An amino-acid-grown maize cell line for use in investigating nitrate assimilation.

Studies on uptake and assimilation of nitrate in plants are confounded by differences in cell function associated with anatomical features of roots as well as by problems inherent with growing plants without nitrate. To circumvent these problems, a Zea mays L. embryo cell line was grown in suspension culture using an amino-acid-based medium consisting of a Murashige and Skoog medium in which ammonium and nitrate were replaced by aspartic acid (100 mg/l), glycine (100 mg/l), arginine (150 mg/l), and glutamine (1 g/l). The growth, cellular characteristics, and physical appearance of the amino-acid-grown cells were similar to cells grown in the presence of nitrate. The amino-acid-grown cells exhibited the expected induction pattern and inhibitor sensitivity of nitrate uptake. This cell line should facilitate research on the induction of nitrate uptake and the regulation of nitrate assimilation into proteins.

Contamination of Ammonium-Based Nutrient Solutions by Nitrifying Organisms and the Conversion of Ammonium to Nitrate.

Conversion of ammonium to nitrate and contamination by nitrifying organisms are often assumed not to be significant in ammonium-based nutrient solutions. To assess this assumption, maize (Zea mays) and pea (Pisum sativum) were grown under greenhouse conditions in aeroponic, hydroponic, and sand-culture systems containing 2 mM ammonium chloride as the sole nitrogen source and evaluated for the activity of contaminating nitrifying organisms. In all three culture systems, root colonization by nitrifying organisms was detected within 5 d, and nitrate was detected in the nutrient solution within 10 d after seedling transfer. In sand culture, solution nitrate concentration reached 0.35 mM by the end of the 17-d experiment. Consistent with the microbial ammonium oxidation sequence, nitrite was detected earlier than nitrate and remained at lower levels throughout the experiment. Nitrate was found in significant quantities in root and shoot tissues from seedlings grown in ammonium-based nutrient solutions in all of the solution culture systems. Maize seedlings grown in an ammonium-based hydroponic system contained nitrate concentrations at 40% of that found in plants grown in nitrate-based solution. Determination of nitrate (or nitrite) levels in the nutrient solution was the weakest indicator of the activity of nitrifying organisms. A bioassay for the presence of nitrifying organisms in combination with tissue analysis for nitrate was a better indicator of microbial conversion of ammonium to nitrate in nutrient solution culture. The results have implications for the use of ammonium-based nutrient solutions to obtain plants suitable for research on induction of nitrate uptake and reduction or for research using solution culture to compare ammonium versus nitrate fertilization.

Correlated induction of nitrate uptake and membrane polypeptides in corn roots.

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the (35)S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.

Nitrate absorption by corn roots : inhibition by phenylglyoxal.

Nitrate transport in excised corn (Zea mays L.) roots was inhibited by phenylglyoxal, but not by 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) or fluorescein isothiocyanate (FITC). Inhibition of nitrate uptake by a 1-hour treatment with 1 millimolar phenylglyoxal was reversed after 3 hours, which was similar to the time needed for induction of nitrate uptake. If induction of nitrate uptake occurs by de novo synthesis of a nitrate carrier, then the resumption of nitrate uptake in the inhibitor-treated roots may occur because of turnover of phenylglyoxal-inactivated nitrate carrier proteins. All three chemicals inhibited chloride uptake to varying degrees, with FITC being the strongest inhibitor. While inhibition due to DIDS was reversible within 30 minutes, both FITC and phenylglyoxal showed continued inhibition of chloride uptake for up to 3 hours after removal from the uptake solution. Assuming that the anion transporter polypeptide(s) carries a positive charge density at or near the transport site, the results indicate that the nitrate carrier does not carry any lysyl residues that are accessible to DIDS or FITC, whereas the chloride carrier does. Both chloride and nitrate carriers, however, seem to possess arginyl residues that are accessible to phenylglyoxal.

Phenol-acetic acid-urea polyacrylamide gel electrophoresis of membrane proteins.

A phenol-acetic acid-urea polyacrylamide gel electrophoretic system (PAU-PAGE) was simplified by adaptation to a slab gel format, allowing the simultaneous comparison of up to 12 samples. The system fractionated most proteins according to molecular mass, although chemical reduction was required since certain proteins (e.g., bovine serum albumin) showed reduction-dependent shifts in mobility. Sodium dodecyl sulfate-PAGE of partially purified membrane proteins can be adversely affected by protein aggregation and proteolysis. PAU-PAGE, which solubilized aqueous insoluble proteins and rapidly inactivated proteases, was useful for assessing the polypeptide composition of plasma membrane preparations.

Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots.

Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.

A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions.

Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.

Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation.

Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).

Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation.

A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.

Partial characterization of a phosphorylated intermediate associated with the plasma membrane ATPase of corn roots.

The phosphorylated protein associated with a deoxycholate-extracted plasma membrane fraction from corn (Zea mays L. var WF9 x Mol7) roots was characterized in order to correlate its properties with those of plasma membrane ATPase. Its phosphorylation, like that of plasma membrane ATPase, was dependent on Mg(2+), substrate specific for ATP, insensitive to azide, oligomycin, or molybdate, and sensitive to N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, or vanadate. Monovalent cations affected the phosphorylation of the protein in a manner consistent with their stimulatory effects on ATPase. For K(+), this was shown to occur through an increase in the turnover of the phosphoenzyme. Analysis of the phosphorylated protein by NaDodSO(4)/polyacrylamide gel electrophoresis revealed the presence of a single labeled polypeptide with a molecular weight of about 100,000. Phosphorylation of this polypeptide was dependent on Mg(2+), sensitive to K(+), and inhibited by vanadate. It is concluded that this polypeptide represents the catalytic subunit of the plasma membrane ATPase. These results are discussed in terms of a model for the coupling of metabolic energy to H(+) and K(+) transport in higher plants.

Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots.

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K(+). K(+)-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K(+) transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

Isolation and transport properties of protoplasts from cortical cells of corn roots.

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 x MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl(2), 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 x 10(3)/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.Cortical cell protoplasts exhibited energy-dependent uptake of K(+) ((86)Rb), H(2) (32)PO(4) (-), and (36)Cl(-) as well as net H(+) extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K(+) was highest between pH 7.5 and 8.0, whereas the influx of H(2)PO(4) (-) was greatest between pH 4.0 and 5.0. K(+) and H(2)PO(4) (-) influx and net H(+) efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl(-) was low, but not greatly different from that observed for other plant cells. K(+) flux was somewhat high, probably because the K(+) concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.

Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots.

The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 x Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K(+)-stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K(+)-ATPase.

Membrane-mediated decrease in root exudation responsible for phorphorus inhibition of vesicular-arbuscular mycorrhiza formation.

The mechanism responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation in sudangrass (Sorghum vulgare Pers.) was investigated in a phosphorus-deficient sandy soil (0.5 micrograms phosphorus per gram soil) amended with increasing levels of phosphorus as superphosphate (0, 28, 56, 228 micrograms per gram soil). The root phosphorus content of 4-week-old plants was correlated with the amount of phosphorus added to the soil. Root exudation of amino acids and reducing sugars was greater for plants grown in phosphorus-deficient soil than for those grown in the phosphorus-treated soils. The increase in exudation corresponded with changes in membrane permeability of phosphorus-deficient roots, as measured by K(+) ((86)Rb) efflux, rather than with changes in root content of reducing sugars and amino acids. The roots of phosphorus-deficient plants inoculated at 4 weeks with Glomus fasciculatus were 88% infected after 9 weeks as compared to less than 25% infection in phosphorus-sufficient roots; these differences were correlated with root exudation at the time of inoculation. For plants grown in phosphorus-deficient soil, infection by vesicular-arbuscular mycorrhizae increased root phosphorus which resulted in a decrease in root membrane permeability and exudation compared to nonmycorrhizal plants. It is proposed that, under low phosphorus nutrition, increased root membrane permeability leads to net loss of metabolites at sufficient levels to sustain the germination and growth of the mycorrhizal fungus during pre- and postinfection. Subsequently, mycorrhizal infection leads to improvement of root phosphorus nutrition and a reduction in membrane-mediated loss of root metabolites.

Isolation of tonoplast vesicles from tobacco protoplasts.

Vacuoles were isolated from protoplasts of Nicotiana glutinosa by the method of Mettler and Leonard (Plant Physiol 1979 64: 1114-1120) with minor modifications so that the number of intact protoplasts contaminating the vacuole preparation was reduced to less than 1% (by number). Isopycnic centrifugation of a [(3)H]choline-labeled, sonicated vacuole preparation on linear 5 to 40% sucrose gradients indicated that tonoplast vesicles equilibrated at a density of about 1.12 grams per cubic centimeter. When tonoplast vesicles were isolated on discontinuous sucrose density gradients substrate specific ATPase activity was not found to be associated with this membrane fraction. These results are discussed in terms of the energetics of ion transport through the tonoplast membrane.

Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction.

The K(+)-stimulated ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 x Mo 17) by solubilization with 30 millimolar octyl-beta-d-glucopyranoside followed by precipitation with dilute ammonium sulfate. The specific activity of the enzyme was increased about five times by this procedure. The molecular weight of the detergent-extracted ATPase complex was estimated to be at least 500,000 daltons by chromatography on a Bio-Gel A-5m column. Negative staining electron microscopy indicated that the detergent-extracted material consisted of amorphous particles, while the ammonium sulfate precipitate was composed of uniform vesicles with an average diameter of 100 nanometers. The protein composition of the ammonium sulfate precipitate was significantly different from that of the plasma membrane fraction when compared by sodium dodecyl sulfate gel electrophoresis. The characteristics of the partially purified ATPase resembled those of the plasma membrane associated enzyme. The ATPase required Mg(2+), was further stimulated by K(+), was almost completely inhibited by 0.1 millimolar diethylstilbestrol, and was not affected by 5.0 micrograms per milliliter oligomycin. Although the detergents sodium cholate, deoxycholate, Triton X-100 and Lubrol WX also solubilized some membrane protein, none solubilized the K(+)-stimulated ATPase activity. Low concentrations of each detergent, including octyl-beta-d-glucopyranoside, activated the ATPase and higher concentrations inactivated the enzyme. These results suggest that the plasma membrane ATPase is a large, integral membrane protein or protein complex that requires lipids to maintain its activity.

Isolation and partial characterization of vacuoles from tobacco protoplasts.

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5'-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential.

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (+/- standard deviation) for unplasmolyzed tobacco cells was -52 +/- 12 millivolts, for cells in 0.3 molar mannitol, -50 +/- 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, -49 +/- 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was -49 +/- 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics.

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K(+) ((86)Rb), (36)Cl(-), H(2) (32)PO(4) (-) and (45)Ca(2+) from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K(+), Cl(-), and H(2)PO(4) (-) measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N(2) gas. In contrast, Ca(2+) influx was not tightly coupled to respiratory energy production. The influx of K(+) was highest between pH 6.5 and 7.5 whereas the influx of H(2)PO(4) (-) and Cl(-) was greatest between pH 4.5 and 5.5. Influx of K(+) and Cl(-) was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K(+) influx by more than 200% but had no effect on the influx of either Cl(-) or H(2)PO(4) (-). Apparent H(+) efflux, as measured by decrease in solution pH, was enhanced by K(+), stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.