Evidence for thiol-induced enhanced in situ translocation of 14C-sucrose from source to sink in Brassica juncea

The translocation of ¹⁴C-sucrose to the different parts of the mustard (Brassica juncea) crop has been evaluated in the context of understanding the source to sink relationship in the thiol-induced enhanced crop yield. The foliar application of thiols like TU, TGA and DTT to the plant gave maximum sucrose phosphate synthase activity, which was found to have direct correlation with the movement of sucrose. The distribution pattern of ¹⁴C-sucrose follows the path from internode and node to pod via leaf. The translocation of ¹⁴C-sucrose was found to be a light dependent process. Among the nucleotides ATP and GTP, only ATP was able to promote the translocation and GTP was ineffective. In this unique in situ tracer experiment using ¹⁴C-sucrose, we could establish that thiols are able to enhance the translocation of sucrose from source to sink.     

Comparison of upward and downward translocation of C from a single leaf of sunflower

When single leaves attached at a given node were allowed to carry on photosynthesis in ¹⁴CO₂ for 30 min, younger plants showed a higher proportion of upward translocation than did older plants. Downward translocation of ¹⁴C-photosynthate was stimulated by ATP pre-treatment of the translocating leaf, while upward translocation was not affected by ATP. A similar phenomenon was observed in the translocation of ¹⁴C-sucrose infiltrated into a leaf with or without ATP. Downward translocation of photosynthate was inhibited by DNP pre-treatment of a fed leaf. Upward translocation, however, was not affected by DNP. Thirty min after infiltration of ¹⁴C-glucose into a leaf, almost all the ¹⁴C translocated upwards was found to be in the form of glucose, while a great part of the ¹⁴C translocated downwards was in the form of sucrose. In the case of translocation of infiltrated ¹⁴C-sucrose, ¹⁴C found both above and below the fed leaf was mainly in the form of sucrose.     

Evidence for active Phloem loading in the minor veins of sugar beet

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 ¹⁴C-sucrose to approximately 20% of the control. Dinitrophenol at a concentration of 8 mm inhibited rather than promoted CO₂ 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 energy-requiring process for both phloem loading and translocation in the source leaf.     

Long distance translocation of sucrose, serine, leucine, lysine, and carbon dioxide assimilates: I. Soybean

To determine the selectivity of movement of amino acids from source leaves to sink tissues in soybeans (Glycine max [L.] Merr. `Wells'), ¹⁴C-labeled serine, leucine, or lysine was applied to an abraded spot on a fully expanded trifoliolate leaflet, and an immature sink leaf three nodes above was monitored with a GM tube for arrival of radioactivity. Comparisons were made with ¹⁴C-sucrose and ¹⁴CO₂ assimilates. Radioactivity was detected in the sink leaf for all compounds applied to the source leaflet. A heat girdle at the source leaf petiole essentially blocked movement of applied compounds, suggesting phloem transport. Transport velocities were similar (ranged from 0.75 to 1.06 cm/min), but mass transfer rates for sucrose were much higher than those for amino acids. Hence, the quantity of amino acids entering the phloem was much smaller than that of sucrose. Extraction of source, path, and sink tissues at the conclusion of the experiments revealed that 80 to 90% of the radioactivity remained in the source leaflet. Serine was partially metabolized in the transport path, whereas lysine and leucine were not. Although serine is found in greater quantities than leucine and lysine in the source leaf and path of soybeans, applied leucine and lysine were transported at comparable velocities and in only slightly lower quantities than was applied serine. Thus, no selective barrier against entry of these amino acids into the phloem exists.     

Evidence for Phloem loading from the apoplast: chemical modification of membrane sulfhydryl groups

The water-soluble, sulfhydryl-specific, chemical modifier p-chloromercuribenzenesulfonic acid reversibly inhibited the accumulation of exogenously supplied ¹⁴C-sucrose into leaf discs of Beta vulgaris. P-Chloromercuribenzenesulfonic acid treatment did not inhibit photosynthesis or respiration or induce membrane leakage to sucrose, indicating that the site of inhibition was the plasmalemma. The active loading of sucrose and ¹⁴CO₂-derived assimilates into the phloem and their translocation from the source leaf were inhibited by the nonpermeant modifier. Several nonpermeant sulfhydryl group modifiers also inhibited sucrose accumulation into leaf discs while two amino-reactive reagents had no effect. The results indicate that sugars are actively accumulated into the phloem from the apoplast and that membrane sulfhydryl groups may be involved.     

Sucrose Hydrolysis in Relation to Phloem Translocation in Beta vulgaris

Asymmetrically labeled sucrose, ¹⁴C(fructosyl)sucrose, was used to determine whether sucrose undergoes extracellular hydrolysis during phloem translocation in the sugar beet, Beta vulgaris. In addition, the metabolism of various sugars accumulated and translocated was determined in various regious of the plant. These processes were studied in detached regions as well as in the intact, translocating plant in the source leaf, along the translocation path, and in a rapidly growing sink leaf and storage beet. The data show that, unlike sucrose accumulation into the sink tissue of sugarcane, sucrose is neither hydrolzyed prior to phloem loading or during transit, nor is it extracellularly hydrolyzed during accumulation into sink leaves or the storage beet.     

The Function of Phloem Connections in Regenerating In Vitro-Grafts*

In order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant-grafts after application of ¹⁴C-sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls. A segmental analysis was used to determine the tissue distribution of ¹⁴C-sucrose in a graft. The ¹⁴C-profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound-phloem bridges (shown as files of aniline-blue-positive sieve plates) traverse the graft interface. In 7-d-old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, ¹⁴C-profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose. Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound-phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro-grafts is directly shown.     

Chlorophenoxyacetic acid and chloropyridylphenylurea accelerate translocation of photoassimilates to parthenocarpic and seeded fruits of muskmelon (Cucumis melo)

We compared the effect of p-chlorophenoxyacetic acid (p-CPA) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) on parthenocarpic and seeded muskmelon (Cucumis melo) fruits in regards to fruit development and the transport of photoassimilates from leaves exposed to ¹⁴CO₂ to the developing fruits. Ten days after anthesis (DAA), the fresh weight, total ¹⁴C-radioactivity and contents of ¹⁴C-sucrose and ¹⁴C-fructose were higher in the CPPU-induced parthenocarpic fruits than in seeded fruits. However, at 35 DAA, fresh weight and sucrose content in mesocarp, placenta and empty seeds of the parthenocarpic fruits were lower than in seeded fruits. Also, total ¹⁴C-radioactivity and ¹⁴C-sugar content of the parthenocarpic fruits were lower as well as the translocation rate of ¹⁴C-photoassimilates into these fruits. Application of p-CPA to the parthenocarpic fruits at 10 and 25 DAA increased fresh weight and sugar content. Moreover, these treatments elevated the total ¹⁴C-radioactivity, ¹⁴C-sucrose content and the translocation rate of ¹⁴C-photoassimilates. The ¹⁴C-radioactivity along the translocation pathway from leaf to petiole, stem, lateral shoot and peduncle showed a declining pattern but dramatically increased again in the fruits. These results suggest that the fruit's sink strength was regulated by the seed and enhanced by the application of p-CPA.     

Phloem Loading of Sucrose: pH Dependence and Selectivity

Autoradiographic, plasmolysis, and ¹⁴C-metabolite distribution studies indicate that the majority of exogenously supplied ¹⁴C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent K_m of the loading process increases at the alkaline pH while the maximum velocity, V_max, is pH-independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose ¹⁴C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport.     

Effects of light intensity and oxygen on photosynthesis and translocation in sugar beet

The mass transfer rate of ¹⁴C-sucrose translocation from sugar beet (Beta vulgaris, L.) leaves was measured over a range of net photosynthesis rates from 0 to 60 milligrams of CO₂ decimeters⁻² hour⁻¹ under varying conditions of light intensity, CO₂ concentration, and O₂ concentration. The resulting rate of translocation of labeled photosynthate into total sink tissue was a linear function (slope = 0.18) of the net photosynthesis rate of the source leaf regardless of light intensity (2000, 3700, or 7200 foot-candles), O₂ concentration (21% or 1% O₂), or CO₂ concentration (900 microliters/liter of CO₂ to compensation concentration). These data support the theory that the mass transfer rate of translocation under conditions of sufficient sink demand is limited by the net photosynthesis rate or more specifically by sucrose synthesis and this limitation is independent of light intensity per se. The rate of translocation was not saturated even at net photosynthesis rates four times greater than the rate occurring at 300 microliters/liter of CO₂, 21% O₂, and saturating light intensity.     

Sink to source translocation in soybean

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [¹⁴C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [¹⁴C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.     

Source and sink leaf metabolism in relation to Phloem translocation: carbon partitioning and enzymology

The import-export transition in sugar beet leaves (Beta vulgaris) occurred at 40 to 50% leaf expansion and was characterized by loss in assimilate import and increase in photosynthesis. The metabolism and partitioning of assimilated and translocated C were determined during leaf development and related to the translocation status of the leaf. The import stage was characterized by C derived from either ¹⁴C-translocate or ¹⁴C-photosynthate being incorporated into protein and structural carbohydrates. Marked changes in the C partitioning were temporally correlated with the import-export conversion. Exporting leaves did not hydrolyze accumulated sucrose and the C derived from CO₂ fixation was preferentially incorporated into sucrose. Both source and sink leaves contained similar levels of acid invertase and sucrose synthetase activities (sucrose hydrolysis) while sucrose phosphate synthetase (sucrose synthesis) was detected only in exporting leaves. The results are discussed in terms of intracellular compartmentation of sucrose and sucrose-metabolizing enzymes in source and sink leaves.     

Activation of Soluble Acid Invertase Accompanies the Cytokinin-Induced Source–Sink Leaf Transition

Activation of soluble acid invertase by cytokinin was shown using as a model a detached sugar-beet leaf, one half (sink) of which was treated with benzyladenine and the other half (source) of which was sprayed with water. Acid invertase was assumed to mediate the hormone-induced sink properties of the cells. The influx of ¹⁴C-sucrose to the leaf half treated with benzyladenine was induced to much greater extent than that of potassium (⁸⁶Rb). This suggests different pathways or mechanisms of translocation of these substances to the induced sink.     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

Translocation of C Sucrose in Sugar Beet during Darkness

The time-course of arrival of ¹⁴C translocate in a sink leaf was studied in sugar beet (Beta vulgaris L. cultivar Klein Wanzleben) for up to 480 minutes of darkness. Following darkening of the source leaf, translocation rapidly declined, reaching a rate approximately 25% of the light period rate by 150 minutes. Comparison of data from plants that were girdled 1 cm below the crown with data from ungirdled plants indicates that after about 150 minutes darkness the beet root becomes a source of translocate to the sink leaf. After about 90 minutes darkness, starch-like reserve polysaccharide from the source leaf begins to contribute ¹⁴C to ethanol soluble pools in that leaf. Because of a 15% isotope mass effect, sucrose, at isotopic saturation, reaches a specific activity which is about 85% of the level of the supplied CO₂. The source leaf sucrose specific activity remains at the isotopic saturation level for about 150 minutes of darkness, after which time input from polysaccharide reserves causes the specific activity to drop to about 55% of that of the supplied CO₂. Sucrose specific activity determinations, polysaccharide dissolution measurements, and pulse labeling experiments indicate that following partial depletion of the sucrose pool, source leaf polysaccharide contributes to dark translocation. Respired CO₂ from the source leaf appears to be derived from a pool which, unlike sucrose, remains at a uniform specific activity.     

The Effect of Sodium and Calcium on the Translocation of 14C-sucrose in Excised Cotton Roots

The effect of sodium and calcium on the translocation of ¹⁴C-sucrose in excised cotton roots (Gossypium birsutum) was studied. The roots were allowed to elongate in a modified Guinn's medium that was very low in calcium (6.25 × 10^?2 mM) and sodium (8.70 × 10^?3 mM). After a period of six days the roots were transferred to 20 × 100 mm Petri dishes that contained 10 × 40 mm Petri dishes as center wells. The roots were draped over the edge of the center well and extended into the outer dish. The outer Petri dish and its center well contained the same solution except that sucrose was supplied only in the center well. The sucrose used was spiked with uniformally labeled ¹⁴C-sucrose. Four treatments were started which varied in their Na and Ca content. Three and six day harvests were taken and the amount of ¹⁴C that had moved from the distal (in the center well) to the apical root section (in the outer dish) was determined. Increasing substrate Na or Ca caused an increase in ¹⁴C-sucrose translocation and the effects of both ions were additive by the final harvest. These results were found to be independent of treatment effects on growth and respiration of the excised roots. These data support the conclusion that Na may partially substitute for Ca in carbohydrate translocation. Thus, roots supplied the Low Ca-High Na and High Ca-Low Na treatments had equal translocation rates over a six day period. The highest translocation rate was obtained with the High Ca-High Na treatment. Data from the High Ca-High Na treatment on two successive three day periods indicate that Na may have a role in translocation other than that associated with substitution for Ca, or maintenance of tissue hydration.     

The Role of Phloem Transport in the Translocation of Sucrose Along the Stem of Carnation Cut Flowers

The pathway and distribution of ¹⁴C-sugars in flower parts havebeen examined to find out in which tissue sugars are translocatedin the stem of the cut carnation; ¹⁴C-sucrose or ¹⁴C-glucosewas supplied at the base of the cut stem from a feeding solutionand the localization and chemical nature of the carbon-14 recoveredfrom flower parts were investigated. By reducing the rate oftranspiration it was found that the uptake of feeding solutionwas also reduced, but the distribution of absorbed ¹⁴C-sucrosein the flower parts was different from that which would be expectedif sucrose moved exclusively in the transpiration stream. Autoradiographsdemonstrated that ¹⁴C absorbed from the feeding solution as¹⁴C-sucrose appeared in both xylem and phloem but predominantlyin the latter; girdling failed to stop the translocation ofthe absorbed ¹⁴C-sucrose. Results of experiments with ¹⁴C-sucroseand ¹⁴C-glucose showed that sucrose was the mobile sugar andthat glucose was converted to sucrose before it was translocated.It was concluded that the translocation of sucrose absorbedfrom the feeding solution takes place both in xylem and phloemand is regulated by a mechanism involving the loading and translocationof sucrose in the phloem.     

Degradation of olestra, a non caloric fat replacer, by microorganisms isolated from activated sludge and other environments

Olestra is a non-caloric fat substitute consisting of fatty acids esterified to sucrose. Previous work has shown that olestra is not metabolized in the gut and is excreted unmodified in human feces. To better understand the fate of olestra in engineered and natural environments, aerobic bacteria and fungi that degrade olestra were enriched from sewage sludges, soils and municipal solid waste compost not previously exposed to olestra. Various mixed and pure cultures were obtained from these sources which were able to utilize olestra as a sole carbon and energy source. The fastest growing enrichment was obtained from activated sludge and later yielded an olestra-degrading pure culture of Pseudomonas aeruginosa. This mixed culture extensively degraded both ¹⁴C-fatty acid labeled olestra and ¹⁴C-sucrose labeled olestra during 8 days of incubation. Longer-term incubation with pure cultures of P. aeruginosa demonstrated that >98% of ¹⁴C-sucrose labeled olestra and >72% of ¹⁴C-fatty acid labeled olestra was mineralized to CO₂ after 69 days. These results indicate that olestra degraders are present in environments not previously exposed to olestra and that olestra can serve as a sole carbon and energy source. Furthermore, a common bacterial species was isolated from activated sludge and shown to have the ability to degrade olestra.     

AtRBOH I confers submergence tolerance and is involved in auxin-mediated signaling pathways under hypoxic stress

Satsuma mandarin fruit (Citrus unshiu Mark.) photosynthesizes as comparable to leaf at about 100 days after full bloom (DAFB). In this study, translocation and accumulation of fruit-fixed photosynthate were investigated by using ¹⁴CO₂. When fruit at 108 DAFB was exposed to ¹⁴CO₂ for 48 h under 135 photosynthetic photon flux density (PPFD), ¹⁴C-sucrose, ¹⁴C-glucose and ¹⁴C-fructose were detected not only in flavedo but juice sac; more than 50?% of fruit assimilated ¹⁴C-sugars were present in juice sac. Thus, majority of rind-fixed photosynthate are infiltrated into juice sac and accumulated there within 48 h after assimilation. Although ¹⁴C-sucrose was predominant at flavedo where high SS (sucrose synthase) activity toward synthesis was present, the amount decreased gradually from the outside (flavedo) to the inside (juice sac) of fruit. In vascular bundle, strong SS toward cleavage and soluble acid invertase activities were involved, and ¹⁴C-fructose was predominant in juice sac. Accordingly, rind-fixed photosynthate is once converted to sucrose, the translocated sugar in Citrus, at flavedo by SS toward synthesis, and loaded on vascular bundle through symplastic and/or apoplastic movement in the albedo tissue. In the vascular bundle, sucrose may be degraded by SS toward cleavage and invertase, and resulting hexoses transported symplastically to the juice sac through juice stalk.     

Sucrose translocation and storage in the sugar beet

Several physiological processes were studied during sugar beet root development to determine the cellular events that are temporally correlated with sucrose storage. The prestorage stage was characterized by a marked increase in root fresh weight and a low sucrose to glucose ratio. Carbon derived from ¹⁴C-sucrose accumulation was partitioned into protein and structural carbohydrate fractions and their amino acid, organic acid, and hexose precursors. The immature root contained high soluble acid invertase activity (V_max 20 micromoles per hour per milligram protein; K_m 2 to 3 millimolar) which disappeared prior to sucrose storage. Sucrose storage was characterized by carbon derived from ¹⁴C-sucrose uptake being partitioned into the sucrose fraction with little evidence of further metabolism. The onset of storage was accompanied by the appearance of sucrose synthetase activity (V_max 12 micromoles per hour per milligram protein; K_m 7 millimolar). Neither sucrose phosphate synthetase nor alkaline invertase activities were detected during beet development. Intact sugar beet plants (containing a 100-gram beet) exported 70% of the translocate to the beet, greater than 90% of which was retained as sucrose with little subsequent conversions.