Sucrose Transport Activity Research Articles

Sweets – The Missing Sugar Efflux Carriers

One of the last mysteries in plant physiology is the efflux of sugars from mesophyll cells into the apoplasmic space. Depending on the strategy of long-distance assimilate transport, text books divide plants into symplasmic and apoplasmic phloem loaders. In both cases large concentration gradients are built up between mesophyll cells and the sieve element–companion cell complex (SE–CCC) of the phloem. In case of apoplasmic loaders this gradient is achieved by active transport of sucrose from the apoplasm into the SE–CCC. This uptake is mediated by well characterized plasma membrane sucrose–H+ symporters (Kuhn and Grof, 2010). As a consequence of this model, sucrose synthesized within mesophyll cells must be exported into the apoplasmic space, but the efflux mechanism is as yet unknown (Lemoine, 2000; Williams et al., 2000). In addition, sugar efflux must play a crucial role in fuelling pollen and embryo development as well as nectar production and secretion of carbohydrates to the rhizosphere. Furthermore, phytopathogenic bacteria usually colonize the apoplasmic space and are dependent on nutrient uptake from the host. Hence, it is very likely that efflux carriers from the host are required for bacterial growth. Despite these important functions, attempts to isolate and characterize sugar efflux carriers failed so far, probably because of unsuitable screening systems. Using a new and elegant screening system, Chen et al. (2010) were able to isolate and characterize the first sugar efflux carrier from plants and later on could demonstrate that these efflux carriers belong to a novel transporter family with homologs in Caenorhabditis elegans and humans. The screening system was based on a mammalian cell line (HEK293T) co-expressing a genetically encoded glucose sensor (Frommer et al., 2009) and a library of polytopic A. thaliana membrane proteins of unknown function as predicted by the membrane protein database Aramemnon (Schwacke et al., 2003). This system allowed the sensitive detection of glucose uptake and let to the identification of AtSWEET1. In Arabidopsis thaliana, AtSWEET1 is a member of a gene family comprising 17 members. Mutant phenotypes of some members of the SWEET family have already been described in A. thaliana and rice. Mutation of AtSWEET8 (ruptured pollen grain 1, RPG1) for example, prevents microsporocyte development by inhibiting glucose efflux from tapetum cells (Guan et al., 2008). Interestingly, several members of the AtSWEET gene family are inducible by pathogens. This let to the hypothesis that some pathogens might hijack host efflux systems to support their own growth in the apoplastic space of infected host plants. Support for this hypothesis comes from rice. Mutations in OsSWEET11 (Xa13) results in resistance against late blight (Chu et al., 2006). Moreover, in wild-type rice plants, Xanthomonas oryzae, the causative agent of late blight, activates OsSWEET11 expression by binding of TAL effectors to promoter elements of the OsSWEET11 gene (Romer et al., 2010). Mutations in these binding sites lead to the failure of gene induction and hence prevent colonization of the host. Interestingly, mutations in the same gene also result in reduced fertility of pollen. Thus pathogens seem to be able to reprogram host cells to support their nutrient supply by interfering with the host transport system. Taken together, assigning a function to the SWEET gene family opens up exiting perspectives for both plant physiology and plant–pathogen interaction. It could clearly be shown that SWEETS are in involved in glucose efflux and play a crucial role during pollen development and plant–pathogen interaction. Since the SWEETS transport glucose, this still leaves the question which proteins are responsible for sucrose efflux around the phloem or from the seed coat. However the base has been laid – one could use the FRET sucrose sensors (Lager et al., 2006) to use the same principal approach as was used for identifying the SWEETS, namely to screen a library of unknowns for novel sucrose transporters that might be responsible for sugar efflux around the phloem. But it seems to be clear that on the basis of the important role of members of the SWEET family during plant–pathogen interaction, novel strategies in plant protection become feasible. Therefore, the article by Chen et al. (2010) represents an exiting milestone in furthering our understanding of transport processes in plants and their relevance in plant–microbe interactions (for illustration see Figure ​Figure11). Figure 1 Schematic representation of sugar transport processes during plant–bacterium interaction and phloem loading. S, sucrose; H+, proton.

Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H(+) symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification

The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

Sucrose regulates elongation of carrot somatic embryo radicles as a signal molecule.

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5% (145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N -acetyl-glucosamine did not exert any influence on the regulation-deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene ( cSUT ) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Sucrose Fermentation by Saccharomyces cerevisiae Lacking Hexose Transport

Sucrose is the major carbon source used by Saccharomyces cerevisiae during production of baker’s yeast, fuel ethanol and several distilled beverages. It is generally accepted that sucrose fermentation proceeds through extracellular hydrolysis of the sugar, mediated by the periplasmic invertase, producing glucose and fructose that are transported into the cells and metabolized. In the present work we analyzed the contribution to sucrose fermentation of a poorly characterized pathway of sucrose utilization by S. cerevisiae cells, the active transport of the sugar through the plasma membrane and its intracellular hydrolysis. A yeast strain that lacks the major hexose transporters (hxt1–hxt7 and gal2) is incapable of growing on or fermenting glucose or fructose. Our results show that this hxt-null strain is still able to ferment sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene, which encodes a high-affinity sucrose-H⁺ symporter, rendered cells incapable of sucrose fermentation. Since sucrose is not an inducer of the permease, expression of the AGT1 must be constitutive in order to allow growth of the hxt-null strain on sucrose. The molecular characterization of active sucrose transport and fermentation by S. cerevisiae cells opens new opportunities to optimize yeasts for sugarcane-based industrial processes.

Protein phosphorylation plays a key role in sucrose-mediated transcriptional regulation of a phloem-specific proton-sucrose symporter.

Assimilate partitioning refers to the systemic distribution of sugars and amino acids from sites of primary assimilation (source tissue) to import-dependent tissues and organs (sinks). One of the defining questions in this area is how plants balance source productivity with sink demand. Recent results from our laboratory showed that sucrose transport activity is directly proportional to the transcription rate of the phloem-specific proton-sucrose symporter BvSUT1 in Beta vulgaris L. Moreover, symporter gene transcription is regulated by sucrose levels in the leaf. Here we show that sucrose-dependent regulation of BvSUT1 transcription is mediated, at least in part, by a protein phosphorylation relay pathway. Protein phosphatase inhibitors decreased sucrose transport activity, symporter protein and mRNA abundance, and the relative transcription rate of the symporter gene. In contrast, protein kinase inhibitors had no effect or increased sucrose transport, protein and mRNA abundance, and transcription. Furthermore, pre-treating leaves with kinase inhibitors before feeding with sucrose blocked the sucrose-dependent decrease in symporter transcription and transport activity. The latter observation provides direct evidence for a protein phosphorylation cascade operating between the sucrose-sensor and the transcriptional regulator that controls BvSUT1 expression and, ultimately, phloem loading.

Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

A proton-sucrose symporter mediates the key step in carbon export from leaves of most plants. Sucrose transport activity and steady-state mRNA levels of BvSUT1, a sugar beet leaf sucrose symporter, are negatively regulated specifically by sucrose. Results reported here show that BvSUT1 mRNA was localized to companion cells of the leaf's vascular system, which supports its role in the systemic distribution of photoassimilate. Immunoblot analysis showed that decreased transport activity was caused by a reduction in the abundance of symporter protein. RNA gel blot analysis of the leaf symporter revealed that message levels also declined, and nuclear run-on experiments demonstrated that this was the result of decreased transcription. Further analysis showed that symporter protein and message are both degraded rapidly. Taken together, these data show that phloem loading is regulated by means of sucrose-mediated changes in transcription of a phloem-specific sucrose symporter gene in a regulatory system that may play a pivotal role in balancing photosynthetic activity with resource utilization.

Intra- and Intermolecular Interactions in Sucrose Transporters at the Plasma Membrane Detected by the Split-Ubiquitin System and Functional Assays

Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons.

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development.

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the prestorage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.

Identification and functional expression in yeast of a grape berry sucrose carrier

A large increase in hexose accumulation is typical of grape berry ripening. To analyse sugar transport processes during grape berry ( Vitis vinifera L. cv Ugni Blanc) development, a cDNA encoding for a sucrose transporter, designated VvSUT1 was cloned. Sucrose transport activity of the VvSUT1 protein was demonstrated by heterologous expression in yeast. A study of gene expression along berry development showed that VvSUT1 transcripts are present at all stages but accumulate with the onset of ripening. This work shows that sucrose transport at the plasma membrane of grape berry cells may play a role in the accumulation of sugars during maturation.

An Active Sucrose Carrier (Scr1) that is Predominantly Expressed in the Seedling of Ricinus communis L.

During the germination of Ricinus communis seedlings sucrose is released from the endosperm and taken up by the cotyledons for their growth and for export to the rapidly growing hypocotyl and roots. Both the mesophyll cells and the phloem of castor bean possess active sucrose carriers. We isolated one of these carriers by heterologous screening with the spinach sucrose carrier cDNA for further investigation of sucrose transport at the cellular level. The deduced peptide sequence of this sucrose carrier (Scr1) has 12 putative membrane spanning domains and shares homology with other transporters of the major facilitator superfamily (MFS). When expressed in Saccharomyces cerevistae , Scr1 shows active sucrose transport that can be inhibited by PCMBS or CCCP. According to the pH-dependence the carrier seems related to SUC2 of Arabidopsis and Plantago , which are phloem-specific in these plants. It also shows a relatively high specificity for sucrose, compared with maltose or raffinose. Scr1 is expressed in equal amounts in all organs of a 6-day-old castor bean seedling and to a lesser extent in green sink and source leaves.

Affinity purification of sucrose binding proteins from the plant plasma membrane

Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [ 3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.

Inhibitors of the Proton-Sucrose Symport

Sucrose transporters are important components of the assimilate partitioning pathway in many plants. In the results reported here, we examined the effect of several inhibitors on proton-coupled sucrose transport into plasma membrane vesicles isolated from sugar beet leaf tissue. Three compounds that are reversible inhibitors of glucose transporters, phlorizin, cytochalasin B, and forskolin, also inhibited the proton-sucrose symport. Additionally, several reagents that covalently modify specific amino acid residues, including p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide (NEM), diethyl pyrocarbonate (DEPC), and Hg 2+, were also examined. NEM was not an effective inhibitor of the symport under both energized (pH 6.0) and unenergized (pH 7.7) conditions. In contrast, PCMBS, DEPC, and Hg 2+ blocked sucrose transport activity. However, in control experiments it was discovered that Hg 2+, but not PCMBS or DEPC, dissipated the proton electrochemical potential difference (Δμ̃ H) that drives sucrose accumulation. It was further demonstrated that Hg 2+ dissipated an imposed Δμ̃ + H in protein-free liposomes, thus obscuring its effect on the sucrose symport. In time- and concentration-dependent inactivation experiments, it was shown that DEPC binding was substrate protectable, thereby implicating binding at or near the active site of the carrier. In contrast, PCMBS activity was not linked to substrate binding. DEPC activity was partially reversed with hydroxylamine. This is consistent with specific modification of a histidine residue. Preloading purified vesicles with free histidine did not slow the DEPC-dependent inactivation kinetics. Since these membrane vesicles are predominantly right-side out, the last observation is consistent with a DEPC-sensitive site which is accessible from the outside face of the vesicle. The results with DEPC suggest that a histidine residue is at or near the active site of the sucrose symport and that this amino acid plays a critical role in the reaction mechanism.

Phloem Transport of Amino Acids in Relation to their Cytosolic Levels in Barley Leaves

A comparison of barley (Hordeum vulgare L.) leaves was made between the cytosolic content of amino acids and sucrose as determined by subcellular fractionation and the corresponding concentration in phloem sap, which was collected continuously for up to 6 days from severed aphid stylets. Because amino acids were found to be almost absent from the vacuoles, and because the amino acid patterns in the stroma and cytosol are similar, whole leaf contents could be taken as a measure of cytosolic amino acid levels for a comparison of data during a diurnal cycle. The results show that the pattern of amino acids in the phloem sap was very similar to the pattern in the cytosol. Therefore, we concluded that the overall process of transfer of amino acids from the cytosol of the source cells into the sieve tubes, although carrier mediated, may be a passive process and that the translocation of amino acids via the sieve tubes requires the mass flow of sucrose driven by the active sucrose transport involved by the phloem loading.

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylmaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitition of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport

Reconstitution of active sucrose transport in plant proteoliposomes

The proteins of purified plasma membranes from sugar beet (Beta vulgaris L.) leaf were solubilized and separated on a size exclusion column. The fractions eluted from the column were monitored by ELISA with antibodies directed to a putative sucrose carrier protein. The peak most reactive in ELISA was approximately 120 kDa, and yielded a 40 kDa peak after denaturation by SDS. The 120-kDa peak was recovered and used for reconstitution experiments with asolectin. Upon imposition of an artificial pH gradient and electrical gradient, the obtained proteoliposomes exhibited active transport of sucrose, but not of valine. The active transport of sucrose was inhibited by N-ethylmaleimide and HgCl2.

Triethyllead Inhibits Mitochondrial ATPase of the Endosperm and Sucrose Uptake in the Cotyledons of Ricinus communis

The active uptake of externally supplied sucrose by isolated cotyledons of Ricinus communis L. is characterized by a K m of 17mM and a V max of 5.3 μmoles × 100 mg −1 FW × h −1 . This uptake was completely inhibited by triethyllead in a non-competitive manner, because in the presence of the toxin the cotyledons transported sucrose with a decreased V max while the K m remained unchanged. The IC 50 for isolated cotyledons was 115 μM; in situ , cotyledons were more susceptible to the toxin, as shown by an IC 50 value of 20 μM. The oligomycin-sensitive ATPase activity of mitochondria isolated from Ricinus communis endosperm was susceptible to low concentrations of triethyllead. Distinct inhibition was seen at ca. 0.1 μM triethyllead, and the IC 50 was determined to be 3 μM. Thus, triethyllead inhibits F 0 /F 1 ATPase more efficiently than oligomycin itself. Addition of glutathione (1 mM) decreased the sensitivity of the enzyme to triethyllead by a factor of 3–4 but was unable to provide full protection. It is concluded that the inhibition of ATP synthesis and the subsequent energy depletion as caused by triethyllead is the likely event leading to the inhibition of active sucrose transport. Physiological concentrations of glutathione partially protect the sucrose transport of cotyledons from the toxic effects of triethyllead.

Characterization of the Active Sucrose Transport System of Immature Soybean Embryos

Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [(14)C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35 degrees C, saturating illumination (pH 6), and 21% oxygen, the apparent K(m) for sucrose is approximately 8 millimolar and V(max) is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.