Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

Dissociation and Reassembly of the Vacuolar H-ATPase Complex from Oat Roots

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H⁺-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H⁺-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg²⁺ and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H⁺-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO₃, or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO₃ > KBr > K-acetate > K₂SO₄ > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H⁺-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H⁺ pumping as seen from the recovery of H⁺ transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H⁺-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H⁺-ATPase to form a functional H⁺ pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H⁺-ATPase in vivo.     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots

The sensitivity of the two forms of nitrate reductase, NR_I and NR_II, obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR_II was less sensitive to inactivation than NR_I. When NR_I and NR_II were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R_m value of 0.32 to 0.61 for NR_I and from an R_m of 0.28 to 0.60 for NR_II. For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR_I yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

Effect of Phloem-Translocated Malate on NO(3) Uptake by Roots of Intact Soybean Plants

In soybean (Glycine max L. Merr. cv Kingsoy), NO₃⁻ assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO₃⁻ uptake by roots. The net NO₃⁻ uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO₃⁻ uptake and C excretion rates by roots. These results suggest that NO₃⁻ uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO₃⁻ uptake, and excretion of HCO₃⁻ ions was probably released by malate decarboxylation. NO₃⁻ uptake rate increased when the supply of NO₃⁻ to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO₄²⁻. We conclude that in situ, NO₃⁻ reduction rate in the shoot may control NO₃⁻ uptake rate in the roots via the translocation rate of malate in the phloem.     

Intracellular pH Regulation during NO(3) Assimilation in Shoot and Roots of Ricinus communis

Ricinus communis L. was used to test the Dijkshoorn-Ben Zioni hypothesis that NO₃⁻ uptake by roots is regulated by NO₃⁻ assimilation in the shoot. The fate of the electronegative charge arising from total assimilated NO₃⁻ (and SO₄²⁻) was followed in its distribution between organic anion accumulation and HCO₃⁻ excretion into the nutrient solution. In plants adequately supplied with NO₃⁻, HCO₃⁻ excretion accounted for about 47% of the anion charge, reflecting an excess nutrient anion over cation uptake. In vivo nitrate reductase assays revealed that the roots represented the site of about 44% of the total NO₃⁻ reduction in the plants. To trace vascular transport of ionic and nitrogenous constituents within the plant, the composition of both xylem and phloem saps was thoroughly investigated. Detailed dry tissue and sap analyses revealed that only between 19 and 24% of the HCO₃⁻ excretion could be accounted for from oxidative decarboxylation of shoot-borne organic anions produced in the NO₃⁻ reduction process. The results obtained in this investigation may be interpreted as providing direct evidence for a minor importance of phloem transport of cation-organate for the regulation of intracellular pH and electroneutrality, thus practically eliminating the necessity for the Dijkshoorn-Ben Zioni recycling process.     

Characterization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.)

The peripheral root cap cells of corn (cv. SX-17A) secrete a fucose-rich, high molecular weight, polysaccharide slime via the dictyosome pathway. To study the synthesis of this polysaccharide, a technique for isolating and assaying GDP-fucose:polysaccharide fucosyl transferase activity was developed. Corn roots were excised from germinated seeds, incubated 12 hours at 10 C in water, and ground in 100 millimolar Tris or Pipes buffer (pH 7.0) with or without 0.5 molar sucrose. The membrane-bound enzyme was solubilized by sonication in the presence of 2 molar urea and 1.5% (v/v) Triton X-100 and assayed by monitoring the incorporation of GDP-[¹⁴C]fucose into endogenous acceptors. Optimum enzyme activity is expressed at pH 7.0 and 30 C in the presence of 0.8% (v/v) Triton X-100. The enzyme does not require divalent cations for activation and is inhibited by concentrations of MnCl₂ or MgCl₂ greater than 1 millimolar. Corn root cap slime will serve as an exogenous acceptor for the enzyme if it is first hydrolyzed in 5 millimolar trifluoroacetic acid for 60 minutes at 18 pounds per square inch, 121 C. This procedure prepares the acceptor by removing terminal fucose residues from the slime molecule. Kinetics of fucose release during hydrolysis of native slime and in vitro synthesized product suggests that the two polymers possess similar linkages to fucose.     

Regulation of NO(3) Influx in Barley : Studies Using NO(3)

Short-term (10 minutes) measurements of plasmalemma NO₃⁻ influx (_oc) into roots of intact barley plants were obtained using ¹³NO₃⁻. In plants grown for 4 days at various NO₃⁻ levels (0.1, 0.2, 0.5 millimolar), _oc was found to be independent of the level of NO₃⁻ pretreatment. Similarly, pretreatment with Cl⁻ had no effect upon plasmalemma ¹³NO₃⁻ influx. Plants grown in the complete absence of ¹³NO₃⁻ (in CaSO₄ solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO₃⁻. Based upon the documented effects of NO₃⁻ or Cl⁻ pretreatments on net uptake of NO₃⁻, these observations suggest that negative feedback from vacuolar NO₃⁻ and/or Cl⁻ acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl⁻ was without effect upon ¹³NO₃⁻ influx, but NH₄⁺ caused approximately 50% reduction of influx at this concentration.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Characterization of the interaction between the plasma membrane H-ATPase of Arabidopsis thaliana and a novel interactor (PPI1)

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H(+)-ATPase (EC 3.6.3.6). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H(+)-ATPase. PPI1-induced stimulation of H(+)-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H(+)-ATPase. These results indicate that PPI1 can bind the H(+)-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action.     

Proteome Analysis of Roots in Cucumber Seedlings Under Iso-Osmotic NaCl and Ca(NO3)2 Stresses

The growth and productivity of cucumber are severely affected by salinity. To understand the complex salt response mechanism, the physiological and biochemical responses of cucumber seedlings to iso-osmotic NaCl and Ca(NO₃)₂ stresses were investigated. In this study, the biomass was significantly decreased under iso-osmotic NaCl and Ca(NO₃)₂ stresses, and the inhibitory effect of Ca(NO₃)₂ stress was less than that of NaCl stress. The soluble protein contents were increased under Ca(NO₃)₂ stress, whereas they were decreased after 6 days of NaCl stress. A sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that there were 14 differentially expressed protein bands in roots under iso-osmotic NaCl and Ca(NO₃)₂ stresses at 0, 3, 6, and 9 days, and seven protein bands were little expressed under NaCl stress at 6 and 9 days. Based on these results, 2-D gel electrophoresis was used to separate cucumber root proteins in response to iso-osmotic NaCl and Ca(NO₃)₂ stresses at 3 days. A total of 43 protein spots changed under salt stress. Of these proteins, 33 were successfully identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS) and categorized into classes, including those corresponding to antioxidants and defense-related proteins and energy and metabolism. The functions of the significantly differentially expressed root proteins were analyzed, which may facilitate a better understanding of different salt response mechanisms, and we suggest that cucumber seedlings showed a more powerful ability to resist Ca(NO₃)₂ stress.

     

Isolation and Characterization of a Boron-Polysaccharide Complex from Radish Roots

A boron-polysaccharide complex was purified from a Driselasedigest of cell walls of radish roots. The complex had a molecularweight of 7.5 KDa and contained boron (0.232%, w/w), uronicacid (52.3%, w/w) and neutral sugars (32.4%). ¹¹B-NMR spectroscopicanalysis suggested that the boron was present as a tetravalent1 : 2 borate-diol complex. ¹This work was supported by a Grant-in-Aid (no. 04660069) fromthe Ministry of Education, Science and Culture. ⁴Present address: Kasai Experimental Farm, Sumitomo ChemicalCo., Ltd. Kasai, Hyogo, 675-23 Japan.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Immunofluorescent Localization of Plasma Membrane H-ATPase in Barley Roots and Effects of K Nutrition

The plasma membrane H⁺-ATPase (PM-H⁺-ATPase) of barley (Hordeum vulgare L. cv Klondike) roots was assayed by cross-reaction on western blots and cryosections with an antibody against the PM-H⁺-ATPase from corn roots. Under conditions of reduced K availability, which have previously been shown to increase K influx by greater than 25-fold, there were only minor changes detected in PM-H⁺-ATPase levels. Antibody labeling of cryosections showed the relative distribution of PM-H⁺-ATPase among cell types in root tips and mature roots. Epidermal cells, both protoderm and mature root epidermis, including root hairs, had high levels of antibody binding. In mature roots, the stelar tissue showing the highest antibody binding was the companion cells of the phloem, followed by pericycle, xylem parenchyma, and endodermis.     

Method for Establishing a Bacterial Inoculum on Corn Roots

Few bacteria from the corn rhizosphere grew in media with 50 μg of mancozeb per ml. A mancozeb-resistant Pseudomonas strain from the rhizosphere was serially subcultured in media containing mancozeb and spectinomycin until it was resistant to 175 μg of mancozeb and 850 μg of spectinomycin per ml. The population of the pseudomonad added to soil fell to low numbers in 6 days in unamended or glucose-amended soil, but its numbers exceeded 10⁵/g for at least 12 days if the soil was supplemented with mancozeb. The numbers of this organism remained small on corn roots derived from untreated, inoculated seeds, but the population was two or more orders of magnitude greater on roots derived from mancozeb-coated seeds. The abundance of the inoculum strain on the 3-cm portion of roots nearest the stem declined markedly after about 1 week, but applying urea to the foliage reduced or prevented the decline. The numbers of the pseudomonad on segments of roots 3- to 6- and 6- to 9-cm from the stem were higher on plants derived from the mancozeb-coated seeds. Applying spectinomycin to the foliage did not promote growth of the bacterium. This method is proposed as a means to establish an introduced bacterium on plant roots.