Plant Physiol. (1988) 86, 129-1330032-0889/88/86/0129/05/$O1 .00/0

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Agein Celery (Apium graveolens L.)'

Received for publication February 9, 1987 and in revised form September 25, 1987

JEANINE M. DAVIS, JOHN K. FELLMAN2, AND WAYNE H. LOESCHER*Department ofHorticulture and Landscape Architecture, Washington State University,Pullman, Washington 99164-6414

ABSTIRACT

In celery (Apium giwacokm L.), the two major translocated carbohy-drates are sucrose and the acyclic polyol mannitol. Their metabolism,however, is different and their specific functions are uncertain. To com-pare their roles in carbon partitioning and sink-source transitions, devel-opmental changes in 14 C)2 laIbeling, pooI sizes, and key enzyme activitiesin leaf tissues were examined. The proportion of label in mannitolincase dramatically with leaf maturation whereas that in sucroseremained fairly constant. Mannitol content, however, was high in allleaves and sucrose content increased as leaves developed. Activities ofmannose-6P reductase, cytoplasmic and chloroplastic fructose-1,6-bis-phosphatases, sucrose phosphate synthase, and sucrose synthase in-creased with leaf maturation and decreased as leaves senesced. Ribulosebisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehy-drogenase activities rose as leaves developed but did not decrease. Thus,sucrose is produced in all photosynthetically active leaves whereas man-nitol is synthesized primarily in mature leaves and stored in all leaves.Onset of sucrose export in celery may result from sucrose accumulationin expnding leaves, but mannitol export is clearly unrelated to mannitolconcentration. Mannitol export, however, appears to coincide with in-creased mannitol biosynthesis. Although mannitol and sucrose arise froma common precursor in celery, subsequent metabolism and transport mustbe regulted separately.

Although sink to source conversions have been well character-ized (7, 9, 11), the exact mechanisms for these transitions andhow they are regulated are still poorly understood. Fellows andGeiger (7) suggest that initiation of export in developing leavesis the result of phloem loading of solutes developing sufficientosmotic pressure to reverse the inward movement of solutes andproduce translocation out of the new source. This hypothesis issupported by changes in carbon partitioning observed in expand-ing leaves of a variety of species. For example, in soybean(Glycine max L.) (21) and sugar beet (Beta vulgaris L.) (9) sucroseconcentrations increased during leaf expansion. In squash (Cu-curbita pepo L.) (23) and cucumber (Cucumis sativus L.) (17)the onset of export coincided with an increase in levels of thetranslocate stachyose. Similarly, in apple (Malus domesticaBorkh.) concentrations of the translocate sorbitol rose as leaves

'Supported in part by the National Science Foundation, Grant DMB-8604100. Scientific Paper No. 7688. College of Agriculture and HomeEconomics, Agriculturl Research Center, Washington State University,Pullman, WA 99164-6242.2Present address: USDA/ARS Tree Fruit Research Laboratory, Wen-

atchee, WA 98801.

matured (15). Termination of solute import, however, is notsimply the result of initiation of export nor does it necessarilyrequire a positive carbon balance in the leaf (22). Several studiesindicate that activities ofenzymes involved in translocate biosyn-thesis and degradation are also important in sink-source conver-sions. For example, in apple activities ofthe enzymes responsiblefor sorbitol biosynthesis increased and those responsible for itsoxidation decreased as leaves developed (15). Similarly, activitiesofenzymes involved in stachyose synthesis and degradation werewell correlated with the accumulation of stachyose in expandingcucumber leaves (17).The objective of this study was to investigate leaf-age depend-

ent differences in partitioning between sucrose and mannitol incelery (Apium graveolens L.) leaves. Celery was the system ofchoice for our studies for several reasons. Most importantly,celery produces and translocates two major carbohydrates, su-crose and the acyclic polyol mannitol (RJ Redgwell, WHLoescher, RL Bieleski, unpublished data). Although these sharea common precursor, F6P3 (20), the final biosynthetic steps forthe two compounds require different enzymes. Comparisons ofthe metabolism of the two translocates during leaf developmentshould provide insight into the roles that each play in carbonpartitioning and sink-source transitions. Also, unlike polyol-producing deciduous tree species that have been studied previ-ously, celery can be grown in the greenhouse year round andkept in the vegetative state to provide a continuous supply ofleaves ofvarying ages. Developmental changes in " C02 labeling,pool sizes, and activities of some key enzymes responsible formannitol and sucrose metabolism in celery leaves are reportedhere.

MATERIALS AND METHODS

Plant Material. Celery (Apium graveolens L., 'Giant Pascal')was grown in a greenhouse with temperatures maintained around21°C day and 16°C night. High pressure sodium lamps were usedto supplement lighting and provide a 14 h photoperiod duringthe winter months. Plants were fertlized daily with 100 ppmPeter's 20:20:20 (W. R. Grace and Co., Allentown, PA). Crownsof plants were sprayed till run-off once a week with 0.4%Ca(N03)2 solution. Leaves were sequentially numbered in orderof emergence from the center of the plant with No. 1 being theyoungest visible leaf. In a typical 14 leafplant, leafNo. 3 (young)was light green, still expanding; leaf No. 7 (mature) was dark

3Abbreviations: F6P, fructose-6-P; TMS, trimethylsilyl; GC2/FID,glass-capillary gas chromatography/flame ionization detection; M6PR,mannose-6-phosphate reductase; PVPP, polyvinylpolypyrrolidone; SPS,sucrose phosphate synthase; SS, sucrose synthase; RuBP, ribulose bis-phosphate; FBPase, fructose-1,6-bisphosphatase; NRG3PDH, nonrevers-ible glyceraldehyde-3-phosphate dehydrogenase.

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Plant Physiol. Vol. 86, 1988

green, just fully expanded; and leaf No. 11 (old) was thickenedand leathery.

Pulse-Chase Labeling. The terminal leaflet of leaves of variousages was enclosed in a plastic bag, volume approximately 1 L.The bag was preloaded with NaH'4 CO3 (56 mCi/mmol) whichreleased 20 MCi "'CO2 when 40 MAl lactic acid was injected intothe bag at time zero. After a 10 min pulse the bag was removedand the leaf chased with ambient CO2 for 10 min. Export of labelfrom the blade into the petiolule was undetectable during thislength chase. PAR was approximately 500 Mumol.m-2.s- from aSylvania 300 W incandescent movie light bulb projected througha water bath. All labeling experiments were conducted betweennoon and 3:00 PM. The sample was extracted and fractionatedaccording to the method of Redgwell (18). In short, the leaf wasquick frozen in liquid N2, dropped into cold metha-nol:chloroform:H20:formic acid (12:5:2:1, v/v) and held at-10°C overnight. After extraction and separation into chloro-form and aqueous phases, the aqueous phase was fractionatedinto neutral, amino acid, organic acid, and phosphate esterfractions by ion exchange chromatography. The neutral fractionwas further separated by two-dimensional TLC on 250 Mm thickcellulose (Sigma Chemical Co., St. Louis, MO) layers. Solventsystem for the first dimension was n-butanol:acetic acid:H20(12:5:3, v/v) and methyl ethyl ketone:acetic acid:saturated boricacid (9:1:1.5, v/v) run twice for the second dimension. Spotswere detected by fluor-enhanced autoradiography (En3 Hance,New England Nuclear, Boston, MA). Plates were then coatedwith 'Strip-mix' (2), spots were cut out and counted by liquidscintillation spectrometry.

Identities of compounds were determined by co-chromatog-raphy with radioactive standards or with nonradioactive stand-ards and an indicator spray. Four different aged plants werelabeled as shown in Figures 2 and 3.

Carbohydrate Determinations. TMS derivatives of the neutralcomponents from the fractionation scheme were analyzed byGC2/FID after conversion of sugars to their respective oximederivatives (4).Enzyme Assays. For all assays, extracts were prepared from

the three terminal leaflets of different aged leaves as described inFigures 4 and 5. For chloroplastic and cytoplasmic FBPases,NADPH-requiring NRG3PDH and RuBP carboxylase, 1.0 g

fresh material was ground for1 min in 3.0 ml of the appropriatebuffer with sand and acid-washed PVPP in a cold mortar andpestle and centrifuged at 12,800g for 4 min. For the M6PR assay,the extract from 0.2 g tissue was held on ice for 10 min aftergrinding for1 min in 2.0 ml 100 mM Tris (pH 7.5 at 25C), 10mM DTT, 2 mg PVPP, and a pinch of sand and then centrifugedat 12,800g for1.5 min. The supernatant was used in every case.

Since inconsistent results were obtained using crude single-leafextracts, SPS and SS were extracted from bulk samples of young,mature or old leaves from 17 leaf plants. Although such samplesprovided a less complete picture of age-related events than single-leaf samples, they still indicated mean changes that occur withleaf development. For routine assays, fresh leaves were groundwith a Polytron homogenizer (Brinkmann Inst. Westbury, NY)in Hepes-NaOH buffer (50 mM Hepes, 5mM MgCl2, 2.5 mMDTT,1 mm EDTA, pH 7.0) containing 0.125M KCI(19) andPVPP(1 mg/g tissue). The brei was filtered through Miraclothand centrifuged at 15,000g for 30 min. Aliquots of the superna-

tants were assayed by incubating with reaction substrates, F6Pfor SPS and fructose for SS, forS to 8 min as previously describedby Rufty etal. (19). Sucrose synthesis was linear for 8 min, afterwhich sucrose hydrolysis was evident. After the reactions were

terminated(19), the samples were dried, myo-inositol was addedas an internal standard, and TMS derivatives were prepared andassayed for sucrose via GC2/FID (6). SPS was also partiallypurified from the supernatants by a modification of previously

published techniques (10, 19). Briefly, the supernatants wereloaded onto a Sepharose 4B column (bed volume 18 ml) andabsorbance at 280 nm monitored. When absorbance returned tobaseline levels, SPS was eluted with 0.5 M KCI. Protein-contain-ing fractions were pooled and concentrated with an Amiconultrafiltration device equipped with a PM-10 membrane. Thepooled fractions were dialysed overnight against Hepes-NaOHbuffer and assayed as described above. Protein content wasdetermined by the dye-binding method (3). Typically, specificactivity increased 50- to 55-fold with this partial purificationprocedure and recovery was in excess of 100%.RuBP carboxylase was assayed radiochemically (16). The fol-

lowing enzymes were assayed spectrophotometrically at 28°C.Chloroplastic FBPase was assayed in a linked system at pH 8.8and was not inhibited by 1 to 2,M fructose-2,6-bisphosphate(14). Cytoplasmic FBPase was assayed as described above, exceptthe pH was 7.5 and its activity was substantially inhibited by 0.5to 2,M fructose-2,6-bisphosphate. NRG3PDH was measured asdescribed by Kelly and Gibbs (13). M6PR was assayed by amodified method of Rumpho et al. (20) using 650 Ml 10 mM Tris(pH 7.5), 100 Ml 1 mM NADPH, 100 MAl enzyme extract, and 150Ml 100 mm mannose-6-P. At least three plants were assayed foreach enzyme. Results shown are from representative plants.

RESULTS

To determine if major photosynthetic products changed withleaf maturity, leaves from 6, 12, 14, and 16 leaf plants werepulse-chase labeled with "CO2. The amount of4 C fixed, how-ever, varied from leaf to leaf. For example, total incorporationof "C into leaves of the 14 leaf plant was 5579, 33,352, and7885 dpm-mg-' fresh weight for leaf numbers 3, 7, and 11,respectively. Factors affecting incorporation included theamount of leaf tissue enclosed in the bag and changes in photo-synthetic rates with leaf development (8). For example, photo-synthetic rates in young, mature, and old leaves, under lightconditions similar to those used in the labeling studies, were 20,28, and 40mg CO2 dm2- h-', respectively (8). Because of thesevariations in incorporation efficiencies, all labeling data arepresented as percent of radioactivity in the aqueous phase.

In all leaves, regardless of incorporation efficiency, 78% ormore of the label was in the aqueous phase. As leaves developed,photosynthetic rates increased (8) as did the percent of radioac-tivity in the neutral fraction (Fig. 1). Conversely, the percent oflabel in amino acid, organic acid, and phosphate ester fractionsdecreased with increasing leaf age. In all leaves, 72 to 95% of theneutral fraction radioactivity was in mannitol and sucrosewhereas glucose comprised only 10% of the radioactivity in this

:100cL 90Fractions: Neutral M Organic acid0 16 leaf plant= 80 KID Amino acid = P04-ester8 70 6leaf plant

es60 12 leaf plant

260.

Leafnumber from center ofplantFIG. 1. "'CO2 labeling of soluble metabolites in developing leaves ofdiffierent aged celeryplants following a 10 minipulse/ 10 minichase. For

each plant, the set of bars on the left represents a young developing leaf,the center set a mature leaf and the set of bars on the right an old leaf.

130 DAVIS ET AL.

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BIOSYNTHESIS OF SUCROSE AND MANNITOL IN CELERY

3 59 37 11 3713taf number from center of plant2.5 14.830.3 8.0 19.0 22.5 8.8 3an 6jj Imngitol15.4 20.3 10.2 17.5 16.5 13.9 12.3 26i sucrose

FIG. 2. Incorporation of '4C02 following a 10 min pulse/10 minchase into mannitol and sucrose in leaves of different aged celery plants.For each plant, the bar on the left represents a young developing leaf,the center bar a mature leaf, and the right bar an old leaf which on the16 leaf plant was beginning to senesce.

02

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0

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Po-mg FW 10.78.7610.2 6.37 7.0012.0 12.8 14.7 13.7 11.6 18.5 11.7 manritol0.51 0.69 6.89 0.213.685.86 0.501.605.22 0.263.244096 sucrose

FIG. 3. Pool sizes and ratios of pool sizes of mannitol and sucrosefrom labeled leaves in Figure 2.

fraction and fructose was labeled (9.3%) only in the youngestleaf of the 16 leaf plant. In all plants, 4 C-mannitol:'4 C-sucroseincreased as leaves matured (Fig. 2). This ratio only decreased inthe oldest leaves of the 16 leaf plant which were beginning tosenesce, as indicated by decreasing photosynthetic rates (8).These data indicate that mannitol synthesis increased dramati-cally, relative to sucrose synthesis, with leaf maturation. Theexception was the 16 leaf plant in which the percent of label insucrose also increased with leaf age. In contrast to the labelingdata, mannitol:sucrose pools decreased as leaves developed (Fig.3). Despite the differences in labeling patterns (e.g. ranging from2.5-30.3% of the aqueous phase radioactivity in the 12 leafplant), mannitol content was relatively constant (5.4-12.0 mg.g-' fresh weight in the 12 leaf plant). Although sucrose contentincreased with leaf maturation, mannitol content was alwaysgreater, 1.5- to 44-fold, depending on leaf age.

Activities of enzymes responsible for mannitol and sucrosemetabolism changed with leaf development. Activity of M6PR,cytoplasmic and chloroplastic FBPases, SPS and SS increased asleaves developed, peaked in the first fully expanded leaves anddecreased in the older leaves (Fig. 4 and 5). Although RuBPcarboxylase and NRG3PDH activities increased as leaves ma-tured these did not decrease in old leaves.

DISCUSSION

Developmental differences in translocate metabolism havebeen noted in leaves of several species with two or more major

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FIG. 4. Activities of several enzymes involved in translocate biosyn-thesis as a function ofleafage in 12 leaf celery plants. First fully expandedleaves were leaves 5 and 6. A, Cytoplasmic enzymes; B, chloropausicenzymes.

translocated carbohydrates. For example, in squash (Cucurbitapepo L.) sucrose synthesis was detectable at all stages of leafdevelopment whereas synthesis of stachyose and raffinose coin-cided with initiation of phloem transport in the leaf (23). Severalwoody plants in which the major translocates are both an acyclicpolyol and sucrose have also been examined. For example, inapple sucrose content was low but fairly constant throughoutleaf maturation, decreasing as leaves senesced. Concentrationsof the major translocate, sorbitol, however, increased dramati-cally with leaf expansion and decreased only during leaf senes-cence (15). Thus, there was a clear correlation between sorbitolaccumulation in newly expanded leaves and the onset of export.A different pattern, however, was found in apricot (Prunusarmeniaca L.) where throughout leaf growth sorbitol levels were

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Plant Physiol. Vol. 86, 1988

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FIG. 5. Activities of sucrose metabolizing enzymes from supernatantsof crude extracts as a function of leaf age in a 17 leaf celery plant. Thepair of bars on the left represents developing leaves, the center set matureleaves, and the set on the right older and senescing leaves.

high and sucrose levels were low, decreasing slightly in olderleaves. A 10 min pulse with 4 C02 followed by an unspecifiedchase in ambient air showed that the amount of [14 C]sorbitolincreased and [14 C]sucrose decreased (when expressed as percentof total sugar) as these apricot leaves developed (1). Thus, al-though translocate pools remained unchanged, there was a goodcorrelation between the leafs ability to synthesize sorbitol andthe apparent onset of export.

Partitioning between polyol and sucrose during celery leafmaturation was different from that found in either apricot orapple. In celery, sucrose levels rose as leaves developed but theproportion of 4 CO2 partitioned into sucrose was fairly constant,indicating rapid utilization of sucrose for growth in young leaftissue as reported in sugar beet (9). The gradual accumulation ofsucrose in expanding celery leaves may be related to the onset ofsucrose export by developing sufficient osmotic pressure to ini-tiate phloem loading as has been shown in sugar beet (7). Incontrast, mannitol levels were high even in young celery leavesand remained relatively constant during leaf maturation eventhough mannitol biosynthesis increased as leaves matured. Thissuggests that the onset of mannitol export was correlated withthe leaf's increasing capability to produce mannitol.Changes in activities of enzymes involved in translocate syn-

thesis and degradation may play important roles in sink-sourceconversions. SPS is the principal enzyme in sucrose biosynthesisin mature leaves (12) and SS is considered a key enzyme forsucrose utilization in sink tissues such as developing leaves (24).In eggplant (Solanum melongena L.), cassava (Manihot esculentaCrantz), grapevine (Vitis vinifera L.), sugar cane (Saccharumofficinarum L.), and maize (Zea mays L.) SS activities werehighest in growing leaves (5), suggesting the importance ofSS insink growth. In sugar beet, however,SS activities were similar insource and sink leaves while SPS activity was detected only insource leaves suggesting a difference in intracellular compart-mentation of sucrose and sucrose metabolizing enzymes in sinkand source leaves (9). In celery, although the proportionof 14 C02partitioned into sucrose was similar in most leaves, sucrose poolsizes increased with leaf maturation. Activities ofSS and SPSalso appeared unrelated to the labeling data, with both exhibitingthe highest activities in mature leaves.SS activity was lowest inyoung leaves which exhibited the highest sucrose utilization rateswhen leaf discs were floated on [14 Cjsucrose (6). This suggests adifference with leafdevelopment in compartmentation ofsucroseand sucrose metabolizing enzymes or that invertase activity ismore important than SS activity for sucrose hydrolysis in celery

sink tissue. Another possibility is that crude extract activities(Fig. 5) do not reflect in vivo activities since partially purified(50- to 55-fold) extracts resulted in recoveries of SPS in excess of100%. For example, following partial purification the SPS valuesin Figure 5 increased by 41, 17, and 135% for young, mature,and old leaves, respectively. These values also do not appearrelated to the labeling data.

In celery, development of the capability to produce the trans-locate mannitol (Figs. 2 and 4A) and loss of the ability to utilizemanmtol (6) are associated with sink-source transitions in grow-ing leaves. Increased activities of the key enzymes involved inmannitol biosynthesis, M6PR, cytoplasmic FBPase, andNRG3PDH (20), are consistent with 4C02 labeling in celery,i.e. activities were barely detectable in young leaves, increasedrapidly as leaves developed, and decreased again as leaves beganto senesce. Mannitol can only be utilized and the degradativeenzymes are only present in sink tissues such as young developingleaves (6). Pharr and Sox (17) found similar changes in activitiesof enzymes involved in stachyose metabolism in cucumber:increased synthesis and decreased degradation were well corre-lated with sink to source conversions. Onset of export and lossof ability to import are frequently accompanied by increasedphotosynthesis, e.g. in sugar beet (9). Although we did notsimultaneously measure photosynthetic gas exchange and trans-locate metabolism in celery, we have shown that photosyntheticrates (8) and RuBP carboxylase activities (Fig. 4B) were low inyoung leaves and highest in first-fully expanded leaves. Further-more, export of 4 C from lamina to petiole (following a 10 minpulse of 4 CO2 and a 1 h chase) was high in first-fully expandedleaves (JM Davis, WH Loescher, unpublished data).

In celery, mannitol and sucrose are similar in many respects:both are primary photosynthetic products and nonreducing sug-ars; both are synthesized in the cytoplasm from a commonprecursor (20) and each makes up about 50% of the translocatedcarbohydrate in petioles of filly expanded leaves (JM Davis, WHLoescher, unpublished data). There are, however, mijor differ-ences in metabolism and partitioning of these two compoundsduring leaf development. Sucrose is produced and utilized inleaves of all ages, but mannitol is synthesized primarily in matureleaves and utilized only in young leaves (6). Mannitol levels,however, are high in all leaves. This indicates that whereassucrose is very active metabolically, mannitol serves primarily asa long distance transport and storage compound. Although onsetof sucrose export in expanding celery leaves may be the result ofan accumulation of sucrose, as has been reported for other species(9, 21), initiation of mannitol export is clearly unrelated to leafmannitol content. Mannitol export does, however, appear tocoincide with increased mannitol biosynthesis. Separate regula-tory mechanisms must exist for mannitol and sucrose biosyn-thesis and translocation in celery.

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6. FELLMAN JK, WH LOESCHER 1987 Comparative studies of sucrose and man-nitol utilization in celery (Apiumgraveolens L.). Physiol Plant 69: 337-341

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BIOSYNTHESIS OF SUCROSE

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