintillation spectrometer, using Luma Safe as scintil-lant (obtained from Lumac-LSC). Radioactivity of all thefractions corresponding to one carbohydrate was summed,after verifying on a few samples using a Flo-one beta liquidradioactivity detector (Radiomatic Instrument & Chemical Co,Inc.) that radioactivity detection and HPLC refractometrydetection ran parallel. Apparent turnover times for sorbitoland sucrose were calculated, after fitting the data to monoex-ponential curves, using Fig.P software. For each carbohy-drate, the equation was y = Ie-kt, where y was the percent ofthe initial specific radioactivity, t was in hours, k was theexponential coefficient, and I was a constant. The apparentturnover time was calculated as k-' for each leaf labeled 7 hafter the beginning of photoperiod.

Collection of Pure Phloem Exudate

The collection of phloem sap from the main stem, abovethe source leaves, was performed by stylectomy (7) usingradio frequency microcautery (high-frequency radio micro-cautery type CA-20 from Murphy Developments, Hilversum,The Netherlands) operating at 48 MHz (26), with a micro-manipulator. The aphids used were apterous adults of Myzuspersicae Sulz. The volume of each exudate droplet was deter-mined from its diameter before collection. Exudate dropletswere collected in microcapillaries (15-50 ,um diameter) untila total volume of approximately 10 nL was obtained, andthen samples were frozen. The exudates were diluted to afinal volume of 50 yL, 20 ,L being used for carbohydrateanalysis by HPLC.

RESULTS

Partitioning of Photosynthetic Carbon

Typical photosynthetic rates in a mature leaf during a singleday/night cycle are shown in Figure 2A. Photosynthetic ratepeaked approximately 4 h after the beginning of the photo-period and then decreased very slowly until the end of thephotoperiod. Photosynthetic rate was measured during eachlabeling experiment (Fig. 2B).

In illuminated peach leaves, the major soluble sugars weresorbitol and sucrose followed by hexoses (Fig. 3). Similarresults were obtained for the petiole, where sorbitol andsucrose represented 1.39 ± 0.38 and 0.55 ± 0.10 mmol of Cg-' dry weight, respectively. The sorbitol to sucrose ratio washigher in the petiole than in the leaf blade 15 h after thebeginning of the photoperiod (Table I). In the leaf blade,during the second part of the photoperiod soluble sugarcontents were quite constant and starch content increasedlinearly. Therefore, chemical steady state was met for thesecarbohydrates. Starch, the level of which was the highestamong the nonstructural carbohydrates in the leaf blade, wascontinuously stored throughout the photoperiod.The partitioning of photosynthetic carbon was determined

at four different labeling times during the second half of thephotoperiod (Fig. 4). Among the soluble sugars, most of thecarbon was partitioned into sorbitol. Partitioning into starchwas nearly equivalent to that into sorbitol. The sorbitol to

sucrose ratio for carbon influx was between 1.9 and 1.2(Table I) and declined from 9 to 15 h after the beginning ofthe photoperiod.

Phloem Sap

The analysis of phloem sap showed that both sorbitol andsucrose were translocated. Concentrations in the collectedphloem sap were 1.16 ± 0.50 and 0.68 ± 0.19 M for sorbitoland sucrose, respectively (mean ± SD of four replicates). Noglucose or fructose were detected by HPLC. Therefore, foreach hexose, the content was less than 0.5% of sucrosecontent, owing to the accuracy limit of the method. Thesorbitol to sucrose carbon ratio in phloem sap (Table I)

' 12

E 10 A

E o

E

4)

0o -20. 0 4 8 12 16 20 2ATime(h after begIn of pwotpiod)

Time (h after beginning of photoperiod)9-

I4 12

E 10C

0 8

E

-C=n

0 6

0.0

0-C0.

C 2

oOA

6 9 12 15

TIME (h after beginning of photoperiod)

Figure 2. Changes in the photosynthetic rate of mature peachleaves. A, A typical continuous recording of photosynthetic rate ofa single leaf during one day/night cycle. B, Photosynthetic rateduring labeling experiments at four different times during the sec-ond half of the photoperiod. These data represent the mean ± SDof four leaves.

1 880 MOING ET AL.




starchF

F _~ sorbitol

0 1% ----Q sucrose

;&~ z alucoseo tt + fructose

_

6 9 12 15

TIME (h after beginning of photoperiod)Figure 3. Changes in mature leaf carbohydrate content measuredafter extraction and analysis by HPLC. These data represent themeans ± SD of 16 leaves. The mean leaf dry weight per unit area +SD of 16 replicates was 38.2 ± 2.5 g m-2 at 15 h.

showed that in the stem as much assimilated carbon was

transported via sorbitol as via sucrose.

._a

0

= 50c

In0

0.0 40

0 20

0 10c0

0.

0 00.

0.CL6 7 9 12 15

TIME (h after beginning of photoperiod)

Figure 4. Changes in the distribution of photosynthetic carbonbetween soluble carbohydrates and starch, determined after 14Clabeling, in mature peach leaves. The total radioactivity fixed was

calculated using the photosynthetic rate measured during labelingand C02-specific radioactivity. 'Others" were calculated as thedifference between calculated radioactivity fixed by photosynthesisand the sum of radioactivity in sucrose, hexoses, sorbitol, andstarch. These data represent the means ± SD of four replicates.

Compartmentation and Export of Photoassimilates

To check the value of the export rate calculated by thecompartmental model, an estimation of export rate was cal-culated as the difference between photosynthetic rate andthe rate of accumulation of carbohydrate, mainly starch. Themean rates of carbohydrate accumulation and photosynthesiswere obtained by fitting linear regressions to the data fromFigure 3 and Figure 2B, respectively. The export rate, calcu-lated as the difference between photosynthetic rate and therate of carbohydrate accumulation, decreased linearly from 8to 6 ,umol of C m-2 s-' during the second half of thephotoperiod.A typical curve of photoassimilate export is shown in Figure

1. Constants and fluxes, calculated from similar curves, are

indicated in Figure 5, where a schematic model is proposed.The calculated carbon ratio between the exportable pool andthe transient storage pool was 4.4 ± 2.7 mol of C mol-1 of C(mean ± SD of six replicates). Considering the hypothesis thatthe sum of the exportable pool and the transient storage poolwas the sum of the soluble sugars, it followed that the ex-

port rate calculated from this model was 3.2 ± 2.1 ,umol ofC m-2 S-1.The relative contribution of sorbitol and sucrose to export

was estimated by direct measurement of the decrease inspecific radioactivity of individual carbohydrates in an illu-

Table I. Sorbitol to Sucrose Ratio during Carbon Assimilation in Mature Leaf, Petiole, and PhloemExudate, Collected with Aphid Stylets, of Peach

These data represent the means ± SD. The number of replicates used is indicated in parentheses.nd, Not determined.

mol C Sorbitol * molV' C Sucrose

Parameter Time after beginning of photoperiod7h 9h 10h 12 h 15 h

Photosynthesisa (4) 1.81 ± 0.29 1.89 ± 0.50 nd 1.28 ± 0.31 1.17 ± 0.07Leaf mesophyllb (16) 2.55 ± 0.53 2.39 ± 0.50 nd 2.32 ± 0.48 2.21 ± 0.38Petioleb (16) nd nd nd nd 2.79 ± 0.54Phloem sapb (4) nd nd 0.87 ± 0.17 nd nd

a Ratio of 14C in sorbitol to 14C in sucrose after labeling and a chase of 2 min followed by extractionand separation of carbohydrates by HPLC. b Ratio of carbon in sorbitol to carbon in sucrose, afterextraction and analysis by HPLC.

E 300U

E 250E*c 2000

o 150

0

X 100l-

o 50.0

0 00-

0

I

1881




Figure 5. Simplified model for carbon exportout of the leaf in mature peach leaves duringthe second half of the photoperiod. These datarepresent the means ± SD of six replicates forcalculated compartment sizes, rate constants,and fluxes (in parentheses).

TRANSITORY 34*10-@ * 19*10-* *'4-

STORAGE

(4.3*2.5 pmotC m-2 *-1)POOL _

40*21 mmolC m-2 189o10- * 207*10- *-

EXPORTABLEPOOL

127 * 15 mmolC mr'

(3.2*2.1 jJmolC mrn- s-1)

26*10-1 * 18*10- 8-1

minated leaf labeled with "4CO2 7 h after the beginning ofthe photoperiod (Fig. 6 for the mean curves). Specific radio-activity in the leaf was higher for sorbitol and sucrose thanfor the other carbohydrates during 5 h following labeling.Specific radioactivity of sucrose decreased immediately afterlabeling. Specific radioactivity of sorbitol was stable duringthe first 3 h after labeling. Sucrose was apparently turnedover more rapidly than sorbitol (Table II). For calculatedexport fluxes (Table II), the sorbitol to sucrose carbon ratiowas 0.75 ± 0.11 mol of C mol' of C (mean ± SD of fourreplicates).The specific radioactivity of carbohydrates in the petiole

was determined 15 h after the beginning of the photoperiodfor leaves labeled 7, 9, 12, and 15 h after the beginning ofthe photoperiod. The results were expressed as a function of

a-

4~400 sorbitol

~-starch

.0 fructose200

± ,~~~ glucoseo L *I-

CL 0 2 4 6 8C0

Time (h after pulse labeling)

Figure 6. Changes in the specific radioactivity of soluble carbohy-drates and starch in mature peach leaves measured after a pulse-chase experiment. The leaves were labeled using 14CO2 for 4 min,7 h after the beginning of the photoperiod; the chase lasted 8 h.Soluble carbohydrates and starch were extracted and specific ra-

dioactivity was determined after HPLC separation as described in"Materials and Methods." These data represent the means ± SD offour replicates.

time after labeling (Fig. 7). Three hours after leaf labeling,the specific radioactivity for sucrose was significantly higherthan that for sorbitol. Later, specific radioactivities for sucrose

and sorbitol were similar. The specific radioactivities of sor-bitol and fructose were identical for all sampling times.

DISCUSSION

Partitioning of Photosynthetic Carbon

In mature peach leaves, the partitioning of photosyntheticcarbon into nonstructural carbohydrates versus unidentifiedfraction (called 'others' in Fig. 4) increased during the pho-toperiod. The radioactivity of this fraction, which was notstudied in the present experiment, is due mainly to aminoacids followed by anionic soluble compounds (data notshown). The partitioning of photosynthetic carbon into sol-uble sugars versus starch increased slightly during the pho-toperiod (Fig. 4). However, the partitioning of photosyntheticcarbon between sucrose and sorbitol remained nearly con-

stant for the duration of photosynthesis during the secondhalf of the photoperiod (Fig. 4). This implies a precise regu-

lation of the amount of sucrose and sorbitol in mesophyllcells. Therefore, sorbitol synthesis should be subject to finecontrol as shown previously for sucrose synthesis (24). Itshould be noted that glucose-6-P, the substrate of aldose-6-P reductase, i.e. the precursor for sorbitol (10, 18, 20), is an

activator of sucrose-phosphate synthetase (24), a key enzymeof the sucrose pathway.

Table II. Calculated Apparent Turnover Time and Export Flux forSorbitol and Sucrose in Mature Peach Leaves during the SecondHalf of the Photoperiod

Turnover time was calculated as indicated in 'Materials andMethods." Export flux was calculated as (turnover time)-1 (sucroseor sorbitol content) in the leaf blade 7 h after the beginning of thephotoperiod. Values represent the means ± SD of four replicates.

Parameter Sorbitol Sucrose

Turnover time (s) 65500 ± 22000 22930 ± 3600aExport flux (imol C m2 S-1) 1.35 ± 0.28 1.82 ± 0.30a Statistically significant difference between columns (t-test a =

0.05)

I1 882 MOING ET AL.




0 2 4

TIME (h after leaf pulse

Figure 7. Changes in the specific radioactivity o

drates and starch in the petiole of a maturelabeling was performed at 7, 9, 12, and 15 h aftethe photoperiod. The chase lasted until the end o

Soluble carbohydrates and starch were extractedioactivity was determined after HPLC separatic"Materials and Methods." These data represent tfour replicates.

Carbon Export

Phloem Sap

To our knowledge, this is the first time Fhas been analyzed for a species in which sorlare both primary end products of photosyntotal solute concentration obtained is probaloration, a factor that is difficult to controsucrose concentration is similar to that relspecies (21). In our experimental conditiosorbitol and sucrose appear equivalent in t4of translocated carbon.

Export Rate

Total radioactivity loss curves are represenexport. The amount of "4CO2 lost by respirprolonged chase period can be neglected be(in the light is sustained mainly by recently as

(14). Moreover, a preliminary experiment (Ishowed that the leaf blade thickness of fixe4mature leaves was between 0.14 and 0.18we can assume that the Geiger-Muller tub(activity in the whole thickness of the leafredistribution of radioactivity among indivihad created little artifact. Comparison of t(loss curves of peach leaves (Fig. 1) with thospecies such as Poa spp. (3) and maizeapparent turnover time in peach is significai

The compartmental analysis indicates two pools involvedin phloem loading (Fig. 5), which are usually attributed tocytoplasmic sucrose and vacuolar sucrose in species wheresucrose is the only exportable carbohydrate (16). In peach,this model may be questioned because each of the two poolsmay represent two different molecular forms, i.e. sucrose andsorbitol. The sorbitol to sucrose carbon ratio was about 2.5in the leaf blade (Table I) and the fast-exportable compart-ment to transient-storage compartment carbon ratio was

about 4.5 (Fig. 5). In peach, the size of the fast-exportableel > compartment was larger than that of the transient-storage

compartment (Fig. 5), whereas it is usually the opposite inherbaceous species. If the latter compartment referred to thevacuole, then the vacuole would play a secondary role in thecarbon management in peach leaf. To obtain a more realistic

II picture of carbon compartmentation in peach leaf, a model* .0___ accounting for four exportable pools (i.e. vacuolar sorbitol,

cytoplasmic sorbitol, vacuolar sucrose, and cytoplasmic su-6 8 crose) is required. The mathematical curve fitting used in the

present paper did not allow characterization of more than!labeling) two exponential functions.

Three methods were used to estimate carbon export ratef soluble carbohy- out of the leaf: measurements of photosynthesis and carbo-peach leaf. Pulse hydrate contents, compartmental study, and examination ofXr the beginning of changes in radioactivity in sorbitol and sucrose. Net photo-ifthe photoperiod. synthetic rate minus carbohydrate accumulation rate gave a!d and specific ra- high estimate of export rate with a value between 8 and 6)fn as described in Mmol of C m-2 s-1. This method of calculating the export ratethe means ± SD of involves fewer assumptions than methods derived from com-

partmental analysis. However, carbon storage in compoundsother than nonstructural carbohydrates is neglected. Thecompartmental study gave a value of approximately 3 ,umolof C m-2 s-1, in agreement with the study of sorbitol andsucrose radioactivities, which also gave a sum of sorbitol plus

Dure phloem sap sucrose export rate of approximately 3 ,umol of C m-2 s-1.bitol and sucrose The discrepancy between the carbon balance sheet and thethesis. The high carbohydrate export using the 14C technique suggests anbly due to evap- export of unlabeled or uncounted carbon. Amino acidso1. However, the (mainly asparagine, glutamine, and glutamic acid), whichported for other were found in phloem sap in the present study, representns, the roles of possible candidates.erms of quantity Davis and Loescher (6) have shown that sucrose is exported

at a faster rate than mannitol in celery. They suggest that thealditol may be preferentially stored in the vacuole. However,one reason for the lower apparent turnover time of sorbitolthan of sucrose is related to the two times higher pool size

*tative of phloem for sorbitol than for sucrose. Nevertheless, in peach the^ation during the observed difference in pool size does not adequately explaincause respiration the difference in quantity of radioactivity exported for these;similated carbon two carbohydrates. In the present experiment, the changedata not shown) with time in specific radioactivity of sorbitol indicated clearlyd and embedded that it continued to incorporate much more radioactivity thanmm. Therefore, did sucrose after the end of pulse labeling. The fact that

e detected radio- sucrose, which was heavily labeled, may and sorbitol mayblade and that not be metabolized in the mature leaf blade (2, 12) gives rise

idual tissues (19) to the hypothesis that the synthesis of labeled sorbitol from)tal radioactivity labeled hexoses originates partly from sucrose, as suggestedse of herbaceous by the present results. The futile cycle, consisting of a cyclic(22) shows that transformation of sucrose and hexoses, demonstrated in otherntly greater. species (4, 9) may provide additional glucose-6-P for sorbitol

7C)

E 200M.E0.

150

0Cioo4._

c4)0

.' 50

0

'0h._

0.

Cn

I i

1883




synthesis. The decrease in radioactivity in sucrose then results

from both export and metabolism for sorbitol synthesis. The

sorbitol to sucrose ratio is higher in petiole than in leaf

mesophyll, which is in agreement with a conversion of part

of the sucrose pool into sorbitol. In the petiole, fructose was

significantly labeled. Because hexoses did not seem to be

moving in the phloem sap, 4C-labeled fructose in the petiole

must have come from hydrolysis of sucrose or sorbitol. It is

suggested that fructose in the petiole is derived from sorbitol

conversion, involving sorbitol dehydrogenase (28), because

the specific radioactivity of fructose (Fig. 7) is different from

that of glucose but is equal to that of sorbitol. The sorbitol to

sucrose ratio for mesophyll or petiole is higher than for the

phloem sap collected by stylectomy of aphids, suggesting a

preferential utilization of sorbitol by young sink leaves (12).

In conclusion, sucrose and sorbitol are both exported via

phloem from illuminated peach leaves. Sucrose may contrib-

ute to supply the sorbitol pool. The analysis of radioactivity

loss curves under chemical steady state shows an apparent

export rate slower than that usually found for herbaceous

species and a limited contribution of the transient storage

compartment.

ACKNOWLEDGMENTS

We gratefully acknowledge Dr. G. Massonie for supplying

aphids and for his help and advice for the stylectomy experiments,

and Dr. P. Raymond for critical reading of the manuscript. We thank

A. Bonnet for his technical assistance in growing the seedlings.

are especially grateful to an anonymous reviewer who greatly helped

us to improve the manuscript.

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eds, Plant Carbohydrates I. Intracellular Carbohydrates. En-

cyclopedia of Plant Physiology, New Series, Vol 13A. Sprin-

ger-Verlag, Berlin, pp 158-1922. Bieleski RL, Redgwell RJ (1985) Sorbitol versus sucrose

photosynthesis and translocation products in developing apri-

cot leaves. Aust J Plant Physiol 132: 657-668

3. Borland AM, Farrar JF (1988) Compartmentation and fluxes

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5. Davis JM, Felmann JK, Loescher WH (1988) Biosynthesis

sucrose and mannitol as a function of leaf age in celery (Apium

graveolens L.). Plant Physiol 86: 129-133

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stylet technique in phloem physiology. Planta 161: 385-393

8. Gaudillere BernoudJJ,JardinetJP, Euvrard M (1987)

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