Immunolocalization of the PmSUC1 sucrose transporter in Plantago major flowers and reporter-gene analyses of the PmSUC1 promoter suggest a role in sucrose release from the inner integument

This paper presents a detailed analysis of the PmSUC1 gene from plantago major, of its promoter activity in Arabidopsis, and of the tissue specific localization of the encoded protein in Plantago. PmSUC1 promoter activity was detected in the innermost layer of the inner integument (the endothel) of Arabidopsis plants expressing the gene of the green fluorescent protein (GFP) under the control of the PmSUC1 promoter. This promoter activity was confirmed with a PmSUC1-specific antiserum that identified the PmSUC1 protein in the endothel of Plantago and of Arabidopsis plants expressing the PmSUC1 gene under the control of its own promoter. PmSUC1 promoter activity and PmSUC1 protein were also detected in pollen grains during maturation inside the anthers and in pollen tubes during and after germination. These results demonstrate that PmSUC1 is involved in sucrose partitioning to the young embryo and to the developing pollen and growing pollen tube. In the innermost cell layer of the inner integument, a tissue that delivers nutrients to the endosperm and the embryo, PmSUC1 may catalyze the release of sucrose into the apoplast.     

Expression of the PmSUC1 sucrose carrier gene from Plantago major L. is induced during seed development

A cDNA clone of the plasma membrane sucrose-H⁺ sym- porter PmSUC1 from Plantago major L. has been isolated and expressed in Saccharomyces cerevisiae . The PmSUC1 protein was characterized in transgenic yeast and in proteoliposomes with an artificial proton-motive-force (pmf) generator. PmSUC1 catalyzes the active uptake of sucrose or maltose in the presence of pmf and is sensitive to uncouplers. Unlike the extremely pH-dependent PmSUC2 sucrose-H⁺ symporter, PmSUC1 is relatively insensitive to changes of the extracellular pH. In leaves and petioles of P. major , expression of PmSUC1 mRNA is restricted to the vascular system. The important new feature about PmSUC1 is that the highest mRNA levels are found in non-vascular tissue of P. major flowers where the gene is transiently expressed during the early stages of seed development. In situ hybridization experiments show that PmSUC1 is expressed only in young ovules; the putative physiological role of PmSUC1 is discussed.     

Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells

High levels of mRNA for the sucrose-H+ symporter PmSUC2 have been found in the vascular bundles of petioles from Plantago major. The possible role of PmSUC2 in phloem loading was studied with antiserum raised against the recombinant PmSUC2 protein. This antiserum labeled a single 35-kD protein band in detergent extracts of P. major vascular bundles. It showed no cross-reaction with the P. major sucrose carrier PmSUC1, which was tested with detergent extracts from plasma membranes of transgenic yeast strains containing either the P. major sucrose transporter PmSUC1 or PmSUC2. The antiserum was used to determine the site of PmSUC2 expression in leaves, petioles, and roots of P. major. In cross-sections and longitudinal sections, the PmSUC2 protein was found in only one single cell type. These cells were identified as companion cells because they are nucleated, contain a dense cytoplasm, and are always adjacent to a sieve element. The labeled cells had the same longitudinal extension as did their sister sieve elements and always ended next to the sieve plates, which were characterized by specific staining. PmSUC2 mRNA and PmSUC2 protein were also detected in P. major roots. The function of PmSUC2 in the different organs and its role in phloem loading are discussed.     

Companion cell-specific inhibition of the potato sucrose transporter SUT1

In many plants, translocation of sucrose from mesnsophyll to phloem for long-distance transport is carrier-mediated. The sucrose H⁺-symporter gene SUT1 from potato is expressed at high levels in the phloem of mature, exporting leaves and at lower levels in other organs. Inhibition of SUT1 by expression of an antisense gene in companion cells under control of the rolC promoter leads to accumulation of high amounts of soluble and insoluble carbohydrates in leaves and inhibition of photosynthesis. The distribution of in situ localized starch does not correspond with areas of reduced photosynthesis as shown by fluorescence imaging. Dissection of antisense effects on sink and source organs by reciprocal grafts shows that inhibition of transporter gene expression in leaves is sufficient to produce chlorosis in leaves and reduced tuber yield. In contrast to the arrest of plasmodesmal development found in plants that express yeast invertase in the apoplast, in mature leaves of sucrose transporter antisense plants plasmodesmata are branched and have median cavities. These data strongly support an apoplastic mode of phloem loading in potato, in which the sucrose transporter located at the plasma membrane of the sieve element/companion cell complex represents the primary route for sugar uptake into the long-distance translocation pathway.     

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H⁺ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The K_M-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The V_MAX but not the K_M-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.     

SUT2, a putative sucrose sensor in sieve elements

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.     

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.     

Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast

Large amounts of sugar are imported into grape berries from source leaves during ripening, and sucrose transporters play a key role during this process. In this study, a putative grape sucrose transporter gene VvSUC27, primarily expressed in sink tissue, was transformed into a yeast strain to characterize its function as a sucrose transporter. Sucrose was taken up by yeast transformed with VvSUC27 at an optimum pH of 4.0–5.0 and a K _m of 8.0–10.5 mM, indicating VvSUC27 is a LAHC (low-affinity/high-capacity) sucrose transporter. The ability of sucrose uptake in transformed yeast was activated by monosaccharides and inhibited by maltose and DEPC. Ya Li Zhang and Qing Yong Meng contributed equally to the paper.     

Identification and characterization of a sucrose transporter isolated from the developing cotyledons of soybean

Reduced carbon produced in mature leaves is distributed throughout plants in the form of sucrose. Sucrose transporter proteins (SUT) play a crucial role in transporting sucrose. We isolated a cDNA encoding a sucrose transporter, GmSUT1, which is expressed in the developing cotyledons of soybean (Glycine max). [14C]sucrose uptake assays demonstrate that GmSUT1 has a K(m) of 5.6mM and a V(max) of 5.8 nmol sucrose min(-1)(mg cells)(-1), which are similar to those of the low-affinity-high-capacity sucrose transporter family. GmSUT1 protein accumulates gradually during cotyledon development, correlating with increasing sucrose levels in the maturing cotyledons. Collectively, these data suggest that GmSUT1 plays an active role in the movement of sucrose into the developing seeds.     

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

Background

Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results

In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion

A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.     

Cross-species amplification of primers developed from Plantago major and P. intermedia in two Hawaiian Plantago species from the section Plantago

Thirty-nine primers, developed from the sister species Plantago major and P. intermedia, were tested in two Hawaiian Plantago species from the section Plantago. Eight primers were polymorphic, of which three were published earlier, and five are new ones presented here. Amplification and polymorphism levels appeared to be high in these Hawaiian species. These markers will be valuable for further mating system and evolutionary studies in species from the section Plantago that are closely related to P. major and P. intermedia.     

Erythrocyte/HepG2-type glucose transporter is concentrated in cells of blood-tissue barriers

In search of possible diverse roles of glucose transporters (GT's), we examined whether any GT's are present in blood-tissue barriers where selective flow of glucose from blood to tissue cells is critically important. We found in rat that the erythrocyte/HepG2-type GT is localized in all the limiting plasma membranes known to serve as blood-tissue barriers, whether the barriers are endothelial type (brain, iris, inner retina, peripheral nerve) or epithelial type (choroid plexus, ciliary body, outer retina, peripheral nerve, placenta), except for plasma membranes in testis and thymus where no appreciable amount of the GT was found. The erythrocyte/HepG2-type GT may play a vital role for the entry of glucose into these firmly guarded tissues.     

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.     

Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues

The Arabidopsis AtSUC3 gene encodes a sucrose (Suc) transporter that differs in size and intron number from all other Arabidopsis Suc transport proteins. Each plant species analyzed so far possesses one transporter of this special type, and several functions have been discussed for these proteins, including the catalysis of transmembrane Suc transport, and also Suc sensing and regulation of other Suc transporters. Here, we show that the AtSUC3 protein is localized in the sieve elements of the Arabidopsis phloem and is not colocalized with the companion cell-specific AtSUC2 phloem loader. Even stronger AtSUC3 expression is observed in numerous sink cells and tissues, such as guard cells, trichomes, germinating pollen, root tips, the developing seed coat, or stipules. Moreover, AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissue. The physiological role of AtSUC3 in these different cells and tissues is discussed.     

Differential regulation of sorbitol and sucrose loading into the phloem of Plantago major in response to salt stress

Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major.     

Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus

Sucrose transporters in the SUT family are important for phloem loading and sucrose uptake into sink tissues. The recent localization of type III SUTs AtSUT4 and HvSUT2 to the vacuole membrane suggests that SUTs also function in vacuolar sucrose transport. The transport mechanism of type III SUTs has not been analyzed in detail. LjSUT4, a type III sucrose transporter homolog from Lotus japonicus, is expressed in nodules and its transport activity has not been previously investigated. In this report, LjSUT4 was expressed in Xenopus oocytes and its transport activity assayed by two-electrode voltage clamping. LjSUT4 transported a range of glucosides including sucrose, salicin, helicin, maltose, sucralose and both alpha- and beta-linked synthetic phenyl glucosides. In contrast to other sucrose transporters, LjSUT4 did not transport the plant glucosides arbutin, fraxin and esculin. LjSUT4 showed a low affinity for sucrose (K (0.5) = 16 mM at pH 5.3). In addition to inward currents induced by sucrose, other evidence also indicated that LjSUT4 is a proton-coupled symporter: (14)C-sucrose uptake into LjSUT4-expressing oocytes was inhibited by CCCP and sucrose induced membrane depolarization in LjSUT4-expressing oocytes. A GFP-fusion of LjSUT4 localized to the vacuole membrane in Arabidopsis thaliana and in the roots and nodules of Medicago truncatula. Based on these results we propose that LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole.