Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V_max only with no apparent change in K_m. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-driven sucrose transport were used to examine proton binding to the symport and the apparent K_m for H⁺ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.     

Role of phloem in sucrose transport by Ricinus cotyledons

Sucrose is taken up and accumulated by cotyledons of Ricinus communis L. Autoradiographic studies reveal a predominant accumulation of sucrose in the phloem of the cotyledons. The export of sucrose from the cotyledons to hypocotyl and roots proceeds in the phloem by mass flow. These results, taken together with previous data, are experimental evidence for proton-sucrose symport as the mechanism of phloem loading.     

Diurnal periodicity of assimilate transport shapes resource allocation and whole‐plant carbon balance

Whole‐plant carbon balance comprises diurnal fluctuations of photosynthetic carbon gain and respiratory losses, as well as partitioning of assimilates between phototrophic and heterotrophic organs. Because it is difficult to access, the root system is frequently neglected in growth models, or its metabolism is rated based on generalizations from other organs. Here, whole‐plant cuvettes were used for investigating total‐plant carbon exchange with the environment over full diurnal cycles. Dynamics of primary metabolism and diurnally resolved phloem exudation profiles, as proxy of assimilate transport, were combined to obtain a full picture of resource allocation. This uncovered a strong impact of periodicity of inter‐organ transport on the efficiency of carbon gain. While a sinusoidal fluctuation of the transport rate, with minor diel deflections, minimized respiratory losses in Arabidopsis wild‐type plants, triangular or rectangular patterns of transport, found in mutants defective in either starch or sucrose metabolism, increased root respiration at the end or beginning of the day, respectively. Power spectral density and cross‐correlation analysis revealed that only the rate of starch synthesis was strictly correlated to the rate of net photosynthesis in wild‐type, while in a sucrose‐phosphate synthase mutant (spsa1), this applied also to carboxylate synthesis, serving as an alternative carbon pool. In the starchless mutant of plastidial phospho‐gluco mutase (pgm), none of these rates, but concentrations of sucrose and glucose in the root, followed the pattern of photosynthesis, indicating direct transduction of shoot sugar levels to the root. The results demonstrate that starch metabolism alone is insufficient to buffer diurnal fluctuations of carbon exchange.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

The effects of increased atmospheric carbon dioxide and temperature on carbon partitioning, source-sink relations and respiration

Abstract. Herbaceous C₃ plants grown in elevated CO₂ show increases in carbon assimilation and carbohydrate accumulation (particularly starch) within source leaves. Although changes in the partitioning of biomass between root and shoot occur, the proportion of this extra assimilate made available for sink growth is not known. Root:shoot ratios tend to increase for CO₂-enriched herbaceous plants and decrease for CO₂-enriched trees. Root:shoot ratios for cereals tend to remain constant. In contrast, elevated temperatures decrease carbohydrate accumulation within source and sink regions of a plant and decrease root:shoot ratios. Allometric analysis of at least two species showing changes in root: shoot ratios due to elevated CO₂ show no alteration in the whole-plant partitioning of biomass. Little information is available for interactions between temperature and CO₂. Cold-adapted plants show little response to elevated levels of CO₂, with some species showing a decline in biomass accumulation. In general though, increasing temperature will increase sucrose synthesis, transport and utilization for CO₂-enriched plants and decrease carbohydrate accumulation within the leaf. Literature reports are discussed in relation to the hypothesis that sucrose is a major factor in the control of plant carbon partitioning. A model is presented in support.     

Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) tubers

Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.     

Movement of [14C]sucrose along the stolon of Saxifraga sarmentosa

Summary The characteristics of ¹³⁷Cs transport along the stolon of Saxifraga previously reported have been confirmed for applied sucrose and natural assimilate. Long-distance transport is strictly unidirectional, with a symmetrical short-distance spread from the point of application. Only the latter takes place in a long piece of excised stolon. Transport is readily reversed when the parent plant is darkened and the daugther, plantlet allowed to photosynthesise. These findings strongly support a mass-flow mechanism for the stolon. They also confirm the value of ¹³⁷Cs as a tracer for assimilate movement, though in contrast to assimilate it suffers appreciable lateral leakage. Pulse labelling of the subtending leaf failed to produce a sharp peak of activity in the stolon. A flattening with time of the ¹⁴C profile is considered to be due to differing linear velocities in parallel sieve tubes.This work formed part of that submitted for the degree, of Ph. D. of the University of London.     

Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

Plant sucrose-H+ symporters mediate the transport of vitamin H

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.     

Ammonium transport and CitAMT1 expression are regulated by light and sucrose in Citrus plants

Here the isolation and characterization of CitAMT1 cDNA from citrange Troyer (Citrus sinensis L. OsbeckxPoncirus trifoliata Blanco) is reported, suggesting that this belongs to the AMT gene family, which is involved in the high-affinity transport system (HATS). Results show that in Citrus plants, the HATS is much more dependent on the light conditions and C status of the roots than the low-affinity transport system. Most importantly, a strong correlation was found between the regulation of both HATS activity and CitAMT1 expression. CitAMT1 expression is sucrose-stimulated and may account for the regulation of NH(4)(+) HATS. Furthermore, a similar link was also recorded with photosynthetic activity in the shoots, suggesting that the variations in production and transport of photosynthates to the roots are responsible for the diurnal changes of both CitAMT1 expression and NH(4)(+) HATS activity. On the other hand, results indicate that the effect of stimulating light on CitAMT1 expression and NH(4)(+) HATS activity is independent of the circadian rhythm. Finally, CitAMT1 expression seems to be specifically stimulated by sucrose, suggesting that sucrose is a pivotal signal governing both assimilate partitioning from source organs and assimilate utilization in sink organs.     

Effects of treatments potentially influencing the supply of assimilate on its partitioning in sugarcane.

Two pot experiments and one field experiment were conducted on sugarcane to assess the effects of treatments expected to change total carbon assimilation on the partitioning of assimilate. In the first experiment pots of cultivars CP and N14 were arranged to simulate normal field spacing. At 5 months, plants were partially defoliated or left intact. In the subsequent four months, defoliation resulted in a small (not significant) decrease in total dry mass increment; it increased the proportional partitioning of assimilates to leaves in N14, whilst in CP it increased the proportional partitioning to stems. In both cultivars defoliation increased proportional allocation to non-structural dry matter, and thus sucrose, in the stem. In the second experiment pots of cv. CP were grown at normal spacing for 4 months, then left untreated, shaded, or placed further apart. During the subsequent four months shading decreased total dry matter increment, but increased proportional partitioning to the stems, and within stems to non-structural dry matter, and so sucrose. Widened spacing increased total assimilation, but decreased proportional allocation to stems; partitioning within the stems was not affected. In the field experiment plants of both cultivars were partially defoliated at 6 months, or left intact. Defoliation resulted in only a very small decrease in stem dry mass increment during the subsequent four months (leaves were not measured). Within the stem partial defoliation caused proportionally increased partitioning to non-structural dry matter, hence to sucrose. The results suggest that sucrose storage receives priority in the allocation of assimilate, rather than representing the accumulation of assimilate not required for vegetative growth.     

Sucrose Transport into Plasma Membrane Vesicles from Tobacco Leaves by H<Superscript>+</Superscript> Symport or Counter Exchange Does not Display a Linear Component

Uptake of [¹⁴C]sucrose by plasma membrane vesicles from leaves of tobacco (Nicotiana tabacum L.) was measured after the imposition of an inwardly directed proton gradient (ΔpH = 2) and an electrical gradient (Δψ = −68 mV, inside negative) across the vesicle membrane. The vesicles were isolated from a microsomal fraction by two-phase partitioning using media that contained 330 mM of either sorbitol or sucrose. Sucrose transport into vesicles isolated using the sorbitol-containing media showed the hallmarks of electrogenic H⁺ -symport, as it was highly dependent on ΔpH, could be increased three- to four-fold by Δψ, and was abolished by carbonylcyanide m-chlorophenylhydrazone (CCCP). Transport of [¹⁴C]sucrose into vesicles that were isolated using the sucrose-containing media apparently occurred by counter exchange. Its initial influx also depended on a low external pH, but it was insensitive to CCCP and hardly stimulated by Δψ. Both symport and counter exchange obeyed simple Michaelis-Menten kinetics. Transport that depends linearly on the external sucrose concentration could not be detected, indicating that the ‘linear’ component that has been observed in sucrose uptake by leaf tissues does not represent a transport route that is provided by the sucrose symporter. The potential role of H⁺/sucrose-symporters in phloem unloading is briefly discussed.This revised version was published online in August 2005 with a corrected cover date.     

Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-β-lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. ΔpH-Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N′-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate-sensitive K⁺-Mg²⁺-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.     

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.     

Effect of nitrate infusion into the shoot apoplast on photosynthesis and assimilate transport in symplastic and apoplastic plants

The shoots of fireweed (Chamerion angustifolium (L.) Holub) and common flax (Linum usitatissimum L.) were infused with 50 mM KNO₃ solution to compare the influence of nitrate on photosynthesis and assimilate export from leaves in plants with the symplastic and apoplastic phloem loading, respectively. The infusion of nitrate in the shoots of both plant species lowered ¹⁴CO₂ fixation and enhanced the assimilate transport in the upward direction. Irrespective of the phloem loading type, the incorporation of ¹⁴C into sucrose decreased in nitrate-treated seedlings exposed to assimilation for short (3 min) periods. However, when shoots were sampled 3 h after ¹⁴CO₂ fixation, the content of ¹⁴C-labeled sucrose was higher in treated plants than in control seedlings infused with water. In fireweed, in contrast to flax, a similar temporal pattern was also characteristic for ¹⁴C incorporation into oligosaccharides. Within 3 h after nitrate infusion into the fireweed apoplast, the mitochondria and the cell vacuolar system underwent ultrastructural changes indicative of the increase in cytosolic osmotic pressure. At the same time, we observed accumulation of fibrillar inclusions in cell vacuoles of vascular bundles. It is concluded that the mechanisms of nitrate influence on photosynthesis and sugar export in leaves of symplastic and apoplastic plants are similar to a certain extent and involve the blocking of pores in phloem tubes, initiated by the NO-signaling system.     

The kinetics of fructose utilization byS. cerevisiae IGC 4261 in sucrose adjunct brewers wort fermentations

Summary Fructose utilization in laboratory-scale sucrose adjunct brewers wort fermentations, using the brewing strainS. cerevisiae IGC 4261, is predicted by a mathematical model based on the kinetic parameters of the membrane transport proteins which affect fructose uptake into the cell. These include biphasic fructose transport via a proton symport and the constitutive hexose facilitated diffusion system, plus the competitive inhibitory effect that glucose has on this latter component. Also the non-competitive inhibitory effects of a) maltose on fructose uptake via its proton symport and b) ethanol on biphasic fructose transport are incorporated within the model, as well as the inoculum size.     

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H⁺ symport system and via facilitated diffusion.To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H⁺ binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H⁺ binding first, yielding six sets of kinetic parameters.Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H⁺ symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H⁺.In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.     

Regulation of Assimilate Partitioning in Soybean : Initial Effects following Change in Nitrate Supply

Increased concentrations of nitrate in a nutrient solution (2, 5, and 10 millimolar KNO₃) were correlated with increased shoot:root ratios of non-nodulated soybeans (Glycine max [L.] Merr.) grown in sand culture. While altering the pattern of C and N partitioning, the N treatments did not affect whole plant photosynthesis over the study period. To determine the mechanism responsible for the observed changes in assimilate partitioning, detailed C and N budgets were worked out with plants from each N treatment over three consecutive 4-day periods of midvegetative growth. The information for the C and N budgets from the 2 and 10 millimolar NO₃⁻ treatments was combined with data on the composition of xylem and phloem exudates to construct a series of models of C and N transport and partitioning. These models were used to outine a `chain-reaction' of cause-and-effect relationships that may account for the observed changes in assimilate partitioning in these plants. The proposed mechanism identifies two features which may be important in regulating the partitioning of N and other nutrients within the whole plant. (a) The concentration of N in the phloem is highly correlated with the N concentration in the xylem. (b) The amount of N which cycles through the root—from phloem imported from the shoot to xylem exported by the root—is regulated by the root's requirement for N: only that N in excess of the root's N requirements is returned to the shoot in the xylem. Therefore, roots seem to have the highest priority for N in times of N stress.     

Manipulation of sucrose phloem and embryo loading affects pea leaf metabolism,carbon and nitrogen partitioning to sinks as well as seed storage pools

Seed development largely depends on the long‐distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source‐to‐sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this ‘Push‐and‐Pull’ approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source‐to‐sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1‐overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild‐type plants. Together, the results demonstrate that the SUT1‐overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push‐and‐Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.