Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

Involvement of sucrose synthase in sucrose catabolism

Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo. SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca²⁺. In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.     

Sucrose leakage from the maize scutellum

Sucrose accumulated in the cytoplasm of mesophyll, parenchyma cells when maize scutella (whole or sliced) were put in concentrated (e.g. 1·0 M) fructose solutions. This accumulated cytoplasmic sucrose leaked from the tissue when the fructose solution was replaced with water or with a more dilute hexitol solution. The amount of leakage was proportional to the concentration difference between the fructose solution bathing the scutellum slices during the sucrose accumulation period and the hexitol solution bathing the slices during the leakage period. Only small amounts of cytoplasmic sucrose leaked from the whole scutellum into water until the root-shoot axis was removed. Other substances also leaked, with sucrose, from the scutellum. Sucrose, nitrogenous compounds, K⁺ and phosphorous compounds leaked in greatest amounts. The results presented are consistent with the ideas of the mass flow hypothesis. In the scutellum system a pressure flow of solution originates in the mesophyll cells, flows from cell to cell through plasmodesmata, into and through the phloem sieve tubes, and, finally, into the bathing solution.     

Dinitrophenol-induced efflux of sucrose from maize scutellum cells

Maize scutellum slices accumulated sucrose during incubation in glucose, fructose or sucrose. Sucrose was accumulated in two compartments, tentatively     

Sucrose transport and hexose release in the maize scutellum

Since hexoses readily diffuse from maize scutellum cells, it should be possible to detect them if they are produced during sucrose transport at the tonoplast or the plasmalemma. To test this idea, scutellum slices were placed in dinitrophenol (DNP) (which inhibits hexose utilization while greatly increasing utilization of vacuolar sucrose), and the utilization, uptake and leakage of sugars were measured. Only negligible amounts of hexose appeared in the DNP solution during a 5-hr incubation during which the slices metabolized 72μmol of sucrose. Glucose and fructose, added at a concentration of 2 mM, were taken up by the slices at rates 33% and 14% (respectively) of the rate of vacuolar sucrose utilization. It is suggested, therefore, that sucrose transport at the tonoplast does not release free hexose into the cytoplasm. Sucrose transport at the plasmalemma was studied using DNP- and mannose-treated slices. During incubation of these slices in sucrose, the disappearance of sucrose resulted in the appearance of significant quantities of glucose and fructose in the bathing solution. Evidence is presented that sucrose is split into glucose and fructose during transport across the plasmalemma. It is concluded that free hexose is not normally a product of this splitting but is a result of an uncoupling in the transport system caused by the DNP or mannose treatments.     

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.     

Glucose control of sucrose synthase in the maize scutellum

The in vivo amounts of UDPG, UTP, UDP and UMP, metabolites known to influence the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS), were measured throughout 5 hr incubations of scutellum slices in fructose or water, i.e. under conditions of sucrose synthesis or breakdown. Cytosolic concentrations were estimated assuming that these metabolites were confined to the cytosol. Within the estimated in vivo concentration ranges, UDPG, UTP and UDP had little effect on the in vitro SS activity, but glucose (100 mM) inhibited SS in the synthesis direction by 63–70% and in the breakdown direction by 86–93%. Glucose inhibition of SS was considerably less when saturating levels of substrates were used. Sucrose did not inhibit SS. It is concluded that during germination the glucose produced from starch breakdown in the maize endosperm enters the scutellum and inhibits SS, preventing a futile cycle and limiting SS participation in sucrose synthesis.     

Sucrose transport at the tonoplast

Sucrose that leaked from maize scutellum slices upon transfer of slices from a hexose or hexitol solution to water or upon placing the slices in a buffered EDTA solution was considered to be cytoplasmic in origin; residual (after leakage) tissue sucrose was considered to be stored in the vacuoles. This paper presents a study of the movement of sucrose across the tonoplast between the vacuoles and the cytoplasmic compartment. It is concluded that; (a) sucrose transport into the vacuoles is directly linked to sucrose synthesis in such a way that free sucrose is not an intermediate in the coupled process, (b) cytoplasmic sucrose is not (cannot be?) stored, (c) sucrose transport out of the vacuoles is linked to the metabolic demand for sugar, and (d) the transport process removing sucrose from the vacuoles does not release free sucrose into the cytoplasm. The sucrose fluxes at the plasmalemma and at the tonoplast are calculated, and the transport processes at the two membranes are compared.     

Nicotine Production by Tissue Cultures of Tobacco as Influenced by Various Culture Parameters

Assimilate efflux from vacuum-infiltrated leaf slices (spinach, barley) into a buffered solution was examined in relation to Ca^{+ +} -activity and osmotic conditions. Efflux from isolated mesophyll protoplasts and from a unicellular green alga (Eremosphaera viridis de Bary) was also measured.In the presence of Ca^{+ +}, assimilate efflux from leaf slices was small (1 to 5 % of the total carbon fixation rate, depending on osmotic conditions). Efflux was drastically stimulated by addition of Ca^{+ +} -chelators. If expressed as µmol carbon mg^-1 chlorophyll h^-1, it reached 50 % of the assimilation rate. Efflux from protoplasts or algae was slow and insensitive to Ca^{+ +} chelators at concentrations which caused fast efflux from leaf slices.Assimilate efflux from leaf slices was rather unspecific. Both in the tissue and the surrounding medium, sucrose was the most abundantly labelled compound (70 to 80 % of total soluble labelled material).A 50 % decrease of efflux was observed when turgor pressure was lowered by addition of sorbitol (200 to 300 mosmol kg^-1). At extremely high sorbitol concentrations (> 1500 mosmol kg^-1) efflux increased again and was relatively less stimulated by EDTA.It is suggested that assimilate efflux from leaf slices is mainly diffusion through open veins and/or plasmodesmata. When these symplastic connections are closed by addition of Ca^{+ +}, the remaining transmembrane flux into the apoplast is small. Thus, assimilate movement from the mesophyll to the phloem appears to be symplastic, not apoplastic as suggested in the literature.     

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

¹⁴C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of ¹⁴C among the soluble sugars extracted from endosperm slices incubated in ¹⁴C-sugars. Competing hexoses reduced the incorporation of ¹⁴C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.     

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     

Utilization of exogenous sugars by excised maize embryos in culture

Sucrose was markedly superior to fructose and glucose in promoting growth of plantlets from immature maize embryos. The elongation of roots is shown to be more sucrose dependent than that of shoots. On the other hand, the exogenous sucrose was less effective than fructose as substrate for carbohydrate catabolism and for the synthesis of alcohol-insoluble compounds at the beginning of embryo cultivation. The absorbed fructose was found to be rapidly converted to sucrose and the level of endogenous sucrose derived from sugar supplied to the medium was higher in fructosethan in sucrose-fed embryos. The preferential utilization of fructose over sucrose, however, declined with the progress of germination which may be related to the decrease in proportion of scutellum in total mass and physiological activity of the embryo.     

Characteristics of the phloem path: analysis and distribution of carbohydrates in the petiole of Cyclamen

Compartmentation fluxes of carbohydrates along the phloem path were analysed in the petiole of Cyclamen persicum (L.) Mill. Sucrose represented the dominant fraction (58-75% of soluble carbohydrates in the vascular symplast). Planteose (12-22%), glucose (3-8%) and fructose (3-13%) occurred in lower amounts (data from liquid chromatography, percentages of the total peak area). Starch was not detectable. Upon feeding leaves with ¹⁴CO2, 98% and 90% of radiolabel was recovered as sucrose in the vascular symplast after 3 h and 24 h, respectively. Thus, sucrose appeared to be the exclusive transport sugar in Cyclamen. Experiments with asymmetrically labelled sucrose revealed that there was no metabolism of translocated sucrose. Analysis of six consecutive petiole segments (each 2 cm in length) showed a homogeneous longitudinal distribution of these sugars differed markedly. On average, the sucrose concentration amounted to 4.7 and 0.4 mg g^-1 FM in the vascular apoplast and petiole parenchyma, respectively. Sucrose was unloaded with out hydrolysis and stored in the periphery of the phloem path. Planteose was identified as another storage saccharide. Sucrose synthesis by sucrose phosphate synthase occurred when isolated vascular bundles were incubated with [¹⁴C]glucose or [¹⁴C]fructose. These data suggest that the phloem path is characterized by both source and sink like activity.     

Inorganic ions modulate the path of phloem loading of sucrose in Ricinus communis L. seedlings

The impact of inorganic ions on sucrose fluxes in the cotyledons and on the pathway of phloem loading was studied in Ricinus communis L. seedlings. The cotyledons were incubated in defined solutions which contained either potassium, sodium, magnesium or calcium as chloride salts, or the sodium salts of sulphate or phosphate. Sucrose uptake from the medium into the cotyledons was only slightly affected by the salts. Sucrose efflux to the medium was increased by phosphate and sulphate and to a lesser extent by sodium and potassium. Phloem loading from the apoplasm and the symplasm was analysed by addition of labelled sucrose to the medium, determination of the specific radioactivity of sucrose in sieve-tube exudate and quantification of export into the seedling axis. Potassium and sodium stimulated the apoplasmic route of phloem loading of sucrose, mostly at the expense of loading from cotyledon sucrose pools. In contrast, sulphate and phosphate strongly inhibited the apoplasmic route whereas the (small) symplasmic flux from the cotyledon sucrose pools was less affected. Magnesium ions inhibited phloem loading by both pathways. The potential of ions in modulating the pathways of sucrose export in day to day operation of plants is discussed.     

Localization of a phosphatase (ATPase) on the plasmalemma of the maize scutellum

A phosphatase (ATPase) was demonstrated on the surface of the maize scutellum cell by showing that (1) when exogenous ATP was hydrolysed by intact scutellum cells, ADP, AMP and P_i appeared in the bathing solution in stoichiometric amounts, (2) the rate of hydrolysis was sensitive to bathing solution pH; (3) exogenous Mg²⁺ increased the rate of hydrolysis and (4) when the ATPase reaction was carried out in the presence of lead nitrate, TEM photographs showed lead phosphate deposits located almost exclusively in the plasmalemma. The ATPase was tightly bound to the plasmalemma and was not destroyed by freezing and thawing scutellum slices, a treatment which disrupted the plasmalemma. Acid treatment (10 mM HCl) of fresh or frozen-thawed scutellum slices destroyed acid phosphatase activity but had little effect on ATPase activity at pH 6.5. Following acid treatment of the scutellum slice preparations, a definite Mg²⁺ requirement for ATPase activity could be demonstrated.     

Invertase and maltase in the free space of the maize scutellum

When maize scutellum slices were incubated in solutions of sucrose or maltose, there was a release of glucose into the bathing solution. The pH optima for glucose release were 2.5 for sucrose and 3.5 for maltose. From measurement of rates of glucose uptake into slices in the presence or absence of sucrose, it is calculated that glucose uptake will introduce errors of 3–9%, depending on the sucrose concentration, in estimates of free-space sucrose-hydrolase activity at pH 2.5. At their respective pH optima, maltose was hydrolysed at a rate 2.5 times that of sucrose. When frozen-thawed slices were used the same pH optima were obtained, but rates of hydrolysis were increased. Raffinose and melezitose also were hydrolysed with pH optima of 2.5 and 3.5, respectively. α-Methyl glucose was not hydrolysed. A 60-min HCl treatment (pH 2) of scutellum slices destroyed 69% of the sucrose-hydrolase activity and 100% of the maltose-hydrolase activity. In contrast, sucrose uptake and sucrose synthesis from exogenous fructose were not affected by HCl treatment. It is concluded that there are two hydrolases, acid invertase and maltase; that they are either on or outside the plasmalemma (in the free space); and that they are not necessary to the disaccharide uptake processes either by supplying exogenous hexose or by acting as transporters.     

Effect of sugar concentration on camptothecin production in cell suspension cultures ofCamptotheca acuminata

Studies for the effects of sugar concentration on camptothecin production in suspension cultures ofCamptotheca acuminata were made with different concentrations of sucrose, glucose, and fructose. Sucrose among tested carbon sources increased the camptothecin production. The highest camptothecin, 29×10⁴ mg/L, was obtained at 6% of sucrose that was 11 times higher than that at 2% of sucrose. Kinetics of camptothecin production with 6% of sucrose showed the camptothecin production was increased up to 3 days and then decreased after 6 days from inoculation. The highest camptothecin was obtained on the third day from inoculation.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday