Cell-density-dependent Changes in the Metabolism of Chloronema Cell Cultures: I. Relationship between Cell Density and Enzymic Activities

In the growing chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw., activities of several enzymes have been found to be cell-density-dependent. Cyclic nucleotide phosphodiesterase (cNPDE), nitrate reductase (NR), and protein kinase showed highest activity at a low cell density (1 to 2 milligrams per milliliter) while indoleacetic acid (IAA) oxidase and peroxidase were highest at a high cell density (>10 milligrams per milliliter). 3′-Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.     

Parameters influencing the liposome-mediated insertion of fluorescein diacetate into plant protoplasts

Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 × 10⁶ protoplasts per milliliter were incubated with 2.4 × 10⁷ liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 × 10⁵ protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions.     

Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[³⁵S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[³⁵S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

Structural Development during Germination of Different Populations of Mitochondria from Pea Cotyledons

The crude mitochondrial fraction from pea cotyledons can, from days 1 to 7 of germination, be separated into three fractions by sucrose density gradient centrifugation. When seeds were grown in water (control) or cycloheximide (120 micrograms per milliliter of medium) for 4 days, the originally different populations of mitochondria acquired a uniform density and separated together in band 1 (density, 1.205 grams per milliliter). The oxidative and phosphorylative activities of mitochondria obtained from 4-day-old control and 4-day-old cycloheximide-treated pea seeds were the same. However, mitochondria from pea seeds that were grown in d-threo-chloramphenicol (1.5 milligrams per milliliter of medium) or erythromycin (0.5 milligram per milliliter of medium) for 4 days separate into three bands (fully developed mitochondria in the top band [band 1] and partially developed mitochondria in the lower two bands [bands 2 and 3]). Separation patterns and oxidative and phosphorylative activities were the same for mitochondria separated from 4-day-old cotyledons treated with d-threo-chloramphenicol or erythromycin and from 1-day-old cotyledons grown in water. This indicated that these inhibitors prevented the partially developed mitochondria originally in bands 2 and 3 from developing further. In contrast, cycloheximide did not seem to interfere with the mitochondrial structural development. These results along with those obtained from the experiments on the effects of d-threo-chloramphenicol, erthromycin, and cycloheximide on ¹⁴C-leucine incorporation into mitochondrial membrane proteins suggest that the increase in mitochondrial activity during germination may be a result of structural development (membrane synthesis) in pre-existing mitochondria.     

THE LABELING OF CULTURED CELLS OF ACER WITH [14C]PROLINE AND ITS SIGNIFICANCE

The distribution of the radioactivity from [¹⁴C]proline that is bound in cultured cells of Acer has been determined by electron microscope autoradiography. In this way proline may be related to the cell wall as a morphological entity rather than as a fraction in a biochemical separation of a heterogeneous crop of cells. The cells in culture may vary greatly. Some are active growing, turgid cells, with thin protoplasts tightly pressed against their walls; in others the protoplasts may spontaneously withdraw from the wall; in still others the protoplasts disorganize, and walls thicken and become sculptured as the cells differentiate and even senesce. Different culturing practices may affect the status of the cells, and this, in turn, affects the distribution of radioactivity from proline in the cells. Cells which are actively growing, turgid, and nucleated have the highest grain density in their protoplasts and nuclei; as the protoplasts of such cells withdraw from their walls, they retain the bulk of the radioactivity. On the other hand, in cells which have thickened walls and sparse protoplast contents, the radioactivity is accumulated in their walls. A high content of proline and hydroxyproline-rich protein is, therefore, not a necessary or invariable feature of the cell walls of cultured Acer cells but depends on the state of development of these cells.     

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.     

Occurrence and biosynthesis of auxin in protonema of the moss Funaria hygrometrica

We have investigated the presence of auxin and the ability of chloronema cells to synthesize indole-3-acetic acid (IAA) in axenic protonema cell cultures of the moss Funaria hygrometrica. The endogenous level of auxin activity was 4 and 7μg-IAA equivalents/kg in caulonema and chloronema cell types, respectively. Based on an indole-α-pyrone fluorometric assay, the level of putative IAA was observed to be 5.0 and 1.9.μg/kg in caulonema and chloronema cells, respectively. [³H]Tryptophan was metabolized into IAA via the indole-pyruvate pathway by intact chloronema cells and also by the cell free homogenates. More [³H]IAA accumulated when homogenates from cells pre-grown at low cell densities (< 0.5 mg/ml) as compared to those at high cell densities ( > 0.5 mg/ml) were used. Since the activities of peroxidase and IAA-oxidase are known to be high at high cell densities, the lack of accumulation of radioactivity in IAA at high densities can be attributed to a high level of IAA-oxidizing enzymes. Our results suggest a possible relationship between IAA accumulation and caulonema differentiation.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 × 10⁻⁵ molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23°C) for 7 days acclimated to −28°C (LT₅₀) when treated with ABA, or to −5°C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H₂O) or as grams water per gram dry weight (gram H₂O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H₂O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H₂O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H₂O/10⁶ cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10⁶ cells increased to 3.3 milligram/10⁶ cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10⁶ cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur.     

Subcellular localization of proteases in wheat and corn mesophyll protoplasts

Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that “differentiated” between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O₂ per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O₂ per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for “marker” enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Malate and Dihydroxyacetone Phosphate-dependent Nitrate Reduction in Spinach Leaf Protoplasts

Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO₃⁻. A similar stimulation of nitrate reduction in the absence of CO₂ fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light-independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO₂ fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship.     

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⁺ (⁸⁶Rb), ³⁶Cl⁻, H₂³²PO₄⁻ and ⁴⁵Ca²⁺ 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 H₂PO₄⁻ 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 N₂ gas. In contrast, Ca²⁺ 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 H₂PO₄⁻ 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 H₂PO₄⁻. 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.     

Mechanism of naphthaleneacetic Acid conjugation: no effect of ethylene

Formation of naphthaleneacetic acid-glucose (NAGLu) in detached leaves, floating on α-naphthaleneacetic acid-1-¹⁴C (NAA, 0.05 microcurie per milliliter, 3.1 μm)-buffer solution (phosphate-citrate, pH 4.2) began immediately while there was a 2- to 4-hour lag before NAA-asparatate (NAAsp) could be detected. Subsequent increase in the NAAsp conjugate reflected a decrease in free NAA to 1 to 2% of the total radioactivity taken up. Pretreatment with 31 μm¹²C-NAA for 18 hours doubled NAAsp formation after transfer for 4 hours to ¹⁴C-NAA. Pretreatment with ethylene, as ethephon (up to 400 milligrams per liter) or ethylene gas (10 microliters per liter), did not induce NAAsp formation. In the presence of NAA, ethylene had no effect on NAA conjugation. Similarly, CO₂ (5%) did not modify the formation of the conjugates. Rhizobitoxine (1.87 μm) inhibited NAA-induced ethylene production but did not prevent NAA-induced formation of NAAsp. We concluded that the conjugation of NAA with aspartic acid is not mediated by ethylene.     

Endosperm protein of wheat seed as a determinant of seedling growth

Seed of a Mexican semidwarf wheat (Triticum aestivum L. cv. Inia 66), was obtained from a nitrogen fertilizer field trial grown in Mexico. A high positive correlation was obtained between seed protein content and seedling dry weight after 3 weeks growth (r = +0.92^**). The seedling dry weight was positively related to the protein content of the aleurone layer and endosperm, but not to the embryo. Small, 35 milligrams, high protein seeds (4.7 milligrams protein per seed) produced larger seedlings than large, 45 milligrams, low protein seeds (4.3 milligram protein per seed). There was no difference in the weight or protein content of embryos from low and high protein seeds and their growth was similar. Composite seeds of the two protein levels were produced by transferring embryos from one endosperm type to the other. After 4 weeks, there was no difference between the different embryo types grown on the same endosperm type. High protein endosperm produced more vigorous seedlings regardless of the embryo type grown on it, indicating that the factor(s) responsible for the greater growth of high protein seed is in the endosperm.     

Sexual Interaction in Heterothallic Strains of Closterium peracerosum-strigosum-littorale: Partial Characterization of a Male-Specific Sexual Pheromone, Protoplast-Releasing Substance

A sexual pheromone, named the protoplast releasing substance (PRS), was formed by mating type minus cells. PRS activates mating type plus cells and results in the release and fusion of protoplasts within distended conjugation-papilla in paired cells and in the release and disruption of protoplasts in unpaired mating type plus cells. In an agar barrier system, the activation of mating type plus cells was markedly inhibited by treatment with pronase (5-10 micrograms per milliliter), proteinase (100 micrograms per milliliter), and α-mannosidase (10 micrograms per milliliter). Trypsin (10-100 micrograms per milliliter) had no effect on the activation in the agar barrier system. The results suggest that PRS is a glycoprotein with pronase-sensitive and trypsin-insensitive structure.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.