N-1-naphthylphthalamic Acid Research Articles

Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue

Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10−5 mol·dm−3 monensin did not affect uptake of the ΔpH probe [2-14C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[14C-phenyl]phosphonium bromide (TPP+), did not affect the response of IAA net uptake to external Ca2+ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [3H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (≤10−5 mol·dm−3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP+.

Inhibition by 2,4‐D of somatic embryogenesis in carrot as explored by its reversal by difluoromethylornithine

The development of somatic embryos is, in many plants, inhibited by 2,4‐dichlorophenoxyacetic acid (2,4‐D) and other auxins. The finding that difluoromethylornithine (DFMO) can counteract this inhibition has been used to test some of the hypotheses for the mechanism of inhibition.Inhibition of somatic embryogenesis in carrot (Daucus carota L.) by exogenous ethylene (from ethephon), antioxidants (ascorbic acid and glutathione), ethanol/acetaldehyde and abscisic acid was not counteracted by DFMO, indicating that the inhibitory effect of 2,4‐D is not manifest through the formation of these compounds. Embryogenesis was abolished by micromolar concentrations of the polar auxin transport inhibitors 2, 3, 5‐triiodobenzoic acid (TIBA), N‐1‐naphthylphthalamic acid (NPA) and 9‐hydroxyfluorene‐9‐carboxylic acid (HFCA). This inhibition was counteracted to a considerable extent by DFMO. Inhibition by relatively high concentrations of the antiauxin 2‐(p‐chlorophenoxy)‐isobutyric acid (CPIB), which does not affect polar auxin transport, was in contrast not counteracted by DFMO. These findings indicate that exogenous auxins may inhibit embryogenesis by interfering with the ability of postglobular embryos to set up internal auxin gradients necessary for polarized growth.

5?-Isothiocyanato-N-1-naphthylphthalamic acid: a chemically reactive site-directed irreversible ligand for phytotropin receptors

A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5′-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 μM resulted in a dose-dependent inhibition of basipetal [14C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 μM or greater. NCS-NPA inhibited the saturable binding of [3H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [3H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.

NPA Inhibits Secretion of Amylases by Barley Aleurone Cells and Auxins can Overcome this Inhibition

AbstractPolar cell growth is regulated by auxin (Klämbt et al., 1992). It is essentially dependent upon exocytosis. Is exocytosis directly regulated by auxin?Aleurone cells secrete amylases, as well as other hydrolases, by exocytosis. In preliminary experiments, with 22 h of incubation, increasing concentration of N‐1‐naphthylphthalamic acid (NPA) parallels the inhibition of amylase secretion.A model system for secretion was designed. Barley aleurone layers, previously incubated in 1 μM GA3 for 16 h, then washed extensively in the presence of 10 μM NPA for 10 min, were incubated finally for 2 h in GA3, NPA and/or auxins. NPA inhibits amylase secretion. Auxins completely abolished secretion inhibition at the optimal concentration of 1 μM.Aleurone layers contain 1.5 to 3 μM endogenous indole‐3‐acetic acid (IAA) and have a high capacity to accumulate and retain IAA. A 7‐fold accumulation occurred within 3 h after application of 20 μM radiolabeled IAA. A loss of about 10% could be measured within the following 4 h period in fresh media, independent of the presence of NPA.The inhibition of amylase secretion by NPA is not due to an inhibition of protein biosynthesis which was tested by the use of [35S]‐methionine.

The discovery of novel auxin transport inhibitors by molecular modeling and three-dimensional pattern analysis

Molecular modeling techniques and three-dimensional (3D) pattern analysis have been used to investigate the chemical and steric properties of compounds that inhibit transport of the plant hormone auxin. These compounds bind to a specific site on the plant plasma membrane characterized by its affinity for the herbicide N-1-naphthylphthalamic acid (NPA). A 3D model was derived from critical features of a set of ligands for the NPA receptor, a suggested binding conformation is proposed, and implications for the topographical features of the NPA receptor are discussed. This model, along with 3D structural analysis techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the criteria of the model, 77 representative molecules were evaluated for their ability to compete for the binding of [3H]NPA to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, representing chemical classes not included in the original compound set, were also found to inhibit polar auxin transport through corn coleoptile sections. Thus, this study demonstrates that 3D analysis techniques can identify active, novel ligands for biochemical target sites with concomitant physiological activity.

Biochemical Bases for the Loss of Basipetal IAA Transport with Advancing Physiological Age in Etiolated Helianthus Hypocotyls

Basipetal transport of [(14)C]IAA in hypocotyl segments isolated from various regions of etiolated Helianthus annuus L. cv NK 265 seedlings declines with increasing physiological age. This decline was the result of a reduction in both transport capacity and apparent velocity. Net IAA uptake was greater and the abilities of auxin transport inhibitors to stimulate net IAA uptake were reduced in older tissues. Net IAA accumulation by microsomal vesicles exhibited a similar behavior with respect to age. Specific binding of [(3)H]N-1-naphthylphthalamic acid (NPA) to microsomes prepared from young and older hypocotyl regions was saturable and consistent with a single class of binding sites. The apparent affinity constants for NPA binding in microsomes prepared from young versus older tissues were 6.4 and 10.8 nanomolar, respectively, and the binding site densities for young versus old tissues were 7.44 and 3.29 picomoles/milligram protein, respectively. Specific binding of [(3)H]NPA in microsomes prepared from both tissues displayed similar sensitivities toward unlabeled flurenol and exhibited only slight differences in sensitivity toward 2,3,5-triiodobenzoic acid. These results demonstrate that the progressive loss of basipetal IAA transport capacity in etiolated Helianthus hypocotyls with advancing age is associated with substantial alterations in the phytotropin-sensitive, IAA efflux system and they suggest that these changes are, at least partially, responsible for the observed reduction of polar IAA transport with advancing tissue age.

Calcium deficiency and auxin transport in Cucurbita pepo L. seedlings

Hypocotyls of Cucurbita pepo L. (zucchini) seedlings grown at low calcium levels (0.1 mM) showed reduced basipetal polar transport of the auxins indole-3-acetic acid and 1-naphthylacetic acid in comparison with control plants grown at 5 mM Ca(2+). The contribution to overall transmembrane auxin transport of the symport uptake carrier was unchanged by calcium deficiency, but the efflux carrier of the auxin-anion uniport was less active and the sensitivity of auxin transport to N-1-naphthylphthalamic acid (NPA) inhibition was thereby reduced. These changes could partially be reversed by short treatments with Ca(2+) or La(3+). The NPA receptor was unaltered in level and affinity for NPA by calcium deficiency. It is suggested that the major lesion responsible for diminished polar auxin transport in calcium-deficient tissue is in flux through the auxin efflux carrier which could be subject to control by Ca(2+) by as yet unestablished mechanisms.

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells.

Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenylacetic acid

Phenylacetic acid (PAA) significantly stimulated the elongation of isolated Phaseolus vulgaris internodal segments and prevented the decline in acid invertase specific activity observed in segments incubated in the absence of growth substances. Unlike IAA, which stimulated both elongation and invertase activity over a very wide range of concentrations (<10-4 - 1 mol.m-3; optimum 10-2 mol.m-3), the response to PAA was restricted to a much narrower range of concentrations (3 × 10-2 - 1 mol.m-3; optimum ca. 1–2 × 10-1mol.m-3). At the optimum concentration of PAA, the stimulation of both responses was about 63–75% of that induced by the optimum concentration of IAA. The differences in the concentration range and magnitude of the responses to IAA and PAA were not due to differences in uptake of the two compounds. The stimulation of elongation by both compounds was prevented by 3.6 × 10-2mol.m-3 cycloheximide (CH), and acid invertase activites were greatly reduced compared with samples treated with growth substances alone. A saturating concentration of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA) slightly promoted the growth of control segments, probably by reducing the loss of residual endogenous auxin to the incubation medium. The elongation induced by PAA at its optimum concentration was considerably greater than the elongation induced by NPA, indicating that PAA did not cause growth by preventing the loss of endogenous auxin from the segments. Elongation responses to combinations of IAA and PAA suggested that the compounds were acting additively and that they were affecting growth by the same mechanism.

Induction of acropetal 14C-photosynthate transport and radial growth by indole-3-acetic acid in Pinus sylvestris shoots

The relationship between radial growth and assimilate movement was determined in one-year-old Pinus sylvestris (L.) cuttings collected during the dormant period and reactivated for 1-27 days under environmental conditions favorable for growth. The cuttings were either left with buds intact, defoliated along the distal 8 cm, or debudded and treated apically with 0 or 1 mg g(-1) indole-3-acetic acid (IAA) in lanolin. Radial growth was measured as tracheid production and bark radial width. The distribution of (14)C-photosynthate 1 or 5 days after exposure to (14)CO(2) was used to indicate assimilate movement. Debudding inhibited tracheid production and decreased acropetal (14)C-photosynthate transport, whereas applying IAA to debudded cuttings promoted both these processes and, in addition, induced vigorous callus growth within the bark immediately below the application point. Distal defoliation markedly increased the amount of (14)C-photosynthate transported toward the apex without altering how debudding and exogenous IAA affected tracheid production and the distribution of (14)C-photosynthate. The production of tracheids induced by apically applied IAA in debudded and distally defoliated cuttings increased progressively in material collected on September 9, October 14 and November 24, whereas bark radial width and acropetal (14)C-photosynthate movement increased only between the first two collection dates. Ringing midway between the IAA source and the bottom of the distally defoliated region with a lanolin mixture of 10 mg g(-1)N-1-naphthylphthalamic acid (NPA), 1 mg g(-1) methyl-2-chloro-9-hydroxy-fluorene-9-carboxylic acid (CF) or 10 mg g(-1) phenylacetic acid (PAA) reduced radial growth and [1-(14)C]IAA transport below the ring and locally promoted radial growth and accumulated radioactivity above. (14)C-Photosynthate transport into the region above the ring either was not altered (CF, PAA) or was increased (NPA). The results indicate that apically applied IAA induces the acropetal movement of (14)C-photosynthate in debudded P. sylvestris shoots by locally promoting activity correlated with tracheid and callus production rather than by affecting radial growth or phloem transport processes, or both, at a distance below the IAA source.

Aryl-Substituted α-Aminooxycarboxylic Acids

Certain herbicidal aminooxyisovalerate analogs were noted in whole plant phytotoxicity bioassays to cause disoriented roots. Since this symptom is often characteristic of interference with the transport of the plant hormone auxin, the ability of several of these compounds to compete for the N-1-naphthylphthalamic acid (NPA) binding site in corn (Zea mays L.) coleoptile membranes was measured. Significant NPA binding activity was found, expecially for the 2,4-dichlorophenyl analog. Application of structure-activity principles from traditional auxin transport inhibitors to this new class of molecules led to the synthesis of the naphthyl analogue. This molecule was extremely active in competing for NPA binding and in eliciting whole plant growth regulator effects. Possible relationships between these molecules and the mode of auxin transport are discussed.

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-(14)C]-or [5-(3)H]IAA occurred at velocities (approx. 8-15 mm·h(-1)) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-(14)C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-(14)C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m(-3)). Transport velocity increased with concentration up to 83 mmol·m(-3) IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-(14)C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m(-3) NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-(14)C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m(-3) NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.

Bisulfite interacts with binding sites of the auxin-transport inhibitor N-1-naphthylphthalamic acid

The affinity of the auxin-transport inhibitor N-1-naphthylphthalamic acid (NPA) for membrane particles as well as for solubilized binding sites from Cucurbita pepo L. hypocotyls was reduced by low concentrations of bisulfite (half-maximal inhibition at 2·10(-3)-3·10(-3) M). Two membrane fractions obtained by sedimentation aided with polyethylene glycol showed differential sensitivity to bisulfite. Other oxidizing or reducing substances tested at 1 mM had no effect, except for N-ethylmaleimide (80% inhibition) and iodine (complete inhibition), both of which reduced the number of binding sites but not their affinity. Addition of bisulfite to either the isoalloxane ring of flavoproteins or to pyridoxal phosphate or quinones is proposed as a possible mechanism of action. Sulfur dioxide, at concentrations measured in polluted air, can lead to bisulfite concentrations in plant tissue sufficient to interfere with NPA-binding sites and hence with auxin transport.

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [(3)H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [(3)H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K(d)) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [(3)H]NPA binding.

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 μM solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 μM, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 μM) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 μM and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3-100 μM unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-(14)C]IAA (external concentration 0.2 μM). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3-100 μM, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.

5′-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [(3)H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K(0.5) = 2.8 x 10(-7) molar. The K(0.5) for NPA under these conditions is 2 x 10(-8) molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [(3)H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [(3)H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i 50= 0.3 μM) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i 50=3 μM). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H(+) symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [(3)H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [(3)H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [(3)H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [(3)H]NAA net uptake was much less sensitive to NPA stimulation than was [(14)C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.

Auxin carriers in Cucurbita vesicles

The association at 0° C of [(3)H]indole-3-acetic acid (IAA) with membrane vesicles prepared from Cucurbita pepo L. hypocotyls at pH 7.9 and resuspended at pH 6.0 was greatly diminished by osmotic shrinkage. Nonradioactive IAA inhibited a large proportion of this association thus revealing a saturable component, also decreased by osmotic shrinkage, which mainly represents operation of an auxin uptake carrier, with saturable binding having only a minor role. The contribution of this carrier to the steady state of IAA transport changed in the same direction as the proton motive force (transmembrane pH and electrical potential difference) which was manipulated using the K(+) ionophore valinomycin with differing K(+) concentration gradients over a range of assay pH values. We conclude that the uptake carrier is electroimpelled with IAA(-)/2H(+) (or IAAH/H(+)) symport as a possible mechanism. The same procedures were used to examine the behaviour of the IAA efflux carrier, whose inhibition by N-1-naphthylphthalamic acid (NPA) causes an increase in IAA accumulation by the vesicles. The extent of NPA-stimulation was linked to the magnitude of the electrical potential difference (negative inside) across the vesicle membranes. We conclude that IAA transport by the efflux carrier is electroimpelled, with IAA(-) a likely substrate.

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free‐flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free‐flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free‐flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N‐1‐naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross‐contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo‐induced leaves by free‐flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.

Isolation of Highly Purified Fractions of Plasma Membrane and Tonoplast from the Same Homogenate of Soybean Hypocotyls by Free-Flow Electrophoresis

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K(+)-stimulated, vanadate-inhibited Mg(2+) ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.