Chapter 16 Biochemical Transport
Characterization Protein
Using
of
Auxin
Phytotropins
S. Brunn, M. V. Subramanian, E. Walters, B. Patel, and J. D. Reagan
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Auxin transport inhibitors (phytotropins) are presumed to bind to the IAA-efflux protein and prevent auxin efflux. Membrane preparations from zucchini and Arabidopsis each showed only one specific binding site for the phytotropin, naphthylphthalamic acid (NPA) with aK =7.12nM,23.8 nM and B = 6.70 pmol/mg, 2.55 pmol/mg, respectively. Several other phytotropins such as semicarbazone (SCB1), pyrenoylbenzoic acid (PBA), 2,3,5 triiodobenzoic acid (TIBA) and quercetin completely displaced bound 3H-NPA from zucchini membranes with I50 values of 16, 0.15, 12850 and 47560 nM, respectively. Hill plot analyses of NPA displacement by these phytotropins also indicated only one binding site on the membrane. Free indole-3-acetic acid (IAA), ethyl ester of IAA, as well as, amino acid and sugar conjugates of IAA, had no effect on 3H-NPA bound to microsomal membranes (MM). Likewise, various auxin agonists like 2,4-dichlorophenoxy acetic acid (2,4-D), dicamba and α-naphthaleneacetic acid (NAA) also had no effect. Various cytokinins, calcium ions, calcium effectors such as chlorpromazine, flunarizine and verapamil as well asflavins,had no effect in the 3H-NPA dissociation assay. Of the various phytotropins tested, only NPA, PBA, TIBA and SCB-1 showed significant IAA accumulation in the stem segment assay and loss of gravitropism in the tomato root bioassay. SCB-1 was very effective in the above bioassays. Quercetin, a weak phytotropin, and calcium effectors were not active in the bioassays. Solubilization of the NPA binding protein from zucchini M M was achieved with 0.4% CHAPS. The detergent solubilized protein exhibited similar 3H-NPA saturation kinetics to that of the membrane bound protein (Kd = 2.0 nM, B = 0.2 pmol/mg). d
max
max
Auxins are a group of weak organic acids that have dramatic and diverse effects on plant growth and development (1). Indole-3-acetic acid (IAA), the most common natural auxin is synthesized and transported unidirectionally away from growing
0097-6156/94/0557-0202$08.00/0 © 1994 American Chemical Society In Bioregulators for Crop Protection and Pest Control; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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16. BRUNN ET AL.
Characterization of Auxin Transport Protein
203
apical meristems (2). This transmembrane polar transport has been reviewed extensively in the literature (2-5) and can be characterized by two stages: 1) simple diffusion across the membrane or via an influx carrier into the cell, and 2) basal efflux via a basally-located efflux carrier (2,6). This transport occurs primarily in parenchymatous cells associated with, but not of the vascular bundles themselves (2). Auxin transport is described by the chemiosmotic hypothesis (3) which outlines the factors driving polar auxin transport. Transport is dependent upon a pH gradient across the membrane which is maintained by a proton electrochemical gradient. The less polar nature of IAA at the prevailing apoplast pH of 5.5, allows it to cross the lipid bilayer passively following a concentration gradient. Direct measurement of auxin uptake and transport into cells and vesicles is a difficult task. More readily measurable is the efflux process and inhibition of IAA efflux by auxin transport inhibitors (7). These inhibitors, referred to as phytotropins, are able to affect apical dominance, geotropic and phototropic responses (8). The most extensively studied phytotropin is 1-N-naphthylphthalarnic acid (NPA) and the binding protein is often referred to as the NPA binding protein. Competition studies using 3H-NPA are useful in determining the relative affinities of other phytotropins for the NPA binding site (8,9). The structural requirements for binding have been defined to consist of an aromatic carboxylic acid (five- or sixmemberedringsystem), which in turn is attached via the ortho position, by either a planar or conjugated bridge, to another aromatic ring. The minimum distance between the two rings has been proposed to be 7.3 Â (8). Several naturally occurring flavonoid compounds have been shown to exhibit phytotropin effects (10) and some, such as quercetin, compete with NPA at the binding site. This has led researchers to consider the flavonoids as natural phytotropins for the control of auxin transport since they are ubiquitous and under stress, can approach high, physiologically effective concentrations. Their levels are also tightly regulated and known to be environmentally-responsive (11). In the present paper, we have described several biochemical properties of the NPA binding protein and compared NPA with several other phytotropins and related natural ligands. The effects of phytotropins on IAA uptake by pea stem segments and tomato root gravitropism are also discussed. MATERIALS AND METHODS Binding Studies. The isolation of total membranes and plasmalemma from zucchini (Curcubita pepo L.) has been previously described (9). Solubilization of the NPA binding protein was achieved by incubating the membranes with 0.4% CHAPS in test buffer ( 0.25 M sucrose, 1 mM MgCl , 20 mM citrate, pH 5.3) for 2 hours on ice. An ultracentrifugation step for 1 hour at 100 000 · g separated the unsolubilized pellet from the solubilized proteins. Quantitation of phytotropin binding (3H-NPA) was achieved by either GF/B filter assay or by using the newly developed Millipore Multiscreen plate assay (12). Radiolabeled NPA, 2,3,4,5(n)-3H naphthylphthalamic acid (48.9 Ci/mmol) was purchased from RPI Corp, Mt Prospect, Π. For the GF/B assay, filters presoaked in 0.1% PEI (polyethylenimine) for 30 minutes were used. Different concentrations of inhibitors in test buffer in a volume of 5 μΐ were added to microcentrifuge tubes. To a membrane solution of 0.1 mg/ml total protein in test buffer (0.2 mg/ml for Arabidopis), was added 3H-NPA to a final concentration of 5 nM. Aliquots of 195 μΐ of membrane-ligand mixture were added to the microfuge tubes for a final volume of 200 μΐ/assay. The samples were incubated at 4«C for 40 minutes on a gyratory shaker and then filtered over vacuum through the PEI treated membrane. Each tube was thenrinsedwith 1 ml test buffer and this was added to the filter. The filter was further washed with 7 ml test buffer to reduce background binding and left to dry on the vacuum for an additional 2 2
In Bioregulators for Crop Protection and Pest Control; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
204
BIOREGULATORS FOR CROP PROTECTION AND PEST CONTROL
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minutes. Filters were placed in 7 ml scintillation vials with 5 ml of Beckman ReadySafe cocktail and counted for 2 minutes in a Beckman LS 5000CE counter. A novel Millipore multiscreen assay system has been developed (12) for 3HNPA dissociation analysis of large numbers of samples. The system consists of a 96 well plate fitted with a mixed cellulose esterfilterat the base which is supported by a removable plastic sheet. The filters were pre-wetted with 200 μΐ distilled water and vacuum filtered using a Millipore vacuum manifold. Like the GF/B assay, inhibitor or buffer were added to the wells in a 5 μΐ volume followed by addition of 195 μΐ mixture of membranes and 3H-NPA and incubated at 4 C. After 45 minutes the plate was vacuum aspirated and the filters washed with 200 μΐ test buffer. The plastic support was removed and the plate dried at 70 C for 20 minutes. The filters were punched out using either the Millipore Multiscreen Punch or a cork borer into 7 ml scintillation vials and soaked in 200 μΐ water prior to the addition of scintillation cocktail. Physiological Studies. To demonstrate inhibition of auxin efflux and gravitropism inhibition caused by phytotropins, we have utilized the 14C-IAA accumulation assay in pea (Pisum sativum) stem segments, and tomato root gravitropism bioassay response (Lycopersicon esculentum variety Celebrity) respectively. The pea stem assay has been previously described (9). About 2 week old, light grown seedlings were the source of stem segments. Accumulation of l O I A A in 5 mm stem segments at 1 μΜ (57 mCi/mmol, American Radiolabeled Chemicals Inc.), in the presence and absence of 1 and/or 10 μΜ phytotropins, was used to measure inhibition of the efflux process. The tomato root assay was adapted from the descriptions of Brunn et al (9). Pre-germinated seedlings were grown for 24 hours in a square petri dish oriented normal to gravity. The plate was then reoriented 90° with respect to gravity for another 24 hours. After a total of 48 hours the tomato roots were rated for root length and angle of curvature as a result of the reorientation to gravity. Various phytotropins were added as necessary to the growth medium to determine GI50 values. RESULTS AND DISCUSSION Binding Assays. Membrane vesicles isolated from etiolated zucchini hypocotyls can provide either total microsomal (MM) or purified plasma (PM) membranes. Either system is appropriate for 3H-NPA binding studies; however, PM preparations yield higher specific activity, albeit with only about 10-12% recovery of total binding activity. The saturable binding of 3H-NPA to zucchini M M shown in Figure la, gave a binding constant of 7.12 nM with a B of 6.7 pmol/mg. GF/B filtration assay gave very low nonspecific binding even at high concentration of the labelled ligand (15-20%). Scatchard analysis of the above data revealed only one population of binding site for NPA (Figure la, inset). Binding studies with Arabidopsis M M also revealed only one binding site (Figure lb, K4, flunarizine and verapamil were also inactive in the dissociation assay. Thein and Michalke (28) have proposed that the NPA binding protein may be a flavoprotein, based on the inhibitory action of bisulfite on NPA binding. Indeed, bisulfite readily displaces 3H-NPA (Figure 2) with an I50 of 9 μΜ. Bisulfite is also known to inhibit flavoprotein oxidases, although it is not a specific inhibitor of flavoproteins. The activity of certain flavoproteins can be enhanced by the addition of exogenous flavins to replace the dissociated cofactor during partial purification (29, 30). However, FAD (Figure 3) did not enhance 3H-NPA binding or had any dissociation effect on the bound radioligand. The effect of FMN and riboflavin was likewise at 0.25 mM (not shown in Figure 3). The flavoprotein nature of NPA binding protein remains equivocal. In order to characterize the NPA-binding protein, attempts were made to solubilize it from microsomal membranes. As stated earlier, PM was not used for solubilization studies due to the low recovery level in the phase separation step involved in PM preparation. Many different detergents were tried to solubilize the protein including Triton X-100, deoxycholate and taurodeoxycholate at various concentrations. The best recovery obtained was using 0.4% CHAPS, but it accounted for only 1-3% of the initial total binding activity. The majority of the binding activity was still retained in the MM. Repeated extraction did not solubilize any more binding activity. The solubilized protein exhibited similar 3H-NPA saturation kinetics to that of the membrane bound protein with K
