A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. II. Effects on uptake and efflux of IAA in maize roots

An explanation is sought for the inhibition of maize root growth and gravireaction brought about by treatment with 3,5-diiodo-4-hydroxybenzoic acid (DIHB). The effects of DIHB and 2,3,5-triiodobenzoic acid (TIBA) on the uptake and efflux of [³H]-indol-3yl-acetic acid (IAA) were tested using segments prepared from the elongation zone (2 to 7 mm region) of maize (Zea mays L. cv. LG11) roots. The uptake of [³H]-IAA (21 nM) by root segments incubated in buffered solutions (pH 5.0) was measured over a 5-min time-course. No significant effect of DIHB at 100 μM was observed, whereas TIBA at 10 μM slightly stimulated the uptake of [³H]-IAA. This experiment was repeated with the addition of non-radioactive IAA (total IAA concentration 1.0 μM). Up to 3 min DIHB (100 μM) had no significant effect, but thereafter a slight stimulation of IAA net uptake was observed. Treatment with TIBA (10 μM) stimulated the accumulation of IAA in the segments. The effects of DIHB (10, 50, 100 μM) and TIBA (10 and 50 μM) on the efflux of [³H]-IAA from segments that had been pretreated in [³H]-IAA (22 nM) were then tested. Treatment with DIHB or TIBA at pH 5.0 inhibited IAA efflux; the inhibition by TIBA was more marked than that produced by DIHB. This experiment was repeated using DIHB (10, 50, 100 μM) buffered at pH 6.0, and an inhibition of IAA efflux was again observed. Both DIHB (10 μM) and TIBA (10 μM) inhibited the binding of [³H]-NPA to a 5000–48000 g membrane fraction prepared from whole maize roots. The effects of the two substances were similar: 40% inhibition of specific binding by DIHB and 41% inhibition by TIBA. This indicates that DIHB, like TIBA, binds to the N-1-naphthyl-phthalamic acid-sensitive carrier for IAA efflux. It is concluded that DIHB, like TIBA, inhibits IAA transport at the level of efflux. The similarity between DIHB and TIBA as regards chemical structure and their inhibitory effects on IAA efflux and NPA binding strongly suggest that they act on the same carrier for IAA efflux across the plasmalemma.     

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Elucidation of an a and L system for amino acid transport in the human lymphoblast using a membrane filtration technique

Summary Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find α-AIB to be a poor competitor of alanine and phenylalanine uptake. This work was supported by Grant No. CA18644, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and from a grant from the National Science Foundation under Grant No. PCM 76-24328.     

Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na⁺. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl₂, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The K_m for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.     

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency

The effect of external inorganic nitrogen and K⁺ content on K⁺ uptake from low-K⁺ solutions and plasma membrane (PM) H⁺-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K⁺ and inorganic nitrogen as NO₃^-, NO₃^-/NH₄⁺ or NH₄⁺ and then starved of K⁺ for 24, 48 and 72 h. NH₄⁺ in full nutrient solution significantly affected the uptake efficiency and accumulation of K⁺, and this effect was less pronounced at the high K⁺ concentration. In contrast, the translocation rate of K⁺ to the shoot was not altered. Depletion assays showed that plants grown with NH₄⁺ more efficiently depleted the external K⁺ and reached higher initial rates of low-K⁺ uptake than plants grown with NO₃^-. One possible influence of K⁺ content of shoot, but not of roots, on K⁺ uptake was evidenced. Enhanced K⁺-uptake capacity was correlated with the induction of H⁺ extrusion by PM H⁺-ATPase. In plants grown in high K⁺ solutions, the increase in the active H⁺ gradient was associated with an increase of the PM H⁺-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K⁺, only the initial rate of H⁺-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H⁺-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO₃^--grown plants. The results suggest that K⁺ homeostasis in NH₄⁺-grown sorghum plants may be regulated by a high capacity for K⁺ uptake, which is dependent upon the H⁺-pumping activity of PM H⁺-ATPase.