Phloem loading in source leaves of sugar beet (Beta vulgaris, L.) was studied to determine the extent of dependence on energy metabolism and the involvement of a carrier system. Dinitrophenol at a concentration of 4 mM uncoupled respiration, lowered source leaf ATP to approximately 40% of the level in the control leaf and inhibited translocation of exogenously supplied '4C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mM inhibited rather than promoted C02 production, indicating a mechanism of inhibition other than uncoupling of respiration. The 8 mM dinitrophenol also reduced ATP to approximately 40% of the level in the control source leaf and reduced translocation of exogenous sucrose to approximately 10% of the control. Application of 4 mM ATP to an untreated source leaf promoted the translocation rate by approximately 80% over the control, while in leaves treated with 4 mM dinitrophenol, 4 mM ATP restored translocation to the control level. No recovery of translocation was observed when ATP was applied to leaves treated with 8 mM dinitrophenol. The results indicate an energyrequiring process for both phloem loading and translocation in the source leaf.Application of '4C-sucrose solutions in a series of concentrations through the upper surface of a source leaf produced a biphasic isotherm for translocation out of the fed region. A similar dual isotherm was obtained for phloem loading with leaf discs floated on 14C-sucrose solutions. The first and possibly the second phases were attributed to active, carriermediated accumulation in the minor vein phloem. Autoradi The source leaf, a sugar-exporting region of a plant, seems to play a key role in providing the driving force for long distance transport of organic solutes throughout the plant. Few translocation studies have been focused on events in the source leaf, and some crucial questions remain unanswered. Is there, for example, an energy-requiring step in the transport of sugar from chloroplasts to sieve elements that is directly related to phloem loading and long distance transport. If an active step is demonstrated, do the kinetics of uptake indicate that this step involves a membrane carrier?In 1939, Curtis and Asai (3) suggested that the osmotic gradient from the mesophyll to the sink tissue was in the opposite direction required for a pressure flow mechanism. Roeckl (26) observed that the mesophyll cells, which produce mobile sugars, have a substantially lower osmotic potential than the phloem exudate. A gradient of increasing concentration toward the sieve tubes would require energy to drive transport in that direction, suggesting the need for an active step between sugar production and translocation out of source leaves. Barrier and Loomis (1) termed the active step "loading," implying that the rate-limiting step is transport into the veins, rather than a simple chemical transformation or synthesis of the translocate. There now seems to be general agreement that an active step does occur in source tissue (10,...

The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze-substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.The sharp concentration increase at the membrane of the sieve element-companion cell complex of the source leaf indicates active phloem loading from the free space at this site. Active loading of the phloem is presumably needed to move the sugar from the chloroplasts of the mesophyll to the sieve tubes against the concentration gradient. The osmotic pressure of the mature sieve element-companion cell complex appears to be approximately the same in source leaf, path, and sink leaf tissue. There is a distinct difference in concentration between the mature sieve element-companion cell complex in the sink and the surrounding mesophyll. The solute distribution suggests that sugar is actively accumulated from the free space by the developing sink leaf tissue.The osmotic values observed in the various cells are consistent with the operation of a mass flow mechanism of translocation driven by active phloem loading and by active accumulation of sugar by sink tissues.

The involvement of the free space in phloem loading of sucrose was studied in sugar beet source leaves (Beta vulgaris, L.). Sucrose, supplied exogenously to the abraded upper surface of leaves at a concentration of 20 mM, was available for translocation at rates siniilar to those obtained with photosynthesis. The exogenous sucrose substituted as a source of translocate for assimilate derived from photosynthesis when the latter process was disrupted by plasmolysis of the leaf with 0.8 M mannitol. The mesophyll symplast was not completely disrupted by this treatment, however. Data from the sugar uptake experiments indicate that phloem loading can occur from the free space.Isotope trapping of labeled sugars derived from "CO2 was used to intercept and identify sugars passing through the free space prior to phloem loading. Increased translocation rates induced by 4 mM ATP or increased light intensity were accompanied by increased trapping of sucrose but not of glucose. The data support the view that sucrose passes into the free space prior to phloem loading.In previous studies, it was shown that phloem loading is an active process which accompanies translocation of sucrose and, at least in some part, is responsible for causing it (3, 13). Active uptake of sucrose was found to occur when exogenous sucrose was supplied (13)

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Petiole cooling is known to cause a temporary decline in mass transfer rate in sugar beet (2,8). The observed recovery of mass transfer rate may be explained in a number of ways. Increased loading in the source leaf might compensate for low velocity by increased solute concentration, or translocation velocity might return to the original rate because of a steepened pressure gradient, a reversal of the cause of increased resistance to flow, or a recovery in the metabolic process which supplies the energy to drive the translocation process. This report provides evidence that inhibition of mass transfer during petiole cooling is a result of decreased translocation velocity and that recovery occurs mainly because of a restoration of velocity.Sugar beet plants (Beta vulgaris, monogerm hybrid [SL129 X 133] ms X [SP6322-0]) and bean plants (Phaseolus vulgaris, var. Black Valentine) were grown in a controlled environment cabinet as described previously (3). Steady state labeling with 14CO2 was used to measure mass transfer rate (3). Labeled CO2 was supplied to a mature sugar beet leaf, and arrival was followed in a young leaf whereas in bean the 14CO2 was supplied to a primary leaf, and arrival of translocate was monitored using the terminal leaflet of a young trifoliate leaf (4).Velocity was measured by a pulse labeling technique for labeling photosynthate (5). As a consequence, it was necessary to devise a pulse labeling method for studying mass transfer in the same plants used for velocity measurements. The proportion of a 14CO pulse which was translocated out of the source leaf during the first 45 min was used as a measure of the relative mass transfer rate. The excellent agreement of the time course obtained by the two methods permitted use of pulse labeling to compare both mass transfer rate and apparent velocity in the same plant (Figs. 1, 2). The apparent velocity was based on the time required for first detection of a count rate above background in the sink leaf. The method gives an apparent velocity which includes the time for loading in the source leaf, for transit from source to sink, and the time required for the accumulated 14C translocate to reach the threshold of detection. The time required for the "4C pulse to reach a detectable level in the sink is related directly to the amount of labeled translocate which is exchanged out of the translocation system and is inversely related to the specific radioactivity of the 14C translocate. As the specific radioactivity of the translocate is increased, the time required to reach the detection threshold is shortened and the apparent velocity approaches a limit which includes principally loading time plus transit time (5). In the velocity measurements we used approximately 50 lic 14C per I This work was supported by United States Atomic Energy Commission Grant AT 11-1-2015.pulse, which was found to give a minimal amount of time for reaching the detection threshold. Loading constitutes a constant and presumably relatively small part of the time between supplying o...