Energy-dependent Loading of Amino Acids and Sucrose into the Phloem of Soybean

Radioactive sucrose, l-leucine, l-glutamate, and gamma-aminobutyrate were applied exogenously to abraded areas of soybean leaves. The three amino acids were translocated with similar velocities and mass transfer rates on a molar basis, although they were metabolized differently in the sink tissue. The concentration dependence of leucine translocation showed a triphasic saturation response, while sucrose translocation showed a biphasic saturation response to increasing concentration. Apparent K(m) and V(max) for leucine and sucrose loading in the phloem differed. Both leucine and sucrose translocation were inhibited by uncouplers, high K(+), and p-chloromercuribenzenesulfonic acid. Treatment with 0.8 m sorbitol had little effect on sucrose translocation but stimulated leucine translocation, indicating an apoplastic route of loading for leucine. No effect on mass transfer rates was observed when sucrose and amino acids were applied exogenously together. These data provide evidence that phloem loading of amino acids and sucrose is mediated by different and separate carriers, both being dependent on an energy-requiring mechanism.     

PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major

Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed.     

Involvement of Protons as a Substrate for the Sucrose Carrier during Phloem Loading in Vicia faba Leaves

The effect of pH on uptake of exogenous sucrose by broadbean (Vicia faba L.) leaf discs without the lower epidermis has been investigated at various sucrose concentrations. The concentration dependence of sucrose uptake showed a biphasic saturation response. At high sucrose concentrations (>20 millimolar), sucrose uptake showed no pH dependence. At low sugar concentrations (<5 millimolar), plots of 1/V against 1/H⁺ give straight lines which all intercept at the same point at the left of the ordinal axis. Calculations show that these data agree well with two-substrate kinetics for the carrier, the substrates being the protons and the sucrose molecules. Our results provide further evidence that protonation/deprotonation processes of the carrier are involved in phloem loading, especially for low sucrose concentrations of 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.     

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.     

Ultrastructure of Chloroplasts in Phloem Companion Cells and Mesophyll Cells as Related to the Stimulation of Sink Activity by Cytokinins

Changes in the chloroplast ultrastructure and starch and lipid content in the mesophyll and phloem companion cells of the phloem were studied after induction of source and sink functions in leaf tissues. A detached sugar-beet leaf, one half of which was treated with water (source part) and the other half of which was treated with 10^–4 M benzyladenine (BA) (acceptor part), was used as a model. After 65-h exposure to diffuse light, starch disappeared and lipid content increased in the source part of the leaf, with simultaneous disorganization of the chloroplast structure, which was most pronounced in the companion cells. Changeover from the source to sink function, induced by BA treatment, did not lead to marked destructive changes in the chloroplast structure of companion cells and resulted in the appearance of starch and in further increase in the level of lipids. Smaller amounts of starch also appeared in the mesophyll chloroplasts in the sink part of the leaf. We suppose that: (1) BA promotes the storage of assimilates, which are imported from the source part of the leaf to the companion cells, in the form of starch and lipids within chloroplasts; and this storage contributes to the maintenance of the sucrose concentration gradient in the conducting system between donor and sink parts of the leaf and, thus, activates metabolite inflow and (2) a barrier exists in the sink part of the leaf for assimilates destined to mesophyll cells, which restricts their export from the phloem.     

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes

Nutrients required for the growth of the embryo and endosperm of developing wheat (Triticum aestivum L.) grains are released into the endosperm cavity from the maternal tissues across the nucellar cell plasma membranes. We followed the uptake and efflux of sugars into and out of the nucellus by slicing grains longitudinally through the endosperm cavity to expose the nucellar surface to experimental solutions. Sucrose uptake and efflux are passive processes. Neither was sensitive to metabolic inhibitors, pH, or potassium concentration. p-Chloromercuribenzene sulfonate, however, strongly inhibited both uptake and efflux, although not equally. Except for p-chloromercuribenzene sensitivity, these characteristics of efflux and the insensitivity of Suc movement to turgor pressure are similar to those of sucrose release from maize pedicels, but they contrast with legume seed coats. Although the evidence is incomplete, movement appears to be carrier mediated rather than channel mediated. In vitro rates of sucrose efflux were similar to or somewhat less than in vivo rates, suggesting that transport across the nucellar cell membranes could be a factor in the control of assimilate import into the grain.     

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading

Oncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.     

A Preliminary Investigation into the Genecology of Plantago major L.

Differences between two types of Plantago major L. growing indifferent habitats were investigated. The lengths of petioleand lamina and the width of the lamina were measured; significantdifferences have been found between the types. When grown randomized for two generations in the greenhouse,these significant differences persisted. It was concluded thatthe two types differed in genotypic constitution.     

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.     

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.     

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion.     

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.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.