AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis

This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H(+)/monosaccharide transporter family. The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices. Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K(m) = 20 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes. Analyses of AtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6 expression only during the late stages of pollen development. A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6 mutation on pollen viability, pollen germination, fertilization, and seed production. However, differences between wild-type and mutant plants could not be observed.     

Utilization and transport of glucose in Olea Europaea cell suspensions

Cell suspensions of Olea europaea var. Galega Vulgar grown in batch culture with 0.5% (w/v) glucose were able to transport D-[(14)C]glucose according to Michaelis-Menten kinetics associated with a first-order kinetics. The monosaccharide carrier exhibited high affinity (K(m) approximately 50 micro M) and was able to transport D-glucose, D-fructose, D-galactose, D-xylose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not D-arabinose, D-mannitol or L-glucose. D-[(14)C]glucose uptake was associated with proton uptake, which also followed Michaelis-Menten kinetics. The transport of 3-O-methyl-D-glucose was accumulative (40-fold, at pH 5.0) and the protonophore carbonyl cyanide m-chlorophenylhydrazone strongly inhibited sugar accumulation. The results were consistent with the involvement of a monosaccharide: proton symporter with a stoichiometry of 1 : 1. When cells were grown with 3% (w/v) glucose, the uptake of D-[(14)C]glucose followed first-order kinetics and monosaccharide:proton symporter activity was not detected. The value obtained for the permeability coefficient of hexoses in O. europaea cells supported the hypothesis that the first-order kinetics observed in 0.5% and 3% sugar-grown cells was produced exclusively by passive diffusion of the sugar. The results indicate that in O. europaea cells sugar levels have a regulatory effect on sugar transport, because the activity for monosaccharide transport was repressed by high sugar concentrations.     

A male gametophyte-specific monosaccharide transporter in Arabidopsis

The AtSTP2 gene (sugar transport protein 2) of Arabidopsis thaliana encodes a high affinity, low specificity monosaccharide carrier that can transport a number of hexoses and pentoses at similar rates. AtSTP2 has 12 putative transmembrane helices and a molecular mass of 55.0 kDa. AtSTP2 expression was localized in AtSTP2 promoter-beta-glucuronidase (GUS) Arabidopsis plants showing AtSTP2-driven GUS activity during pollen maturation and also in germinating pollen. Immunohistochemical studies with anti-AtSTP2 antiserum as well as RNA in situ hybridization analyses modified these results and showed that AtSTP2 expression is confined to the early stages of gametophyte development. Both AtSTP2 mRNA and AtSTP2 protein are first seen at the time of beginning callose degradation and microspore release from the tetrades. AtSTP2 mRNA and AtSTP2 protein are no longer detected after the mitotic divisions and the formation of the trinucleate gametophyte. No AtSTP2 mRNA or AtSTP2 protein is seen in fully developed or germinating pollen. The putative role of AtSTP2 in the uptake of glucose units resulting from callose degradation during pollen maturation is discussed.     

AtSTP3, a green leaf-specific, low affinity monosaccharide-H+ symporter of Arabidopsis thaliana

This paper describes the expression analyses of the AtSTP3 gene of Arabidopsis thaliana, the functional characterization of the encoded protein as a new monosaccharide transporter, and introduces the AtSTP gene family. The kinetic properties of the AtSTP3 protein (for sugar transport protein 3) were studied in a hexose transport deficient mutant of Schizosaccharomyces pombe. AtSTP3 represents a new monosaccharide transporter that is composed of 514 amino acids and has a calculated molecular mass of 55·9 kDa. Kinetic analyses in yeast showed that AtSTP3 is a low affinity, energy‐dependent H ⁺ symporter with a K_m for D ‐glucose of 2 m M . RNase protection analyses revealed that AtSTP3 is expressed in leaves and floral tissue of Arabidopsis. This expression pattern of the AtSTP3 gene was confirmed in AtSTP3 promoter‐ β ‐glucuronidase (GUS) plants showing AtSTP3‐driven GUS activity in green leaves, such as cotelydons, rosette and stalk leaves and sepals. Wounding caused an induction of GUS activity in the transgenic plants and an increase of AtSTP3 mRNA levels in Arabidopsis wild‐type plants. Polymerase chain reaction analyses with degenerate primers identified additional new AtSTP genes and revealed that AtSTP3 is the member of a large family of at least 14 homologous genes coding for putative monosaccharide‐H ⁺ symporters (AtSTPs).     

Roles of cell-wall invertases and monosaccharide transporters in the growth and development of Arabidopsis

The hydrolysis of sucrose by cell-wall invertases (cwINV) and the subsequent import of hexoses into target cells appears to be crucial for appropriate metabolism, growth and differentiation in plants. Hexose uptake from the apoplast is catalysed by monosaccharide/H+ symporters (Sugar Transport Proteins or STPs), which have the potential to sense sugars. Import of extracellular hexoses may generate signals to orchestrate cellular activities, or simply feed metabolic pathways distinct from those fed by sucrose. It is predicted that Arabidopsis has six cwINV genes and at least 14 STP genes. These genes show different spatial and temporal patterns of expression, and several knock-out mutants have been isolated for analysis. AtSTP1 transports glucose, galactose, xylose, and mannose, but not fructose. It accounts for the majority of the AtSTP activity in vegetative tissues and its activity is markedly repressed by treatment with exogenous sugars. These observations are consistent with a role in the retrieval of cell-wall-derived sugars, for example, during carbohydrate limitation or cell expansion. The AtSTP1 gene is also expressed in developing seeds, where it might be responsible for the uptake of glucose derived from imported sucrose. The large number of AtcwINV and AtSTP genes, together with complex patterns of expression for each, and the possibility that each protein may have more than one physiological function, provides the plant with the potential for a multiplicity of patterns of monosaccharide utilization to direct growth and differentiation or to respond flexibly to changing environmental conditions.     

Transport kinetics of 6-deoxy-D-glucose inCandida parapsilosis

The strictly aerobic yeastCandida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 °C. the accumulation ratio is not, The uptake is most active at pH 4.5–8.5. It appears not to involve stoichiometric proton symport. The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses. The apparent half-life of the transport system was 3.5–4 h.     

Enhancing effect of saccharides on the mutagenicity of 2-chloro-4-methylthiobutanoic acid

The mutagenicity of 2-chloro-4-methylthiobutanoic acid (CMBA), a nitrite-treated Sanma fish mutagen, in Salmonella typhimurium TA100 was enhanced by addition of D-glucose during the CMBA-treatment. Several other monosaccharides also enhanced the mutagenicity of CMBA, and the order of the enhancing potency was found to be D-mannose, D-glucose>D-fructose, D-ribose, D-galactose. A disaccharide, maltose, showed only little enhancement. No enhancement was found with L-glucose. We investigated whether saccharides affect uptake of [methyl-14C]CMBA into S. typhimurium TA100. Saccharides which enhanced CMBA-induced mutagenesis increased the uptake. L-Glucose did not enhance the uptake. There was good correlation between the enhanced mutagenesis and increased radioactivity in Salmonella, suggesting that the enhancing effect of monosaccharide on the CMBA-induced mutagenesis results from the enhanced uptake of the mutagen into bacteria.     

Diurnal and light-regulated expression of AtSTP1 in guard cells of Arabidopsis

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.     

Structural Specificity of Sugar Transport at the Blood-Nerve Barrier

Abstract: The major component of D-glucose transfer across the membranous sites of the blood-nerve barrier (BNB) occurs via a facilitative mechanism at a rate greater than twice the rate of D-glucose metabolism by nerve. To characterize further properties of monosaccharide transport at the BNB, unidirectional transfer constant (K) values were determined in vivo in tibial nerve of anesthetized rats for radiolabeled mannitol, L-glucose, and a series of D-glucose analogs. K values (× 10⁻⁴ ml s⁻¹ g^−l) equaled 4.8 for 2-deoxy-D-glucose, 3.7 for D-glucose, 2.3 for 3- O -methyl-D-glucose, 1.4 for D-man-nose, 0.6 for D-galactose, 0.2 for mannitol, and 0.19 for L-glucose. The rank order of ratios between K values of a D-hexose and D-glucose, which reflects the rank order of affinity of the system for individual sugars, was 2-deoxy-D-glucose > D-glucose > 3-O-methyl-D-glucose > D-mannose > D-galactose. The results demonstrate that the order of substrate affinity of the monosaccharide carrier at the BNB is similar to that at cerebral capillaries and at erythrocytes. At normal concentrations of plasma D-glucose, the contribution of simple passive diffusion to unidirectional D-glucose influx across the BNB equals 5%, which is greater than that at cerebral capillaries and reflects the greater permeability to hydrophilic nonelectrolytes of the endoneurial vasculature.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.     

三齿草藤(Vicia bungei Ohwi)凝集素糖组分的高效液相色谱分析

本文报道经亲和层析纯化的三齿草藤凝集素(VBL)的糖含量和糖组分的测定结果。经酚-硫酸法测得VBL的总糖含量为4.7％。应用高效液相色谱法对一系列已知标准单糖的定性定量分析条件进行了探索,选用乙腈-水-甲醇=60:30:5体系作流动相,YWG-NH_2作固定相,在高效液相色谱仪中测出VBL含有核糖和鼠李糖,二者摩尔数之比为9.4:1。     

Specificity of the tyrosine-phenylalanine transport system in Bacillus subtilis

l-Tyrosine and l-phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l-tyrosine or l-phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l-tyrosine or l-phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l-phenylalanine and l-tyrosine is strictly competitive. Neither l-tyrosine nor l-phenylalanine uptake was competitively inhibited by other naturally occurring l-amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l-tyrosine and l-phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l-tyrosine and l-phenylalanine to inhibit the uptake of l-[(14)C]tyrosine and l-[(14)C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the alpha-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged alpha-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l-tryptophan and l-histidine, do not compete with l-tyrosine and l-phenylalanine for uptake. The presence of the p-hydroxy substitutent in the side chain (as in l-tyrosine) enhances the uptake of the aryl amino acid analogues investigated.     

Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent K(m) of 2.8 mM and a V(max) of 260 nmol/min per mg protein. Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.     

Reduced Na+ uptake in the NaCl-hypersensitive sos1 mutant of Arabidopsis thaliana.

Sos1 is an Arabidopsis thaliana mutant with > 20 times higher sensitivity toward Na+ inhibition due to a defective high-affinity potassium-uptake system. We report here that sos1 accumulates less Na+ than the wild type in response to NaCl stress. The Na+ contents in sos1 seedlings exposed to 25 mM NaCl for 2 or more d are about 43% lower than those in the wild type. When assayed at 20 mM external NaCl, sos1 seedlings pretreated with low potassium have 32% lower Na+ uptake than the wild type. However, little difference in Na+ uptake could be measured when the seedlings were not pretreated with low potassium. Low-potassium treatment was shown to induce high-affinity potassium-uptake activity in Arabidopsis seedlings. No substantial difference in Na+ efflux between sos1 and the wild type was detected. The results show that the reduced Na+ accumulation in sos1 is due to a lower Na+ influx rate. Therefore, the sos1 mutation appears to disrupt low-affinity Na+ uptake in addition to its impairment of high-affinity K+ uptake.     

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.     

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.