Phloem turgor and the regulation of sucrose loading in Ricinus communis L.

The influence of plant water relations on phloem loading was studied in Ricinus communis L. Phloem transport was maintained in response to bark incisions even at severe water deficits. Water stress was associated with a net increase in the solute content of the sieve tubes, which resulted in maintenance of a positive phloem turgor pressure _p. There was a significant increase in solute flux through the phloem with decreasing xylem water potential (). In addition, sugar uptake by leaf discs was examined in media adjusted to different water potentials with either sorbitol (a relatively impermeant solute) or ethylene glycol (a relatively permeant solute). The limitations in this experimental system are discussed. The results nevertheless indicated that sucrose uptake can be stimulated by a reduction in cell _p, but that it is little affected by cell or solute potential _s. On the basis of these data we suggest that sucrose loading is turgor-pressure dependent. This may provide the mechanism by which transport responds to changes in sink demand in the whole plant.Abbreviations water potential - _s solute potential - _p pressure potential     

Evidence on the mechanism of enhanced sucrose uptake at low cell turgor in leaf discs of Phaseolus coccinius

Plants often tolerate water deficits by lowering the osmotic potential of their cell sap. This may be achieved by accumulation of solutes which results in the maintenance of a positive turgor potential. In this study, the effect of water deficit on sugar uptake was investigated in leaf discs of Phaseolus coccinius L. (cv. Scarlet). Evidence is presented that cell turgor affects the kinetics of sugar transport at the membrane level. Uptake kinetics of sucrose, glucose and 3-O-methyl glucose by tissues equilibrated in solutions of relatively high (200–400 mOsm) osmotic concentration consisted of a sat-urable and a linear component. Low external osmotic concentration i.e., high cellular turgor inhibited the saturating component of sucrose uptake, resulting in a linear uptake profile. However, high cell turgor had no effect on glucose or 3-O-methyl glucose uptake kinetics. The effect of turgor versus osmotic component of water potential was differentiated by comparing responses to non-penetrating (manmtol) or polyethylene glycol, (3350) and penetrating (ethylene glycal) osmotica. Changes in sucrose uptake rates and kinetics were due to changes in cellular turgor and not osmotic potential. Furthermore, at low cellular turgor, a net increase in sucrose uptake occurred as a consequence of enhanced influx rates and not as a result of reduced efflux rates. The data are consistent with previous findings that sugar uptake rates are enhanced under low turgor. We present first evidence indicating that the mechanism by which higher rates of sucrose uptake are maintained underwater deficit conditions is by the activation of the saturable transport system. This mechanism supports previous suggestions that changes in cell turgor are sensed and manifested at the membrane level.     

Influence of cell turgor on sucrose partitioning in potato tuber storage tissues

Sucrose uptake and partitioning in potato (Solanum tuberosum L.) tuber discs were examined under a range of mannitol and ethylene-glycol concentrations. Mannitol caused the same changes in turgor over a wide range of incubation periods (90 min-6 h), indicating that it did not penetrate the tissue. In comparison, ethylene glycol reduced turgor losses but did not eliminate them, even after 6 h. Between 100 mM and 300 mM mannitol, turgor fell by 350 kPa, compared with 35 kPa in ethylene glycol. Uptake experiments in mannitol alone showed that total sucrose uptake was strongly correlated with both osmotic potential and with turgor potential. In subsequent experiments sucrose uptake and partitioning were examined after 3 h equilibration in 100 mM and 300 mM concentrations of mannitol and ethylene glycol. Total sucrose uptake and the conversion of sucrose to starch were enhanced greatly only at 300 mM mannitol, indicating an effect of turgor, rather than osmotic potential on sucrose partitioning. The inhibitors p-chloromercuribenzenesulfonic acid and carbonylcyanide m-chlorophenylhydrazone (CCCP) both reduced sucrose uptake, but in quite different ways. p-Chloromercuribenzenesulfonic acid reduced total sucrose uptake but did not affect the partitioning of sucrose to starch. By contrast, CCCP inhibited total uptake and virtually eliminated the conversion of sucrose to starch. Despite this, sucrose uptake in the presence of CCCP continued to increase as the mannitol concentration increased, indicating an increase in passive transport at higher mannitol concentrations. Increased sucrose uptake above 400 mM mannitol was shown to be the result of uptake into the free space. The data show that starch synthesis is optimised at low but positive turgors and the relation between sucrose partitioning and the changing diurnal water relations of the tuber are discussed.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid     

Direct and indirect measurements of Phloem turgor pressure in white ash

Direct determinations and indirect calculations of phloem turgor pressure were compared in white ash (Fraxinus americana L.). Direct measurements of trunk phloem turgor were made using a modified Hammel-type phloem needle connected to a pressure transducer. Turgor at the site of the direct measurements was calculated from the osmotic potential of the phloem sap and from the water potential of the xylem. It was assumed that the water potentials of the phloem and xylem were close to equilibrium at any one trunk location, at least under certain conditions. The water potential of the xylem was determined from the osmotic potential of xylem sap and from the xylem tension of previously bagged leaves, measured with a pressure chamber. The xylem tension of bagged leaves on a branch adjacent to the site of the direct measurements was considered equivalent to the xylem tension of the trunk at that point. While both the direct and indirect measurements of phloem turgor showed clear diurnal changes, the directly measured pressures were consistently lower than the calculated values. It is not clear at present whether the discrepancy between the two values lies primarily in the calculated or in the measured pressures, and thus, the results from both methods as described here must be regarded as estimates of true phloem turgor.     

Mechanism of sucrose uptake by isolated rat hepatocytes

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.     

The effect of cell turgor on sugar uptake in strawberry fruit cortex tissue

A reduction in cell turgor has been shown to stimulate sugar uptake in several plant sink tissues and it may regulate the import of assimilate into the sink apoplast, as well as maintain cell turgor. To determine whether cell turgor influences sugar uptake by strawberry (Fragaria x ananassa Duch. cv. Brighton) fruit cortex tissue, disks were cut from greenhouse-grown primary fruit at the green-white stage of development and placed in buffered incubation solutions containing either mannitol or ethylene glycol as an osmoticum. Cell turgor of fruit disks was calculated from the difference between the water potential of bathing solution and tissue solute potential after incubation at various osmolarities. Cell turgor increased when tissue disks were placed into mannitol incubation solutions more dilute than the water potential of fresh tissue (about 415 mOsmol kg^?1). The rate of uptake of [¹⁴C]-sucrose or [¹⁴C]-glucose decreased as osmolarity of the incubation solution increased, i.e. as cell turgor declined. Cell turgor and the rate of [¹⁴C]-sucrose uptake were unaffected when rapidly permeating ethylene glycol was used as an osmoticum. A decrease in cell turgor reduced both the V_max of the saturable (carrier mediated) kinetic component of sucrose uptake, and the slope of the linear (diffusional) component. The sulfhydryl binding reagent p-chloromercuibenzenesulfonic acid, an inhibitor of the plasma membrane sucrose carrier, strongly inhibited only the saturable component of sucrose uptake. Increased uptake of the nonmetabolizable sugar, O-methyl-glucose, at high turgor was similar to that of glucose, indicating that carrier activity was influenced by cell turgor, not cell metabolism. Turgor did not influence efflux of [¹⁴C]-sucrose from disks and had no effect on cell viability. Strawberry fruit cells do not possess a sugar uptake system that is stimulated by a reduction in turgor.     

Measurement of turgor pressure and its gradient in the Phloem of oak

A direct method is described for measuring the pressure in secondary phloem sieve tubes of oak trees. One end of a 26-gauge stainless steel tube was shaped such that when it penetrated the outer bark and transected a few sieve elements, it was stopped by the xylem so that small openings in the end allowed phloem sap to enter the tube. The other end of the stainless tube (phloem needle) was joined to a long glass capillary sealed at its other end to form a manometer for measuring phloem sap pressure. A method for measuring the average osmotic and turgor pressures in cells of leaves is also described. Phloem turgor pressures varied greatly in a series of phloem punctures around the trunk at 1.5 and at 6.3 meters. The variation in turgor pressure was always greater than the variation in osmotic pressure. In a series of turgor pressures arranged in descending order, the values in a sequence for the upper level was usually a little (0-3 atm) larger than the values for the lower level. These results may suggest that translocation of assimilate is favored by a small turgor pressure gradient, but they do more to emphasize the complications in measuring gradients in an elastic low resistance distribution system composed of contiguous longitudinal conduits. The results also imply that the sieve tubes are inflated with assimilate fluid under high pressure which can readily move longitudinally and with less pressure drop than would be necessary if the sieve tubes were rigid.     

Sucrose uptake in isolated phloem of celery is a single saturable transport system

The uptake of different sugars was studied in segments of isolated phloem from petioles of celery (Apium graveolens L.) in order to determine the kinetics and specificity of phloem loading in this highly uniform conductive tissue. The uptake kinetics of sucrose and the sugar alcohol, mannitol, which are both phloem-translocated, indicated presence of a single saturable system, while uptake of non-phloem sugars (glucose and 3-O-methylglucose) exhibited biphasic kinetics with lower uptake rates than those for sucrose and mannitol. The presence of unlabeled mannitol, 3-O-methylglucose and maltose in the incubation solution did not cause inhibition of labeled-sucrose uptake, indicating high carrier specificity and lack of sucrose hydrolysis in vivo. The pH optimum for sucrose uptake was 5–6. Furthermore, a rapid and transient alkalinization of the external media by sucrose indicated a sugar/H⁺-cotransport mechanism. Dual-labeling experiments showed that sucrose influx continued at a constant rate (V _max=15 mol·h^-1·(g FW)^-1), whereas sucrose efflux was low and insensitive to external concentration. Therefore, the saturable uptake kinetics for sucrose did not appear to be the result of an equilibrium between rates of sucrose influx and efflux.Abbreviations 3-OMG 3-O-methylglucose - PCMBS p-chloromercuribenzene sulfonate - SE-CC sieve element-companion cell - VB vascular bundle     

Effect of plant hormones on sucrose uptake by sugar beet root tissue discs

Abscisic acid (ABA), auxins, cytokinins, gibberellic acid, alone or in combination were tested for their effects on short-term sucrose uptake in sugar beet (Beta vulgaris cv USH-20) roots. The effect of ABA on active sucrose uptake varied from no effect to the more generally observed 1.4-to 3.0-fold stimulation. A racemic mixture of ABA and its trans isomer were more stimulatory than ABA alone. Pretreating and/or simultaneously treating the tissue with K⁺ or IAA prevented the ABA response while cytokinins and gibberellic acid did not. While the variable sensitivities of beet root to ABA may somehow be related to the auxin and alkali cation status of the tissue, tissue sensitivity to ABA was not correlated with ABA uptake, accumulation, or metabolic patterns. In contrast to ABA, indoleacetic acid (IAA) and other auxins strongly inhibited active sucrose uptake in beet roots. Cytokinins enhanced the auxin-induced inhibition of sucrose uptake but ABA and gibberellic acid did not modify or counteract the auxin effect. Trans-zeatin, benzyladenine, kinetin, and gibberellins had no effect on active sucrose uptake. None of the hormones or hormone mixtures tested had any significant effect on passive sucrose uptake. The effects of IAA and ABA on sucrose uptake were detectable within 1 h suggesting a rather close relationship between the physiological activities of IAA and ABA and the operation of the active transport system.     

Pathway of Phloem unloading of sucrose in corn roots

The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) seedling roots. Several lines of evidence show that exogenous sucrose, unlike translocated sucrose, is hydrolyzed in the apoplast prior to uptake into the root cortical cells. These include (a) presence of cell wall invertase activity which represents 20% of the total tissue activity; (b) similarity in uptake and metabolism of [¹⁴C]sucrose and [¹⁴C]hexoses; and (c) randomization of ¹⁴C within the hexose moieties of intracellular sucrose following accumulation of [¹⁴C] (fructosyl)sucrose. Conversely, translocated sucrose does not undergo apoplastic hydrolysis during unloading. Asymmetrically labeled sucrose ([¹⁴C](fructose)sucrose), translocated from the germinating kernels to the root, remained intact indicating a symplastic pathway for unloading. In addition, isolated root protoplasts and vacuoles were used to demonstrate that soluble invertase activity (V_max = 29 micromoles per milligram protein per hour, K_m = 4 millimolar) was located mainly in the vacuole, suggesting that translocated sucrose entered via the symplasm and was hydrolyzed at the vacuole prior to metabolism.     

Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specifity of the sucrose uptake system

The uptake of sucrose against a concentration gradient into the dextran-impermeable [³H]H₂O space of red beet (Beta vulgaris L.) vacuoles has been studied using silicone-layer-filtering centrifugation on both fluorometric and ¹⁴C-measurement of sucrose. Sucrose transport into vacuoles proceeds partly by an active transport system and partly by passive permeation. The K _M(20°C) for active sucrose uptake was found to be about 22 mM and the V _Max(20°C) was about 174 nmol sucrose x (unit betacyanin)^-1 x h^-1. The temperature dependency of sucrose transport appears to have an activation energy of 35,0 KJ×mol^-1. Among various mono-, di-, and trisaccharides tested, raffinose acts as a competitive inhibitor of sucrose uptake.Abbreviations EDTA ethylenediamine tetraacetic acid - fr. wt. fresh weight - Tris tris-(hydroxymethyl)-aminomethan     

Phloem loading of sucrose: Update and opportunities in molecular biology

Sucrose accumulates in the phloem against a concentration gradient via a presumed sucrose-specific carrier protein located at the plasmalemma of the sieve elements/companion cells. Recent evidence suggests that sucrose carrier in soybean is a 62-kDa protein. Immunocytochemical localization has shown the protein to be exclusively at the plasmalemma, which is also the site of sucrose transport. To enhance our understanding of the phenomenon, the structural gene of the sucrose carrier must be cloned and sequenced. Furthermore, development of appropriate probes should help answer long-standing questions relative to the molecular nature of sugar transport and phloem loading, the mechanism of induction/activation of sugar carriers, and developmental regulation of expression of genes encoding such carriers.     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Interaction of chalones and glucocorticoid hormones in regulation of cell division

The action of hepatic chalone on cell proliferation in inoculated hepatoma 22a of mice was studied in the presence of a changed level of glucocorticoid hormones in experimental animals. Chalone was obtained from the liver of intact rats by ethanol precipitation. The intensity of cell proliferation in hepatoma was evaluated by the colcemide and autoradiography methods. Six hours after chalone injection c-mitosis in the tumor decreased 2.7-fold, and the DNA index 6.8-fold. It may be concluded that the preparation used contains both G1- and G2-chalones. Single or repeated injections of hydrocortisone to mice inhibits cell proliferation to a less degree than administration of chalone alone. Combination of hydrocortisone and chalone produces the same effect as injection of chalone alone. Adrenalectomy diminishes susceptibility of hepatoma cells to exogenous chalone. The degree of tumor proliferative activity in the adrenalectomized animals was half as much after chalone injection, as compared to that in intact animals. Thus, a certain level of glucocorticoid hormones in hepatoma tissue is necessary to reveal the action of chalones.     

Accumulation of phosphate, sulfate and sucrose by excised Phloem tissues

Excised petiolar vascular bundles and excised phloem tissues have been shown to take up phosphate, sulfate and sucrose by a true accumulation process and against high concentration ratios. Phosphate was accumulated principally as inorganic phosphate, and sucrose principally as sucrose. The rates of accumulation of the 3 solutes into the phloem-containing tissues were from 4 to 35 times higher than into comparable parenchyma tissue. It is suggested that this active accumulation mechanism plays an important role in the phenomenon of phloem transport.

The excised vascular, phloem and parenchyma tissues show an aging phenomenon: aerating the excised tissues for 18 hours prior to their use causes marked changes in the accumulatory behavior of the tissue. The data suggest that 1 phosphate accumulation system of low affinity but high capacity exists in fresh tissue, and that aging allows the development of a second, additional phosphate accumulation mechanism of high affinity and low capacity. A possible role in the control of phosphate movement is suggested.

     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Changes in cell turgor pressure related to uptake of solutes by Microcystis sp. strain 8401

Uptake of several naturally occurring organic solutes by the unicellular cyanobacterium Microcystis sp. caused changes in cell turgor pressure (p(t)), which was determined by measuring the mean critical pressure (p(c)) of gas vesicles in the cells. Cells had an initial p(t) of 0.34 MPa, which decreased to 0.08 MPa in 0.15 M sucrose. In solutions of polyols, p(t) gradually recovered as the solutes penetrated the cytoplasmic membranes. From measurements of the exponential rate of turgor increase, cell volume and surface area, the permeability coefficient of the cytoplasmic membrane to each solute was calculated. Permeabilities to amino acids, ammonium ions and sodium acetate indicated little passive movement of these substances across the cell surface from solutions at high concentrations. We looked for evidence of ion trapping of acetic acid: at low pH there was a rapid rise in turgor pressure indicating a rapid uptake of this weak acid. After 20 min the turgor was lost, apparently due to loss of integrity of the cell membranes. For cells in natural habitats, studies of the permeability of cells to solutes is relevant to the problem of retaining substances that are accumulated by active uptake from solutions of low concentrations in natural waters.     

The sucrose transporter of celery. Identification and expression during salt stress

In celery (Apium graveolens L.), long-distance transport of reduced carbon occurs both in the form of sucrose (Suc) and mannitol. The presence of mannitol has been related to the resistance of celery to salt stress. To investigate the transport events occurring during salt stress, we have cloned the H(+)/Suc transporter of celery AgSUT1 (A. graveolens Suc uptake transport 1) from a mature leaf cDNA library. The function of the encoded protein was confirmed by expression in yeast. AgSUT1 is a H(+)/Suc transporter with a high affinity for Suc (K(m) of 139 microM). Another closely related cDNA (AgSUT2) was also identified. AgSUT1 is mainly expressed in mature leaves and phloem of petioles, but also in sink organs such as roots. When celery plants were subjected to salt stress conditions (30 d watering with 300 mM NaCl) favoring mannitol accumulation (J.D. Everard, R. Gucci, S.C. Kann, J.A. Flore, W.H. Loescher [1994] Plant Physiol 106: 281-292), AgSUT1 expression was decreased in all organs, but markedly in roots. The results are discussed in relation to the physiology of celery.     

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.     

锌肥对日光温室西芹硝酸盐及营养品质研究

利用日光温室西芹不同锌水平的试验,研究了以氮,钾肥作基肥时,不同锌水平对西芹的硝酸盐累积,营养品质及产量的影响,结果表明,当施用硫酸锌30kg/hm2,西芹的产量达到最高,而硝态氮含量与CK相比,叶片和叶柄分别降低28．6％和29．5％,施用锌肥可以提高Vc的含量,对可溶性糖的影响不大。