Phloem Unloading in Developing Leaves of Sugar Beet - NCBI

Plant Physiol. (1985) 79, 237-241 0032-0889/85/79/0237/05/$0 1.00/0

Phloem Unloading in Developing Leaves of Sugar Beet' 1. EVIDENCE FOR PATHWAY THROUGH THE SYMPLAST Received for publication January 8, 1985 and in revised form May 28, 1985

J. GOUGLER SCHMALSTIG*2 AND DONALD R. GEIGER Biology Department, University of Dayton, Dayton, Ohio 45469-0001 ABSTRACT

from the terminus of the translocation path would be linked

directly to metabolic conversion in mesophyll cells. Active upPhysiological and transport data are presented in support of a sym- take of solutes into the serve mainly for retrieval plastic pathway of phloem unloading in importing leaves of Beta vulgaris of solutes from the freemesophyll would We know that in developing space (17). L. ('Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene leaves import is decreased by treatments such as low temperature sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake and anoxia which inhibit metabolism (8). of exogenous I'4qsucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of '4C-label into sink leaves during steady state labeling of a source leaf with '4CO2. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, L-1'4Cjglucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, L-j'4q glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.

Partitioning among sink organs is considered a major factor in crop yield (13). Differences in the path followed from the phloem to sink cells are expected from anatomical and physiological differences among sink organs (10, 13) and have been verified experimentally. A path traversing the free space has been demonstrated in sugarcane (14) and bean stems (22), and in Viciafaba L. stems parasitized by Cuscuta (27). Evidence indicates a symplastic path of phloem unloading in developing root tips of pea (2) and corn (12). In soybean and maize, phloem solutes may pass through the symplast into cells in the maternal tissue that surround the embryo and later be released into the free space for transit to embryonic tissue (5, 24). In comparison to these systems, considerably less is known about the path followed by solutes during phloem unloading in young leaves. Two possible paths of exit from the phloem of a young leaf may be envisioned: (a) through the symplast by way of plasmodesmata connecting cells of the phloem and surrounding mesophyll cells; and (b) exit directly into the free space or apoplast, followed by uptake into mesophyll cells. The mechanism and driving force for removal of solutes and water from the terminus of the translocation path will depend on the unloading pathway. If the major portion of the phloem contents move directly into mesophyll cells through plasmodesmata, then removal of solutes

In contrast, phloem unloading through the free space necessitates membrane transfer both from the phloem and into the mesophyll cells. Flux from the phloem into the apoplast would be promoted by keeping the solute concentration in the free space low, either by active transport into the mesophyll cells, or perhaps, by metabolic conversion inside the sink cells. In sugar beet, sucrose is the major translocated sugar and there is evidence for carrier-mediated uptake of sucrose into mesophyll cells of sink leaves (6) as demonstrated by saturation kinetics. To distinguish between the apoplastic and symplastic pathways, we considered the probable role of a membrane barrier in the transfer of material from the phloem to the mesophyll. In the first experimental approach, an inhibitor of sucrose uptake was infiltrated into the free space to examine participation of membranes bounding the free space in phloem unloading. The second approach made use of the distribution of a xenobiotic sugar, L-glucose, to test if there are open plasmodesmata connecting sieve elements with the surrounding sink cells. The results support a symplastic pathway.

MATERIALS AND METHODS Plant Material. Sugar beet plants (Beta vulgaris L. 'Klein E multigerm') were grown in a mixture of equal amounts of Jiffymix and sand; the plants were watered four times daily with the nutrient solution described by Snyder and Carlson (23) except that H3B03 was increased to 20.6 gM. Plants were grown for 4 to 6 weeks using a 14-h light period at 25°C and a 10-h dark period at 17°C. The photon flux density at the leaf-blade level was 430 to 480 ME m-2 s-'. Young leaves were used at an age at which they were strong sinks and were large enough to be treated. The leaf length was 5 to 7 cm, representing 30 to 40% of FLL.3 Leaves of this age received 0 to 10% of their dry weight gain from photosynthesis within the leaf and the rest from import (data not shown). The leaves were beginning the basipetal sinkto-source transition with about 18 to 36% of their length exhibiting vein loading (6; see also Fig. 1). Sucrose Uptake. The upper leaf surface was lightly abraded with cerium oxide abrasive powder using a camel hair brush. Micrographs taken with a scanning electron microscope (not shown) revealed removal of wax but no evidence of damage to epidermal cells after abrasion. Solution containing ['4C]sucrose at a specific radioactivity of 61.7 Bq nmol-' and concentration

'Supported by National Science Foundation Grant DMB-8303957 and a grant from Monsanto Agricultural Products Co. (D. R. G.). 2Present address: Central Research and Development Dept., Experi3Abbreviations: FLL, final laminar length; GM, Geiger-Mueller, mental Station, E. I. du Pont de Nemours and Co., Wilmington, DE PCMBS, p-chloromercuribenzene sulfonic acid; NEM, N-ethylmaleim19898. ide; NCER, net carbon exchange rate. 237

238

SCHMALSTIG AND GEIGER

noted (Mes buffer, 50 mm [pH 5.5] containing 1 mM CaCl2 and 5 mM K2HP04) was either added to the abraded surface of the leaves or supplied to leaf punches floating on it. Uptake time was 60 min unless specified after which the tissue was placed over unlabeled sucrose of the same concentration for 20 min to allow exit of ['4C]sucrose from the free space. Leaf punches were initially 4 mm in diameter. After removal of free space solutes smaller concentric punches were taken for analysis to lessen the edge effect on measured uptake. The tissue was either digested for liquid scintillation counting or frozen over dry ice and dried for autoradiography while frozen. Digestion was done by adding 0.2 ml HCl04 and 0.4 ml H202 to the tissue, then heating at 50°C for 1 to 2 h. For autoradiography, the dried tissue was flattened in a hydraulic press and placed on Kodak Industrex Xray film, type M, for the length of time needed to accumulate 5 x 106 counts from 1 cm2 of leaf tissue as determined by GM tube counting (7). PCMBS Treatment. PCMBS, purchased from Sigma Chemical Co., was prepared in buffer of the same composition as for sucrose uptake and used immediately. To ensure a maximum inhibition without penetration of the cells, a concentration of 10 mM PCMBS and 10-min treatment time followed by three 10min rinses with buffer was used. The uptake time for sucrose was 60 min. PCMBS at higher concentrations resulted in no greater inhibition. There was no difference in PCMBS inhibition of sucrose uptake when the PCMBS was passed through a Dowex chelating resin to remove free Hg, and so this purification step was not used routinely. PCMBS was flooded over the abraded leaf surface on one side on the midvein and held in place with caulking putty. The other half was flooded with buffer alone. Measurement of Import. Steady state 4C02 labeling of one source leaf was performed as described previously (9). Import was measured continuously as accumulation of label in a young leaf held horizontally; a GM tube was fastened beneath it. The upper surface was abraded as for sucrose uptake experiments and Mes buffer of the same composition described for sucrose uptake was held on the leaf surface with caulking putty. Approximately 50% of the total area of the leaf (100% of the leaf area monitored by the GM tube) was flooded with buffer. The buffer solution was circulated between a 2-ml reservoir and the leaf surface by a peristaltic pump at the rate of 1 ml min-'. Solutions were removed and added from the reservoir to keep the solution level over the leaf constant so as not to change the counting efficiency of the GM tube. There were oscillations in the rate of import (Fig. 5) which were similar to those reported for stomatal resistance (1). Changes in stomatal resistance lead to changes in leaf water content and therefore to changes in absorber thickness and counting efficiency of the GM tube. To eliminate the effect of oscillations on the measurement of import rate and obtain values for statistical analysis, the mean of rates before treatment were compared by Student's t test to the mean of rates after treatment. The volume of the solution removed was measured and the solution counted by liquid scintillation to determine radioactivity which was removed from the leaf. of 11-Hgj-PCMBS. ["3Hg]-PCMBS was purchased I from Amersham International and the purity confirmed by electrophoresis against unlabeled PCMBS. The entire abraded leaf surface was flooded with labeled PCMBS as described in "PCMBS Treatment" except that only 35% of the leaf surface was covered. A total of 0.5 ml of 10 mM PCMBS at specific radioactivity 76.6 Bq nmol[' was added. After the three rinses with buffer, the whole leaf was frozen over dry ice, dried, and prepared for autoradiography as described in "Sucrose Uptake." Pieces of tissue were placed over melted embedding paraffin so that only one face and the edges were covered by wax. Paradermal sections, 40 ,m in thickness, were then cut from the tissue pieces with a sliding microtome. The sections were weighed with a

Plant Physiol. Vol. 79, 1985

microbalance and placed in liquid scintillation cocktail for counting. Vislulization of L-{"qGlucose. The source leaf of a plant trimmed to leave only one source and one sink leaf was supplied with 5 mM L-['4C]glucose at specific radioactivity 37 Bq nmolU' by reverse-flap vein feeding. The technique consisted of dissecting free a first order vein and inserting a 50 gl capillary tube over the cut end on the side nearest the midvein. L-['4C]Glucose in Mes buffer, of the same composition as described for sucrose uptake, was then added to the capillary tube with a microsyringe. Four capillary tubes were put on one source leaf such that each fed an area drained by at least one intact major vein. Accumulation of label in the sink leaf was measured with a GM tube fastened next to the leaf. Vein-feeding continued for 10.5 h. The sink leaf was frozen over dry ice, dried, and prepared for autoradiography. After autoradiography, parts of the sink leaf tissue were digested with HC104 and H202 and counted by liquid scintillation. The concentration of L-glucose in the tissue was estimated by assuming 1 g fresh weight equivalent to 1 ml. L-['4C]Glucose was supplied to the free space of a sink leaf through the cut end of the petiole for 6 h. The leaf was the same age as the sink leaf in the previously described experiments. LGlucose concentration was such that, after uptake of 200 1d, the final tissue concentration of L-glucose would be about the same as that of the sink leaf fed from the source leaf. After uptake, these leaves were frozen, dried, and prepared for autoradiography. RESULTS PCMBS Inhibitor Studies. The first approach used to distinguish the two models was to test the effect of an inhibitor of sucrose uptake on phloem unloading. If solutes were passing through the free space during phloem unloading, then the presence of an inhibitor of sucrose uptake should impede phloem unloading by preventing rapid removal from the free space. Such an inhibitor should have the following characteristics: (a) inhibit uptake of exogenous sucrose into mesophyll cells, (b) remain external to the cell, hence not affecting metabolism, and (c) infiltrate the tissue, particularly down to the vicinity of the veins. The sulfhydryl reagent, PCMBS was chosen because of its competitive inhibition of sucrose uptake (18) and because PCMBS was shown to satisfy the three described characteristics when applied to sugar beet sink leaf tissue. There is evidence that its site of action is external to the plasma membrane because it does not affect respiration and photosynthesis nor cause membrane leakage in sugar beet leaves (1 1). PCMBS also does not change the membrane potential difference in Vicia faba L. pod walls

(20). Uptake of exogenous sucrose by both leaf discs and intact leaves was linear with time. Pretreatment with 10 mm PCMBS for 10 min, followed by three 10-min washes with Mes buffer inhibited sucrose uptake into both intact leaves (Fig, 1) and leaf discs (Table I). Uptake into treated disks averaged 24.4% of control rates over the range of sucrose concentrations used (0.10.5 mM). This inhibition was partially reversed by 20 mM DTT (Table II). The inhibition was not restricted to inhibition of uptake into veins, as seen in mature leaves of sugar beet (1 1) and Viciafaba L. (18). At the age of the sink leaves used, extensive vein loading capacity has not developed (6). Vein loading capacity had developed only in the tip of the untreated leaftissue (Fig, 1). In the half of the same leaf which was treated with PCMBS, no vein loading occurred (Fig, 1). Sucrose uptake was inhibited throughout the tissue. These results satisfy the first critera of inhibition of sucrose uptake into mesophyll cells. The requirement that PCMBS does not enter cells nor affect metabolism was demonstrated by lack of an effect on the NCER in either the light or the dark (Fig, 2). PCMBS was also shown

PHLOEM UNLOADING IN DEVELOPING LEAVES

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Table I. Inhibition of Sucrose Uptake by PCMBS Mean and SD of uptake rate are shown for three experiments. Rates as per cent of control do not differ in analysis of variance test at 1% significance level. Sucrose Concentration M 0.1 0.5 1.0 5.0

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to infiltrate through the leaf tissue. [203Hg]PCMBS was imaged in the veins in the margins of the leaf outside of the area to

which labeled PCMBS was added (Fig. 3). This demonstrates that PCMBS did penetrate to the level ofthe veins and was swept through the xylem stream. Pieces of tissue which were treated with [203HgJPCMBS and dried while frozen were sectioned paradermally. Label was found in the lower leaf sections, serving as additional evidence that PCMBS did infiltrate the tissue (Fig. 4). Assuming that PCMBS remained in the free space and the free space is about 10% of the total tissue volume, PCMBS concentrations in the free space ranged from 9 to 1 mm in the upper and lower layers, respectively. Import of labeled carbon was measured before and after PCMBS treatment during steady state 14C02 labeling of a source

FIG. 3. Autoradiograph of one-half of a sink leaf flooded with [(3Hg] PCMBS. The area which was flooded is distinguished by a higher level of radioactivity (lighter areas). Bar represents I cm. Enlarged (x5) regions located by symbols.

leaf(Fig. 5A). With PCMBS infiltrating the free space throughout the several layers of the leaf, one would expect the treatment to inhibit the uptake of any sucrose passing through the free space during phloem unloading. To verify that changing the solution over the leaf surface did not change the counting efficiency of the GM tube, several changes of buffer only were made (indicated by the arrow pairs in Fig. 5), before the inhibitor was added. Approximately 0.3% of the total radioactivity imported into the leaf was collected in the circulating solution. After PCMBS treatment this amount increased to 3.0%. PCMBS had no effect on the rate of import of material from the source leaf (Fig. 5A). To confirm that our measurement system could indicate a decline in the import rate, a penetrating sulfhydryl reagent NEM was tested. NEM treatment did inhibit import (Fig. 5B). Visualization of L-["CjGlucose. A sink leaf was supplied with a xenobiotic sugar, L-glucose, either through the phloem or through the xylem stream to determine if materials exiting the phloem pass through the apoplast before entry into mesophyll cells. L-Glucose is considered to cross the cell membrane pas-

SCHMALSTIG AND GEIGER

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UPPER LOWER SECTION POSITION FIG. 4. Localization of [203Hg]PCMBS in layers of sink leaf by paradermal sections. PCMBS in nmol per mg dry weight is plotted versus arbitrary position from upper to lower leaf surface. Leaf pieces shown here were sectioned from upper to lower surface (x, V, 0) or lower to upper surface ([, A). A A

FIG. 6. Autoradiographs of sink leaves. A, C, D: leaves supplied with

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total amount added to the source leaf was translocated to the sink leaf. Tissue concentration in the sink leaf ranged from 1 t a~~~~~~ mm in the base of the leaf to 0.02 mm in the tip of the leaf. In the sink leaf L-['4C]glucose was distributed throughout, as seen in autoradiographs (Fig. 6A). The image was similar to that of a 400 450 350 300 sink leaf which received ['4C]sucrose through the phloem during TIME (min) steady state 4C02 labeling of a source leaf (Fig. 6B). Both show more label in the basal, less mature regions and a darker image in I, II, and III order veins (classification of Esau [4]) than in surrounding tissue. Chromatography ofsink leaf extracts showed 5mM that at least 85% of the translocated L-['4C]glucose remained in NEM Wnonmetabolized form (data not shown). 9t4 4t I.- -0 3.0 t94 t 94 4 The images of leaves to which L-['4C]glucose was added diCK x xx 2.5 -x x rectly to the free space through the cut end of the petiole (Fig. x x 20~~~~~~x x x x 6C) or by reverse-flap vein feeding (Fig. 6D) were strikingly 21.51 5 different from those of phloem-supplied leaves. L-['4C]Glucose x 1.0 x entering through the cut petiole, was visible near I and II order x x x x x x veins and a few millimeters into their tributaries perhaps repre0.5 ~~~~~~~~~~x O.5 senting the site of water removal from the xylem. Almost no L0 250 300 50 200 [14C]glucose moved more than 5 mm into any vein smaller than TIME (min) II order. Little label entered the surrounding mesophyll as exFIG. 5. Effect of 10 mm PCMBS (A) and 5 mm NEM (B) on carbon pected for the slowly permeating L-glucose. 1.6

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import into a sink leaf during steady state '4C02 labeling of a source leaf. Rates were calculated from slopes of accumulation of label using the mean distribution theorem. The arrow pairs show times when solution circulating over the leaf surface was removed and new solution added. Ten min treatment time is shown on the graph. Data are from one of five experiments with similar results. Means of import rates before and after treatment (A) were not statistically different in Student's t test at

DISCUSSION of material a Flux into sink leaf did not change after treatment with PCMBS (Fig. 5A), despite the inhibition by PCMBS of exogenous sucrose uptake (Fig. 1; Table I). Together these results demonstrated that inhibition of carrier-mediated steps accessible to the free space is unable to slow the import of material into a (a) = 0.05. sink leaf. Conceivably uptake into the mesophyll could be insively in tumor cells (16) and sugar beet leaves (7). The uptake hibited but flux into the leaf could remain constant due to rate of sucrose is 13 times that of L-glucose when both are at a buildup of labeled material in the free space. This is unlikely 10-mM concentration in sugar beet source leaves (7). L-["4C] since only 3% of label imported was sampled in the circulating Glucose was supplied to the free space of a source leaf by reverse- solution after PCMBS treatment. If unloading from sieve eleflap vein feeding. Small patches of the source leaf regions fed by ments occurs by an apoplastic route likely it would depend on L-glucose became wilted after several hours and dried out by the active, carrier-mediated transport for conveying material from end of the experiment (10.5 h). This likely was due to accumu- the sieve elements or for lowering the concentration of free space lation in the free space, at places of high transpirational water solutes by active uptake into surrounding cells or both. Carrierloss, of a high concentration of L-glucose which acted as an mediated efflux of sucrose from the sieve elements is reported to osmoticum. This confirms the slow uptake of L-glucose from the be inhibited by PCMBS (18). PCMBS also inhibits loading and apoplast. Despite the slow uptake, enough L-glucose entered the export of sucrose derived by photosynthesis in source leaves of phloem to allow translocation to the sink leaf which was found sugar beet (1 1), evidence that this agent inhibits uptake, not only to arrive at a constant rate after an initial 2-h lag. About 2% of of exogenous sucrose, but also of endogenous sucrose that passes

PHLOEM UNLOADING IN DEVELOPING LEAVES through the free space prior to being taken up by cells. Further evidence that sucrose carriers are not necessary for phloem unloading in these leaves comes from the fact that phloem import of L-['4C]glucose (Fig. 6A) and ['4C]sucrose (Fig. 6B) produced similar autoradiographic images even though there are no membrane carriers for L-glucose (7, 16). There is carriermediated uptake of sucrose into mesophyll cells of mature (7, 17) and developing leaves (6) of sugar beet but phloem unloading clearly does not depend on it. These pieces of evidence support the operation of a mechanism of phloem unloading in which imported materials need not cross the plasma membrane of the mesophyll cells (2). There was little uptake of L-['4C]glucose into the mesophyll when supplied through the cut petiole (Fig. 6C) or directly into a vein (Fig. 6D), routes that were used to mimic possible unloading through the apoplast. Instead, labeled sugar accumulated in the vicinity of larger veins with little transfer to the mesophyll. The pattern may be the result, in part, of the path taken from the xylem by water supplying transpiration. Images similar to those in Figure 6, C and D were also seen by McNeil et al. (19) when ['4C]-labeled neutral amino acids, valine and asparagine, were pulse-fed to cut shoots of Lupinus albus L. Other amino acids which were pulse-fed, such as aspartate, were readily taken up by the mesophyll. The authors suggested that slower membrane uptake of valine and asparagine accounted for the distribution. In contrast to supply by way of the free space, the phloem path into the mesophyll of developing leaves was open to Lglucose (Fig. 6A). Phloem unloading of L-glucose and other solutes in developing leaves is best explained by mass flow through mature sieve elements and then through plasmodesmata into developing sieve elements and mesophyll cells. Though the clear demonstration of the feasibility of symplastic phloem unloading in a developing sink leaf suggests the presence of open plasmodesmata throughout the importing leaf, their existence in sufficient numbers to account for import, their extent and the state of openness need to be verified. The function of plasmodesmata in symplastic transport is still in question. Recent structural and transport studies suggest that transport through plasmodesmata is feasible. Overall et al. (21) used results from an ultrastructural study of plasmodesmata in Azolla roots to support the presence of a closed desmotubule and a cytoplasmic annulus partially occluded with particles. Dimensions of the inner-particle space were estimated to be 2.6 to 3.6 nm2. These passages are similar in size to those that permitted intercellular movement of dyes smaller than 874 D in Elodea canadensis Michx. leaves (15). Although it is reasonable to postulate symplastic movement through plasmodesmata in young leaves, this topic needs further study. The symplastic route for phloem unloading in developing leaves has important physiological implications for the sinksource transition of leaves. The free exit of solutes from sieve elements that is seen in sink leaves during unloading must be stopped before the high solute concentration and pressure characteristic of source leaf sieve elements can be achieved. There have been suggestions in the literature that a closing off of symplastic connections between phloem cells and mesophyll occurs during the leaf transition from sink to source (25). Evidence for such a scenario in guard cells was observed for plasmodesmata between guard cells and from guard cells to surrounding cells in immature leaves of Beta vulgaris L. and Allium cepa L. (26). The plasmodesmata were truncated on the guard cell side in mature leaves, an arrangement that appears necessary to allow the build up of pressure needed for guard cell function. The question clearly deserves further work. Utilization of a symplastic pathway of unloading in young leaves also has implications for the control of partitioning. A

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regulatory process may change the degree of openness of plasmodesmata as suggested by several authors (3, 21). Another basis of control is the commonly observed role of energy metabolism in sink import. Instead of requiring energy for transport across the plasma membrane, sink leaf unloading by the symplastic route likely utilizes energy to convert sucrose and other solutes in the mesophyll to osmotically less active forms such as protein, starch, cellulose and other macromolecules. These metabolic conversions presumably are linked to continued cell expansion and differentiation. Under these circumstances, unloading is subject both to internal developmental events and environmental factors such as mineral nutrition, water status, and light quality and intensity. Mechanisms linking the above factors and processes to control of import in relation to leaf development need further investigation. LITERATURE CITED 1. BARRS HD 1971 Cyclic variations in stomatal aperture, transpiration and leaf water potential under constant environmental conditions. Annu Rev Plant Physiol 23: 223-237 2. DICK PS, T APREES 1975 The pathway of sugar transport in roots of Pisum sativum. J Exp Bot 26: 305-314 3. ERWEE MG, PB GOODWIN 1983 Characterization of the Egeria densa Planch. leaf symplast, Inhibition of the intercellular movement of fluorescent probes by group II ions. Planta 158: 320-328 4. ESAU K 1967 Minor veins in Beta leaves: structure related to function. Proc Am Phil Soc 11 1: 219-233 5. FELKER FC, JC SHANNON 1980 Movement of '4C-labeled assimilates into kernels of Zea mays L. III. An anatomical examination and microautora-

diographic study of assimilate transfer. Plant Physiol 65: 864-870 6. FELLOWS RJ, DR GEIGER 1974 Structural and physiological changes in sugar beet leaves during sink to source conversion. Plant Physiol 54: 877-885 7. FONDY BR, DR GEIGER 1977 Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol 59: 953-960 8. GEIGER DR, AL CHRISTY 1971 Effect of sink region anoxia on translocation rate. Plant Physiol 47: 172-174 9. GEIGER DR, BR FONDY 1979 A Method for continuous measurement of export from a leaf. Plant Physiol 64: 361-365 10. GEIGER DR, BR FONDY 1980 Phloem loading and unloading: pathways and mechanisms. What's New in Plant Physiology 11: 25-28 1 1. GIAQUINTA RT 1976 Evidence for phloem loading from the apoplast, chemical modification of membrane sulfhydryl groups. Plant Physiol 57: 872-875 12. GIAQUINTA RT, W LIN, NL SADLER, VR FRANCESCHI 1983 Pathway of phloem unloading of sucrose in-corn roots. Plant Physiol 72: 362-367 13. GIFFORD RM, JH THORNE, WD HITZ, RT GIAQUINTA 1984 Crop productivity and photoassimilate partitioning. Science 225: 801-808 14. GLAszIOU KT, KR GAYLER 1972 Storage of sugars in stalks of sugarcane. Bot Rev 38: 471-490 15. GOODWIN PB 1983 Molecular size limit for movement in the symplast of the Elodea leaf. Planta 157: 124-130 16. HATANAKA M 1974 Transport of sugars in tumor cell membranes. Biochim Biophys Acta 355: 77-104 17. MAYNARD JW, WJ LUCAS 1982 Sucrose and glucose uptake into Beta vulgaris leaf tissues, A case for general (apoplastic) retrieval systems. Plant Physiol 70: 1436-1443 18. M'BATCHI B, S DELROT 1984 Parachloromercuri-benzenesulfonic acid, a potential tool for differential labeling of the sucrose transporter. Plant Physiol 75: 154-160 19. McNEIL DL, CA ATKINS, JS PATE 1979 Uptake and utilization of xylem-borne amino compounds by shoot organs of a legume. Plant Physiol 63: 10761081 20. MOUNOURY G, S DELROT, JL BONNEMAIN 1984 Energetics of threonine uptake by pod wall tissues of Viciafaba L. Planta 161: 178-185 21. OVERALL RL, J WOLFE, BES GUNNING 1982 Intercellular communication in Azolla roots: I. Ultrastructure of plasmodesmata. Protoplasma II 1: 134-150 22. PATRICK JW, PM TURVEY 1981 The pathway of radial transfer of photosynthate in decapitated stems of Phaseolus vulgaris L. Ann Bot 47: 611-621 23. SNYDER FW, GE CARLSON 1978 Photosynthate partitioning in sugarbeet. Crop Scil 8: 657-661 24. THORNE JH 1981 Morphology and ultrastructure of maternal seed tissues of soybean in relation to the import of photosynthate. Plant Physiol 67: 10161025 25. TURGEON R 1984 Termination of nutrient import and development of vein loading capacity in albino tobacco leaves. Plant Physiol 76: 45-48 26. WILLE AC, WJ LUCAS 1984 Ultrastructural and histochemical studies on guard cells. Planta 160: 129-142 27. WOLSWINKEL P 1978 Phloem unloading in stem parts parasitized by Cuscuta: the release of 'IC and KI to the free space at OeC and 25-C. Physiol Plant 42: 167-172