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.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose 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 ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]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.     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Different Patterns of Vein Loading of Exogenous [C]Sucrose in Leaves of Pisum sativum and Coleus blumei

Vein loading of exogenous [¹⁴C]sucrose was studied using short uptake and wash periods to distinguish between direct loading into veins and loading via mesophyll tissue. Mature leaf tissue of Pisum sativum L. cv Little Marvel, or Coleus blumei Benth. cv Candidum, was abraded and leaf discs were floated on [¹⁴C]sucrose solution for 1 or 2 minutes. Discs were then washed for 1 to 30 min either at room temperature or in the cold and were frozen, lyophilized, and autoradiographed. In P. sativum, veins were clearly labeled after 1 minute uptake and 1 minute wash periods. Autoradiographic images did not change appreciably with longer times of uptake or wash. Vein loading was inhibited by p-chloromercuribenzenesulfonic acid. These results indicate that uptake of exogenous sucrose occurs directly into the veins in this species. When C. blumei leaf discs were floated on [¹⁴C]sucrose for 2 minutes and washed in the cold, the mesophyll was labeled but little, if any, minor vein loading occurred. When discs were labeled for 2 minutes and washed at room temperature, label was transferred from the mesophyll to the veins within minutes. These results indicate that there may be different patterns of phloem loading of photosynthetically derived sucrose in these two species.     

In vitro and in vivo modification of sugar transport and translocation in celery by phytohormones

In an attempt to address the controversy in the literature as to whether phytohormones have any direct effect on phloem loading of sucrose, we investigated the effect of gibberellic acid (GA₃) and indoleacetic acid (IAA) on sugar transport and translocation in celery (Apium graveolens L. cv. Utah 5270). Both hormones enhanced sucrose uptake into isolated vascular bundles and phloem tissue of celery and enhanced the export of ¹⁴C assimilates from leaves of intact plants in vivo. The hormone-induced increase of uptake into isolated vascular bundles or phloem was specific for sucrose and mannitol which are translocated in phloem. Furthermore, the hormone-induced increase in translocation was not due to an increase in sink demand, since neither glucose nor sucrose uptake rates were affected in the storage parenchyma tissue discs (sink region) in the presence of GA₃ or IAA. The evidence suggests that phytohormones may have a direct effect on phloem loading of sucrose. The possibility of short-term GA₃ and IAA effects on processes resulting in membrane transport of sugars in celery is discussed.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

A Reanalysis of the Two-Component Phloem Loading System in Beta vulgaris

Kinetic analysis of [¹⁴C]sucrose loading into sugar beet leaf discs revealed the presence of two transport components. At low exogenous sucrose concentrations, a saturable component, which exhibited Michaelis-Menten characteristics, was the main mode of transport. At concentrations greater than 50 millimolar, phloem loading was dominated by a linear component which appeared to operate as a first order kinetic transport process. Over the exogenous sucrose concentrations employed, influx could be described by the equation v = V_maxS/(S + K_m) + kS. Influx via both processes was strongly pH-dependent. Evidence is presented that the linear component was not explicable in terms of simple diffusion, or exchange diffusion, into either mesophyll or minor vein phloem tissue. Extensive metabolic conversion of sucrose was not a factor contributing to influx at high external sucrose concentrations. At present, it is believed that both components operate in parallel at the membrane bounding the sieve element-companion cell complex. The saturable component is identified with sucrose-H⁺ cotransport. While the significance of the linear component has been established, its nature remains to be elucidated.     

Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co²⁺, Ni²⁺, or Zn²⁺ for 1 to 4 days was studied by incubating leaf discs in [¹⁴C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7-millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni²⁺ and Zn²⁺. The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions.     

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.     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.     

Evidence for the presence of a sucrose carrier in immature sugar beet tap roots

The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1'-fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of [³H]sucrose and [¹⁴C]F-sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. [¹⁴C]F-sucrose was translocated and accumulated in the roots at a higher rate than [³H]sucrose. This was due to [³H]sucrose hydrolysis along the translocation path. Presence of [³H]hexose and [¹⁴C]F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue.     

Effect of Exogenously Supplied Foliar Potassium on Phloem Loading in Beta vulgaris L

The effect of foliar application of K⁺ on processes associated with phloem loading was investigated in source leaves of sugar beet (Beta vulgaris L.). KCI was supplied exogenously at concentrations of up to 100 millimolar in the solution bathing the abraded upper epidermis of source leaves. K⁺ added at concentrations below 30 millimolar generally promoted the rate of export of material derived from ¹⁴CO₂ but not from exogenously applied [¹⁴C]sucrose. Paralleling promotion of export, the level of material derived from photosynthesis, which was released into the bathing solution, also increased in response to addition of K⁺ to the free space. Net photosynthetic rate was not affected. K⁺ at 5 and 15 millimolar concentrations did not stimulate uptake of [¹⁴C]sucrose into source leaf discs.     

Uptake and distribution of N-phosphonomethylglycine in sugar beet plants

Glyphosate (N-phosphonomethylglycine) was readily transported in sugar beet plants (Beta vulgaris L., Klein E type, monogerm). Concentrations in sink leaves reached 2.5 to 13.7 micromolar in 10 hours from a 15 millimolar solution supplied to one mature leaf. Distribution of glyphosate followed that of [³H]sucrose used as a marker for materials transported by phloem, indicating that this is the primary means for distribution of glyphosate. Possible mechanisms of entry into the sieve tubes were evaluated using isolated leaf discs. Concentration dependence of uptake and kinetics of exodiffusion from tissue indicate a passive, nonfacilitated mechanism. Uptake was not affected by pH, eliminating the passive, weak acid mechanism. Permeability of the plasmalemma to glyphosate was calculated as 1.7 × 10⁻¹⁰ meters per second. This characteristic would allow slow entry and exit from the phloem, and together with other physiological parameters of the plant, is postulated to allow accumulation and transport in the phloem.     

Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb

Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with ¹⁴CO₂ was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos.     

Efflux of sucrose from minor veins of tobacco leaves

Mature leaves import limited amounts of nutrient when darkened for prolonged periods. We tested the hypothesis that import is restricted by the apoplast-phloem loading mechanism, ie., as sucrose exits the phloem of minor veins it is retrieved by the same tissue, thus depriving the mesophyll of nutrient. When single, attached, mature leaves of tobacco (Nicotiana tabacum L.) plants were darkened, starch disappeared from the mesophyll cells, indicating that the supply of solute to the mesophyll was limited. Starch was synthesized in mesophyll cells of darkened tissue when sucrose was applied to the apoplast at 0.1–0.3 mM concentration. Efflux from minor veins was studied by incubating leaf discs on [¹⁴C]sucrose to load the minor veins and then measuring subsequent ¹⁴C release. Efflux was rapid for the first hour and continued at a gradually decreasing rate for over 13 h. Net efflux increased when loading was inhibited by p-chloromercuribenzene-sulfonic acid, anoxia, isotope-trapping, or reduction of the pH gradient. Neither light nor potassium had a significant effect on the rate of labeled sucrose release. The site of labeled sucrose release was investigated by measuring efflux from discs in which sucrose had previously been loaded preferentially by either the minor veins or mesophyll cells. Efflux occurred primarily from minor veins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - Mops 3(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SE-CC sieve element-companion cell complex     

Sugar transport into protoplasts isolated from developing soybean cotyledons : I. Protoplast isolation and general characteristics of sugar transport

A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Q₁₀ value and high activation energies).     

Inhibition of Loading of C Assimilates by p-Chloromercuribenzenesulfonic Acid : Localization of the Apoplastic Pathway in Vicia faba

The apoplast of mature leaves excised from broadbean (Vicia faba L.) plants was infiltrated with 2 millimolar p-chloromercuribenzenesulfonic acid (PCMBS) via the transpiration stream, and the ability of the tissues to take up sugars was tested. An infiltration time of 75 minutes was sufficient to obtain a maximal (75%) inhibition of exogenous [¹⁴C]sucrose (1 millimolar) uptake. This infiltration affected neither CO₂ assimilation nor the transmembrane potential difference of leaf cells but strongly inhibited phloem loading of endogenous [¹⁴C] assimilates. The study of the symplastic relations between the different cell types of the mature leaf showed that the density of the plasmodesmata is generally very low in comparison with other species investigated so far, particularly when considering the mesophyll/bundle sheath and the bundle sheath/phloem cells connections, as well as the connections of the transfer cell-sieve tube complex with the surrounding cells. These three successive barriers therefore strongly limit the possibilities of symplastic transit of the assimilates to the conducting cells. The comparison of the densities of plasmodesmata in an importing and an exporting leaf suggests that the maturation of the leaf is characterized by a marked symplastic isolation of the phloem, and, within the phloem itself, by the isolation of the conducting complex. As a consequence, these physiological and cytological data demonstrate the apoplastic nature of loading in the mature leaf of Vicia faba, this species undoubtedly presenting a typical model for apoplastic loading.     

Ammonia regulation of carbon metabolism in photosynthesizing leaf discs

Alfalfa (Medicago sativa L., var. El Unico) leaf discs, floating on buffer containing NH₄Cl and photosynthesizing with ¹⁴CO₂, produced more labeled amino acid and less sucrose than did control discs (no added NH₄Cl). The level of pyruvate increased and that of phosphoenolpyruvate decreased. These and other changes in levels of labeled compounds led us to conclude that pyruvate kinase was activated by ammonia, resulting in increased transfer of photosynthetically incorporated carbon to synthesis of amino acid skeletons at the expense of sucrose synthesis. Carbon flow through enzymes catalyzing the anaplerotic reactions was apparently stimulated.