Planta (1992)187:254-260
P l a n t a 9 Springer-Verlag 1992
Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites Wolfgang Michalke 1, Gerard F. Katekar 2, and Art E. Geissler 2 1 Institut ffir Biologie III der Universitiit, W-7800 Freiburg, Federal Republic of Germany 2 CSIRO Division of Plant Industry, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia Received 24 November; accepted 4 December 1991
Abstract. Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in doublelabelling experiments using [2,3,4,5-3H]l-N-naphthylphthalamic acid and 3-indolyl-[2-14C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (KD at 10 -8 mol-1-1) for one site and low affinity (KD at 10 -6 tool.1-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (KD below 10-8 mol. 1-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles. Key words: Auxin transport - Cucurbita (phytotropin binding) Hypocotyl - Membrane vesicles - 1-N-Naphthylphthalamic acid - Phytotropin binding - 2-(1-Pyrenoyl)benzoic acid
Introduction Auxin is known to be transported actively in a polar manner through shoot and root tissue of young plants (for a review see Kaldewey 1984). The process has been described as a transfer from cell to cell across the plasma membranes and intervening free space (Cande and Ray 1976). Control of the transfer of auxin across the plasma membrane is therefore a possible way for the plant to achieve the observed auxin movement. Abbreviations: IAA=Indol-3-yl acetic acid; NPA= 1-N-naphthyl-
phthalamic acid; PBA=2-(1-pyrenoyl)benzoic acid; TIBA=2,3,5-
triiodobenzoic acid
Three possible ways for auxin to cross the membrane can be conceived: 1) diffusion through the lipid component, either by way of a protonated or anionic form, with the latter being less efficient; 2) entering the cell by way of an influx carrier, and 3) by exiting the cell by way of an efflux carrier (see papers by Goldsmith 1977; Benning 1986; Rubery 1987). The latter two routes can be distinguished from the first because the former is saturable by auxin or its analogues, and the latter is sensitive to specific inhibitors. Auxin uptake into either tissue segments or sealed vesicles was found to be decreased by the auxin analogues 2,4-dichlorophenoxyacetic acid and 2-naphthylacetic acid (Sussman and Goldsmith 1981a; Depta and Rubery 1984; Benning 1986). This was construed as evidence in favour of an influx carrier, such a carrier being saturated by the added auxins. Auxin accumulation in sealed membrane vesicles induced by a pH gradient was also larger than could be accounted for by a possible ion-trap mechanism for weak organic acids (Lomax et al. 1985). This also favoured the presence of an influx carrier. However, auxin uptake into osmotically shrunken vesicles was not reduced as would be expected if uptake were based upon an influx carrier. Thus the observed auxin specificity may rather be the result of a presumed auxin-binding site at the plasma membrane, with the binding affinity also depending on pH gradient and membrane potential (Clark and Goldsmith 1986, 1987). Engagement of an efflux carrier by specific inhibitors should result in auxin accumulation. Such accumulation occurs in both tissue and membrane vesicles when this material is treated with 1-N-naphthylphthalamic acid (NPA), 2-(1-pyrenoyl)benzoic acid (PBA), or 2,3,5-triiodobenzoic acid (TIBA) (Thomson et al. 1973; Sussman and Goldsmith 1981a, b; Hertel et al. 1983). This can be regarded as evidence for the presence of an efflux carrier, with the molecules used being specific inhibitors of the efflux mechanism. Both NPA and PBA are members of the phytotropin class of plant growth regulators (for a review of phytotropins see Rubery 1990). Members of the class have ap-
W. Michalke et al.: Phytotropins and auxin transport: Two recognition sites peared separately in the literature for over 60 years (see e.g. Boas and Merkenschlager 1925). Chemically, they generally consist of a benzoic acid moiety which is linked at the ortho position to a second, generally larger, aromatic ring. They have been found to have c o m m o n chemical and stereochemical features, c o m m o n physiological properties - the most striking being their ability to abolish the tropic responses - and a c o m m o n m o d e of action (Katekar 1976; K a t e k a r and Geissler 1977a, b; K a t e k a r et al. 1987a, b). Importantly, [ 3 H ] N P A was found to bind strongly to m e m b r a n e preparations of maize coleoptiles (Lembi et al. 1971). Subsequently, it was shown that other phytotropins could also bind, and their binding ability could also be correlated with their physiological activities (Katekar et al. 1981) including their ability to inhibit auxin transport (Katekar and Geissler 1977b). It has been concluded that the N P A binding site is probably a receptor (Katekar et al. 1981). The N P A receptor appears to be ubiquitous in plants (Venis 1985), and perhaps throughout the plant (Katekar and Geissler 1989; Firn 1987). It is unlikely that the recognition characteristic would be preserved without some evolutionary pressure to maintain it, so that a natural agonist is presumed (Thomson 1972; K a t e k a r et al. 1981; Jacobs and Rubery 1988; Rubery 1990). While it has been proposed that flavonoids m a y be natural ligands (Jacobs and Rubery 1988), binding to the receptor is comparatively weak, so that it cannot be regarded as settled whether they are the only natural ligands. The N P A receptor therefore appears to form an essential part of an efflux carrier which is involved in the auxin-transport process, and the process m a y be modulated by an endogenous ligand(s). An examination of its properties through the effects of phytotropin binding is warranted. In animal physiology, receptors have often been detected through the use of synthetic antagonists which need bear no structural relationship to the natural agonists (AriEns et al. 1979). Further, differing types of receptor have been detected for one agonist. Examples are the adrenergic and histamine receptors, where several different receptors, with different functions, are known for each class (AriEns et al. 1979). An analogous situation is at least possible for the N P A receptor. There is evidence for this possibility: In a correlation between receptor binding in maize coleoptiles and the ability to abolish the gravitropic response in Lepidium, a systematic difference was found between arylphthalamic acids (closely related chemically to NPA) and other phytotropins, in that the antigravitropic activity of the N P A analogues was significantly less for a given degree of binding than for other phytotropins (Katekar 1985). With respect to auxin transport, a quantitative discrepancy was observed between the low concentration of N P A needed to saturate specific binding c o m p a r e d with the higher concentration needed to inhibit transport (Hertel et al. 1983). In accumulation and uptake experiments, eosin, which is a phytotropin but not an N P A analogue, and morphactin (also an inhibitor of auxin transport) were found to stimulate auxin accumulation at lower concentrations than NPA, but inhibited binding of [ 3 H ] N P A only equal to or less than N P A (Sussman
255
and Goldsmith 1981b, their Figs. 4 and 5 and discussion). M e m b r a n e vesicles isolated from Cucurbita hypocotyls and maize coleoptiles were found to accumulate auxin, and their ability to do so was stimulated by phytotropins and T I B A (Hertel et al. 1983; Clark and Goldsmith 1986; Benning 1986; L o m a x 1986; Sabater and Rubery 1987b, c; Heyn et al. 1987). This indicates that such m e m b r a n e preparations can exhibit, in vitro, important features of the plant's auxin-transport system. Since the N P A receptor is on the same membranes, the receptor and its resultant effects can be examined in the same preparation by means of a double-labelling technique, using [ 3 H ] N P A to measure phytotropin binding, and [14C]IAA to measure accumulation of IAA. This technique was therefore used to assess the binding and activity of phytotropins of differing structures, affinity and potency.
Material and methods Plant material. Seeds of zucchini squash (Cucurbita pepo L. cv. Black Jack; Arthur Yates & Co. Pty Ltd., Milperra, N.S.W., Australia) were planted in a moist vermiculite-perlite mixture (50/50, v/v) in plastic trays and grown for 7d at 25~ C in darkness, except for watering once per day in dim green light for a few minutes. Radiochemicals and chemicals. 3-Indolyl-[2-14C]acetic acid ([14C]IAA), 1.81 GBq/mmol, was purchased from Amersham, Melbourne, Australia, and [-2,3,4,5-aH]l-N-naphthylphthalamic acid ([3H]NPA), 2.04TBq.mmol -i, from Centre d'Energie Atomic, Gif-sur-Yvette, France. Unlabelled NPA and other phytotropins were synthesized as previously described (Katekar 1976; Katekar and Geissler 1977a, b). Carbonylcyanide m-chlorophenylhydrazone (CCCP) was obtained from Calbiochem, San Diego, Cal., USA; dithiothreitol (DTT) and phenylmethylsulfonylfluoride (PMSF) were obtained from Boehringer, Mannheim, FRG, and sorbitol from Sigma Chemical Co., St Louis, Mo., USA. All other chemicals were reagent grade, coming from various commercial sources. Buffers. Buffers used, and variations, were as follows. So-1 buffer: 250 mM sorbitol, 25 mM Hepes (4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid), 3 mM EGTA (ethylene glycol- bis (fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid), adjusted to pH 7.8 8.0 by adding BisTrisPropane (1,3-bis[-tris(hydroxymethyl)methylamino]propane); usually about 40 mmol of the latter were added per 11. Bovine serum albumin (0.1%), 10 - 3 M DTT and 10 3 M PMSF were added immediately before use. So-Ia: The pH was adjusted with 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris, base) instead of BisTrisPropane, and 3 mM EDTA (ethylenediaminetetracetic acid) was used instead of EGTA. So-lI buffer: 300 mM sorbitol, 5 mM BisTrisPropane, 1 mM KCI, 2-(N-morpholino)ethanesulfonic acid (Mes) was added to adjust the pH to 5.8-6.0, usually about 10-15 mmol were used per 1 1. So-IIa: 5 mM Mes was used, and pH adjusted with BisTris instead of BisTrisPropane. Preparation of membrane particles. The protocol of Hertel et al. (1983) was followed, with variations as used by Clark and Goldsmith (1986), and some of our own. Sections of Cucurbita hypocotyls (10-15 cm total length) were harvested in daylight as segments of approx. 5 cm length, beginning about 0.5 cm below the hook, and
256
W. Michalke et al.: Phytotropins and auxin transport: Two recognition sites
collected in a glass beaker cooled on ice. All following procedures were conducted at 2-4 ~ C in daylight. Usually 30~35 g of sections were harvested, and So-I buffer was added at 4-5 ml.(g FW) ~. The mixture was then homogenized twice for 5 s with a 5-s interval in a household herb mill. The resulting brei was squeezed through a narrow-mesh nylon cloth to remove small pieces of tissue and cell wall. The filtrate was centrifuged at 5000 rpm for 20 min in an SS34 rotor of a Sorvall centrifuge (DuPont Instruments, Norwalk, Conn., USA) to remove nuclei and most of the mitochondria. The supernatant was centrifuged at 28000 rpm for 30 min in an ultracentrifuge (Beckman Instruments, Palo Alto, Cal., USA) using a Ti60 or Ti70 rotor. The resulting sediment contained vesicles of the cellular membranes such as plasma membrane, endoplasmic reticulum, Golgi, tonoplast and mitochondrial fragments. It was resuspended in the same amount of So-I as before and recentrifuged, giving a sediment of "washed" cellular membranes. The sediment was resuspended in So-II buffer to give a concentration of 1 2 g of FW (of the sections) per 1 ml of final homogenate. The final homogenate was used for the assay 5-10 min after resuspension.
Assay of NPA binding and 1AA accumulation. Two in-vitro properties of the phytotropins - the ability to bind to the NPA receptor in membrane vesicles and to stimulate IAA accumulation into these vesicles were assayed simultaneously using a double-labelling technique. A 0.5-ml sample of final homogenate was added to 0.5ml of So-II buffer containing [3H]NPA (0.05-0.4nM), [i4C]IAA (10-20 nM) and various concentrations of the phytotropins to be tested. The mixture was incubated for 20-30 min on ice, to allow binding of NPA and accumulation of IAA, and then centrifuged at 24000 rpm (about 80000.g) for 30 min in a Beckmann ultracentrifuge using a Ty-25 rotor. The supernatants were aspirated from the centrifuge tubes, and Tris base (0.4 ml, 1 mM) was added to the sediments; these were then allowed to stand for 2 3 h or overnight, depending on their size. The suspensions obtained by this treatment were transferred to scintillation fluid (1 1 toluene, 0.5 1 Triton X-100, 4 g 2,5-diphenyloxazole (PPO)) and their 3H and ~4C contents counted in separate channels of a Beckmann scintillation counter. Separate samples of 3H and 14C were prepared using the same scintillation fluid in order to determine the spillover between the two channels, and the experimental counts were corrected accordingly.
Results Higher N P A concentrations are needed to stimulate I A A accumulation in membrane vesicles than to inhibit [ 3 H ] N P A binding. Binding of [3H]NPA and accumulation of [-lgC]IAA in membrane vesicles from zucchini hypocotyls were measured at various concentrations of NPA. The results are shown in Fig. 1, upper panel. With increasing concentration of non-radioacative NPA, progressively less [3H]NPA remained bound to the membranes. However, it was only at concentrations when the binding of [3H]NPA was reduced to very low values that a stimulation of [14C]IAA accumulation became noticeable. With further increases in NPA concentration, IAA accumulation continued to rise while little change in [3H]NPA binding was observed. It can be seen that the two effects were produced by concentrations of NPA that differ by two orders of magnitude: 50% inhibition of [3H]NPA binding occurred at about 10-8 M, while 50% of maximal stimulation of [14C]IAA accumulation r e q u i r e d 10 - 6 M NPA under the conditions used. It thus appears that occupation of those NPA-binding sites which are saturable with low NPA concentrations (here-
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P l a n t a 9 Springer-Verlag 1992
Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites Wolfgang Michalke 1, Gerard F. Katekar 2, and Art E. Geissler 2 1 Institut ffir Biologie III der Universitiit, W-7800 Freiburg, Federal Republic of Germany 2 CSIRO Division of Plant Industry, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia Received 24 November; accepted 4 December 1991
Abstract. Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in doublelabelling experiments using [2,3,4,5-3H]l-N-naphthylphthalamic acid and 3-indolyl-[2-14C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (KD at 10 -8 mol-1-1) for one site and low affinity (KD at 10 -6 tool.1-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (KD below 10-8 mol. 1-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles. Key words: Auxin transport - Cucurbita (phytotropin binding) Hypocotyl - Membrane vesicles - 1-N-Naphthylphthalamic acid - Phytotropin binding - 2-(1-Pyrenoyl)benzoic acid
Introduction Auxin is known to be transported actively in a polar manner through shoot and root tissue of young plants (for a review see Kaldewey 1984). The process has been described as a transfer from cell to cell across the plasma membranes and intervening free space (Cande and Ray 1976). Control of the transfer of auxin across the plasma membrane is therefore a possible way for the plant to achieve the observed auxin movement. Abbreviations: IAA=Indol-3-yl acetic acid; NPA= 1-N-naphthyl-
phthalamic acid; PBA=2-(1-pyrenoyl)benzoic acid; TIBA=2,3,5-
triiodobenzoic acid
Three possible ways for auxin to cross the membrane can be conceived: 1) diffusion through the lipid component, either by way of a protonated or anionic form, with the latter being less efficient; 2) entering the cell by way of an influx carrier, and 3) by exiting the cell by way of an efflux carrier (see papers by Goldsmith 1977; Benning 1986; Rubery 1987). The latter two routes can be distinguished from the first because the former is saturable by auxin or its analogues, and the latter is sensitive to specific inhibitors. Auxin uptake into either tissue segments or sealed vesicles was found to be decreased by the auxin analogues 2,4-dichlorophenoxyacetic acid and 2-naphthylacetic acid (Sussman and Goldsmith 1981a; Depta and Rubery 1984; Benning 1986). This was construed as evidence in favour of an influx carrier, such a carrier being saturated by the added auxins. Auxin accumulation in sealed membrane vesicles induced by a pH gradient was also larger than could be accounted for by a possible ion-trap mechanism for weak organic acids (Lomax et al. 1985). This also favoured the presence of an influx carrier. However, auxin uptake into osmotically shrunken vesicles was not reduced as would be expected if uptake were based upon an influx carrier. Thus the observed auxin specificity may rather be the result of a presumed auxin-binding site at the plasma membrane, with the binding affinity also depending on pH gradient and membrane potential (Clark and Goldsmith 1986, 1987). Engagement of an efflux carrier by specific inhibitors should result in auxin accumulation. Such accumulation occurs in both tissue and membrane vesicles when this material is treated with 1-N-naphthylphthalamic acid (NPA), 2-(1-pyrenoyl)benzoic acid (PBA), or 2,3,5-triiodobenzoic acid (TIBA) (Thomson et al. 1973; Sussman and Goldsmith 1981a, b; Hertel et al. 1983). This can be regarded as evidence for the presence of an efflux carrier, with the molecules used being specific inhibitors of the efflux mechanism. Both NPA and PBA are members of the phytotropin class of plant growth regulators (for a review of phytotropins see Rubery 1990). Members of the class have ap-
W. Michalke et al.: Phytotropins and auxin transport: Two recognition sites peared separately in the literature for over 60 years (see e.g. Boas and Merkenschlager 1925). Chemically, they generally consist of a benzoic acid moiety which is linked at the ortho position to a second, generally larger, aromatic ring. They have been found to have c o m m o n chemical and stereochemical features, c o m m o n physiological properties - the most striking being their ability to abolish the tropic responses - and a c o m m o n m o d e of action (Katekar 1976; K a t e k a r and Geissler 1977a, b; K a t e k a r et al. 1987a, b). Importantly, [ 3 H ] N P A was found to bind strongly to m e m b r a n e preparations of maize coleoptiles (Lembi et al. 1971). Subsequently, it was shown that other phytotropins could also bind, and their binding ability could also be correlated with their physiological activities (Katekar et al. 1981) including their ability to inhibit auxin transport (Katekar and Geissler 1977b). It has been concluded that the N P A binding site is probably a receptor (Katekar et al. 1981). The N P A receptor appears to be ubiquitous in plants (Venis 1985), and perhaps throughout the plant (Katekar and Geissler 1989; Firn 1987). It is unlikely that the recognition characteristic would be preserved without some evolutionary pressure to maintain it, so that a natural agonist is presumed (Thomson 1972; K a t e k a r et al. 1981; Jacobs and Rubery 1988; Rubery 1990). While it has been proposed that flavonoids m a y be natural ligands (Jacobs and Rubery 1988), binding to the receptor is comparatively weak, so that it cannot be regarded as settled whether they are the only natural ligands. The N P A receptor therefore appears to form an essential part of an efflux carrier which is involved in the auxin-transport process, and the process m a y be modulated by an endogenous ligand(s). An examination of its properties through the effects of phytotropin binding is warranted. In animal physiology, receptors have often been detected through the use of synthetic antagonists which need bear no structural relationship to the natural agonists (AriEns et al. 1979). Further, differing types of receptor have been detected for one agonist. Examples are the adrenergic and histamine receptors, where several different receptors, with different functions, are known for each class (AriEns et al. 1979). An analogous situation is at least possible for the N P A receptor. There is evidence for this possibility: In a correlation between receptor binding in maize coleoptiles and the ability to abolish the gravitropic response in Lepidium, a systematic difference was found between arylphthalamic acids (closely related chemically to NPA) and other phytotropins, in that the antigravitropic activity of the N P A analogues was significantly less for a given degree of binding than for other phytotropins (Katekar 1985). With respect to auxin transport, a quantitative discrepancy was observed between the low concentration of N P A needed to saturate specific binding c o m p a r e d with the higher concentration needed to inhibit transport (Hertel et al. 1983). In accumulation and uptake experiments, eosin, which is a phytotropin but not an N P A analogue, and morphactin (also an inhibitor of auxin transport) were found to stimulate auxin accumulation at lower concentrations than NPA, but inhibited binding of [ 3 H ] N P A only equal to or less than N P A (Sussman
255
and Goldsmith 1981b, their Figs. 4 and 5 and discussion). M e m b r a n e vesicles isolated from Cucurbita hypocotyls and maize coleoptiles were found to accumulate auxin, and their ability to do so was stimulated by phytotropins and T I B A (Hertel et al. 1983; Clark and Goldsmith 1986; Benning 1986; L o m a x 1986; Sabater and Rubery 1987b, c; Heyn et al. 1987). This indicates that such m e m b r a n e preparations can exhibit, in vitro, important features of the plant's auxin-transport system. Since the N P A receptor is on the same membranes, the receptor and its resultant effects can be examined in the same preparation by means of a double-labelling technique, using [ 3 H ] N P A to measure phytotropin binding, and [14C]IAA to measure accumulation of IAA. This technique was therefore used to assess the binding and activity of phytotropins of differing structures, affinity and potency.
Material and methods Plant material. Seeds of zucchini squash (Cucurbita pepo L. cv. Black Jack; Arthur Yates & Co. Pty Ltd., Milperra, N.S.W., Australia) were planted in a moist vermiculite-perlite mixture (50/50, v/v) in plastic trays and grown for 7d at 25~ C in darkness, except for watering once per day in dim green light for a few minutes. Radiochemicals and chemicals. 3-Indolyl-[2-14C]acetic acid ([14C]IAA), 1.81 GBq/mmol, was purchased from Amersham, Melbourne, Australia, and [-2,3,4,5-aH]l-N-naphthylphthalamic acid ([3H]NPA), 2.04TBq.mmol -i, from Centre d'Energie Atomic, Gif-sur-Yvette, France. Unlabelled NPA and other phytotropins were synthesized as previously described (Katekar 1976; Katekar and Geissler 1977a, b). Carbonylcyanide m-chlorophenylhydrazone (CCCP) was obtained from Calbiochem, San Diego, Cal., USA; dithiothreitol (DTT) and phenylmethylsulfonylfluoride (PMSF) were obtained from Boehringer, Mannheim, FRG, and sorbitol from Sigma Chemical Co., St Louis, Mo., USA. All other chemicals were reagent grade, coming from various commercial sources. Buffers. Buffers used, and variations, were as follows. So-1 buffer: 250 mM sorbitol, 25 mM Hepes (4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid), 3 mM EGTA (ethylene glycol- bis (fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid), adjusted to pH 7.8 8.0 by adding BisTrisPropane (1,3-bis[-tris(hydroxymethyl)methylamino]propane); usually about 40 mmol of the latter were added per 11. Bovine serum albumin (0.1%), 10 - 3 M DTT and 10 3 M PMSF were added immediately before use. So-Ia: The pH was adjusted with 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris, base) instead of BisTrisPropane, and 3 mM EDTA (ethylenediaminetetracetic acid) was used instead of EGTA. So-lI buffer: 300 mM sorbitol, 5 mM BisTrisPropane, 1 mM KCI, 2-(N-morpholino)ethanesulfonic acid (Mes) was added to adjust the pH to 5.8-6.0, usually about 10-15 mmol were used per 1 1. So-IIa: 5 mM Mes was used, and pH adjusted with BisTris instead of BisTrisPropane. Preparation of membrane particles. The protocol of Hertel et al. (1983) was followed, with variations as used by Clark and Goldsmith (1986), and some of our own. Sections of Cucurbita hypocotyls (10-15 cm total length) were harvested in daylight as segments of approx. 5 cm length, beginning about 0.5 cm below the hook, and
256
W. Michalke et al.: Phytotropins and auxin transport: Two recognition sites
collected in a glass beaker cooled on ice. All following procedures were conducted at 2-4 ~ C in daylight. Usually 30~35 g of sections were harvested, and So-I buffer was added at 4-5 ml.(g FW) ~. The mixture was then homogenized twice for 5 s with a 5-s interval in a household herb mill. The resulting brei was squeezed through a narrow-mesh nylon cloth to remove small pieces of tissue and cell wall. The filtrate was centrifuged at 5000 rpm for 20 min in an SS34 rotor of a Sorvall centrifuge (DuPont Instruments, Norwalk, Conn., USA) to remove nuclei and most of the mitochondria. The supernatant was centrifuged at 28000 rpm for 30 min in an ultracentrifuge (Beckman Instruments, Palo Alto, Cal., USA) using a Ti60 or Ti70 rotor. The resulting sediment contained vesicles of the cellular membranes such as plasma membrane, endoplasmic reticulum, Golgi, tonoplast and mitochondrial fragments. It was resuspended in the same amount of So-I as before and recentrifuged, giving a sediment of "washed" cellular membranes. The sediment was resuspended in So-II buffer to give a concentration of 1 2 g of FW (of the sections) per 1 ml of final homogenate. The final homogenate was used for the assay 5-10 min after resuspension.
Assay of NPA binding and 1AA accumulation. Two in-vitro properties of the phytotropins - the ability to bind to the NPA receptor in membrane vesicles and to stimulate IAA accumulation into these vesicles were assayed simultaneously using a double-labelling technique. A 0.5-ml sample of final homogenate was added to 0.5ml of So-II buffer containing [3H]NPA (0.05-0.4nM), [i4C]IAA (10-20 nM) and various concentrations of the phytotropins to be tested. The mixture was incubated for 20-30 min on ice, to allow binding of NPA and accumulation of IAA, and then centrifuged at 24000 rpm (about 80000.g) for 30 min in a Beckmann ultracentrifuge using a Ty-25 rotor. The supernatants were aspirated from the centrifuge tubes, and Tris base (0.4 ml, 1 mM) was added to the sediments; these were then allowed to stand for 2 3 h or overnight, depending on their size. The suspensions obtained by this treatment were transferred to scintillation fluid (1 1 toluene, 0.5 1 Triton X-100, 4 g 2,5-diphenyloxazole (PPO)) and their 3H and ~4C contents counted in separate channels of a Beckmann scintillation counter. Separate samples of 3H and 14C were prepared using the same scintillation fluid in order to determine the spillover between the two channels, and the experimental counts were corrected accordingly.
Results Higher N P A concentrations are needed to stimulate I A A accumulation in membrane vesicles than to inhibit [ 3 H ] N P A binding. Binding of [3H]NPA and accumulation of [-lgC]IAA in membrane vesicles from zucchini hypocotyls were measured at various concentrations of NPA. The results are shown in Fig. 1, upper panel. With increasing concentration of non-radioacative NPA, progressively less [3H]NPA remained bound to the membranes. However, it was only at concentrations when the binding of [3H]NPA was reduced to very low values that a stimulation of [14C]IAA accumulation became noticeable. With further increases in NPA concentration, IAA accumulation continued to rise while little change in [3H]NPA binding was observed. It can be seen that the two effects were produced by concentrations of NPA that differ by two orders of magnitude: 50% inhibition of [3H]NPA binding occurred at about 10-8 M, while 50% of maximal stimulation of [14C]IAA accumulation r e q u i r e d 10 - 6 M NPA under the conditions used. It thus appears that occupation of those NPA-binding sites which are saturable with low NPA concentrations (here-
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