Naphthylphthalamic acid is enzymatically hydrolyzed at the hypocotyl-root transition zone and other tissues of Arabidopsis thaliana seedlings

Plant Physiol. Biochem., 1999, 37 (6), 413−430

Naphthylphthalamic acid is enzymatically hydrolyzed at the hypocotyl-root transition zone and other tissues of Arabidopsis thaliana seedlings Angus Murphy, Lincoln Taiz* Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz CA 95064, USA * Author to whom correspondence should be addressed (fax +1 831 459 3139; e-mail [email protected])

(Received September 29, 1998; accepted March 29, 1999) Abstract — During prolonged storage, solid naphthylphthalamic acid (NPA) turns purple due to the formation of the dye, 1,1'-azonaphthylene (ANA). ANA forms oxidatively from two molecules of the NPA degradation product, α-naphthylamine (αNA). At concentrations ≥ 30 µM, solutions of ‘purple NPA’ stained Arabidopsis thaliana seedlings specifically at the hypocotyl-root transition zone and other regions. Staining was caused by the aggregation of ANA and could be reconstituted using undegraded NPA and either αNA or ANA. Studies with [3H]-NPA confirmed that NPA is localized at sites of staining. Genestein, curcumin and quercitin inhibited the staining reaction. Liquid chromatography-mass spectral (LC-MS) analysis of the ANA in the stained tissue indicated that ANA is synthesized at sites of staining. It was postulated that the amide bond of NPA is enzymatically cleaved, producing αNA. The αNA combines to form ANA, which aggregates to yield an insoluble precipitate. Consistent with this hypothesis, NPA amidase activity was detected in purified plasma membranes. The NPA amidase activity was activated by 500 µM MnCl2 and inhibited by phloretin, genestein, quercitin, bestatin and EDTA. © Elsevier, Paris Amidase / aminopeptidase / Arabidopsis seedlings / auxin / naphthylphthalamic acid / histochemistry ANA, azonaphthylene / AONA, azoxynaphthylene / AP, aminopeptidase / αNA, α-naphthylamine / βNA, β-naphthylamine / DEZ, distal elongation zone / DTT, dithiothreitol / ES/MS+, electrospray positive ion mass spectroscopy / HTZ, cotyledon-hypocotyl transition zone / MES-BTP, morpholino ethanesulfonic acid-bis tris propane / MS, Murashige Skoog basal salts / NPA, N,1-naphthylphthalamic acid / NPI, N,1-naphthylphthalamide / PMSF, phenylmethyl sulfonyl fluoride / RTZ, hypocotyl-root transition zone

1. INTRODUCTION 1-Naphthylphthalamic acid (NPA) is a phytotropin herbicide that interferes with growth by inhibiting polar auxin transport [20]. In the course of carrying out experiments utilizing NPA and Arabidopsis seedlings, we inadvertently used an old bottle of NPA that had turned purple with age. At concentrations ≤ 30 µM, no difference was observed between seedlings treated with purple vs. fresh NPA. However, at > 30 µM ‘purple NPA’, seedlings exhibited an unusual ring of magenta staining at the hypocotyl-root transition zone (RTZ) and at other locations. Subsequently, we observed that solutions of fresh NPA (> 30 µM) also induced staining at the RTZ, but the staining took longer to develop and was more diffuse than the ‘purple NPA’ staining. Plant Physiol. Biochem., 0981-9428/99/6/© Elsevier, Paris

Preliminary spectroscopic analysis of the pigment extracted from the stained region indicated that the coloration was not due to anthocyanins. NPA is described by the manufacturer (Chemservices, West Chester, PA) as a light purple powder. In aqueous solution, the amide bond of NPA becomes hydrolyzed, yielding phthalic acid and α-naphthylamine [12, 38]. The reaction is catalyzed by the undissociated carboxyl group of the phthalic acid moiety [13]. Although α-naphthylamine, itself, is a light red color, it is likely that α-naphthylamine generated by NPA hydrolysis gradually oxidizes to nitronaphthylene and subsequently combines with another α-naphthylamine to form the deep magenta-colored dye, 1,1'-azonaphthylene (ANA) [15]. Based on these studies of NPA in solution, we inferred that the dark purple color of old solid NPA is due to the formation of ANA. The two questions we

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set out to investigate were: a) What is the chemical basis of the staining reaction? and b) What causes the localization of the reaction at the hypocotyl-root transition zone and at other regions? Our results suggest that NPA initially binds to, and is slowly cleaved by, amidases (amido hydrolases) localized at the regions that become stained. The release of α-naphthylamine leads to the formation of 1,1'-azonaphthylene in the tissue. Based on the different kinetics of the staining reactions with ‘purple NPA’ vs. fresh NPA, we infer that a trace amount of α-naphthylamine or ANA in the NPA solution is required to act as a nucleating agent for the heavy accumulation of ANA at the sites of NPA cleavage. We also present evidence that a NPA amidase activity is localized on the plasma membrane and that this activity is inversely correlated with the amount of measurable NPA-binding activity of the plasma membrane.

2. RESULTS 2.1. Sites of NPA Staining Figure 1 A and B show examples of a control Arabidopsis seedling and a seedling germinated in the presence of 100 µM ‘purple NPA’, respectively. The seedling treated with ‘purple NPA’ exhibited severe inhibition of growth, especially of the root, which is characteristic of the effects of herbicidal levels of NPA. In addition, a ring of staining occurred at the hypocotyl-root transition zone (RTZ) and at the epicotyl-hypocotyl transition zone (HTZ) of the ‘purple NPA’-treated seedling. Staining of the HTZ was discontinuous. In some cases (only faintly visible in this micrograph), a ring of staining was also visible at the distal elongation zone (DEZ) of the root. Higher magnification views of the ‘purple NPA’ staining reaction are shown in figure 2. No visible

Figure 1. NPA staining of Arabidopsis seedlings. A, Control; B, 100 µM ‘purple NPA’; C, 100 µM fresh NPA; D, 5 µM αNA; E, 100 µM fresh NPA plus 5 µM αNA; F, 30 µM fresh NPA plus 5 µM αNA; G, 1 mM fresh NPA plus 0.05 % Fast Blue B. HTZ, Cotyledon junction; RTZ, hypocotyl-root transition zone; DEZ, distal elongation zone.

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Figure 2. Specific regions stained by fresh NPA (100 µM) plus α-naphthylamine (5 µM). A, Seedling grown for 3 d in the staining medium showing staining near tips of cotyledons; B, surface view of the RTZ of a seedling grown for 2 d in ¼ MS and transferred for 3 d to the staining medium; C, cross-section of the stained RTZ of seedling incubated as in B, showing staining of epidermal and stelar cells (arrow); D, staining around the HTZ and the margins of cotyledons (seedling grown as in B); E, the RTZ of a seedling grown for 2 d in ¼ MS and transferred for 4 d to the staining medium; F, staining of the stele in the lower root of a seedling grown for 4 d in ¼ MS and transferred for 2 d to the staining medium; G, staining of the vascular tissue and bases of root primordia in a seedling grown for 5 d in ¼ MS and transferred for 2 d to the staining medium; H, staining of the vascular tissue in the inflorescence of a 5-week-old plant which was treated with 100 µM fresh NPA plus 5 µM α-naphthylamine in lanolin paste.

staining occurred prior to the end of the second day of germination, even when the embryos were dissected out of their seeds for viewing. Staining of the cotyledons, particularly near the tips, first appeared in 3-d seedlings (figure 2 A). In 5-d seedlings, the cells of the RTZ, which were papillate in shape, became uniformly stained over their entire surfaces (figure 2 B), and staining was primarily in the epidermis and vascular tissue (figure 2 C). At about the same time, the region around the HTZ became stained (figure 2 D) and the staining of the RTZ became more intense and extensive (figure 2 E). By 6 d, the stele of the root became stained (figure 2 F), and by 7-d staining was observed at the base of branch roots (figure 2 G). Seedlings exposed for longer than 3 d developed amorphous dye deposits on cotyledon surfaces (data not shown) similar to those described in peanut and several weed species after simultaneous treatment with paraquat and NPA [36]. We also examined staining of reproductive structures by applying the ‘purple NPA’ in lanolin

paste to the inflorescence. As shown in figure 2 H, the vascular system of the inflorescence stem is stained. In addition, faint staining of papillate cells of the stigma was detected (data not shown). In all tissues examined, green tissues became increasingly chlorotic after the first 48 h of treatment, consistent with the herbicidal levels applied.

2.2. Chemistry of the NPA staining reaction Based on reports in the literature on the degradation of NPA in solution, as well as on our own preliminary spectral analyses of the ‘purple NPA’ and extracts of stained RTZ tissue (see Introduction), we hypothesized that the staining was caused by the aggregation of 1,1'-azonaphthylene (ANA), which forms spontaneously in an oxidative N-coupling reaction between two α-naphthylamines (αNA), as diagrammed in figure 3 A. Subsequent analysis of the ‘purple NPA’ by LC-MS confirmed that the original reagent bottle contained, in addition to NPA and naphthylphthalimvol. 37 (6) 1999

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Figure 3. Diagrams of coupling mechanisms for the histochemical detection of NPA amidase activity. A, Proposed oxidative coupling reactions for the detection of NPA amidase activity with αNA. B, Standard azo dye coupling reactions for the detection of aminopeptidase activity using Fast Blue B [10].

ide (NPI), a trace amount (< 0.01 %) of 1,1'-ANA (figure 4 A), as well as trace amounts of αNA and phthalic acid (table I). The simplest explanation for the NPA staining is that the contaminating ANA in the ‘purple NPA’ solution specifically binds to the hypocotyl-root transition zone and other regions of Arabidopsis seedlings.

If so, the total amount of ANA present in the tissue of the stained seedlings should be less than or equal to the total amount of ANA originally present in the incubation medium. To test this hypothesis, dye was extracted from 200 NPA-stained seedlings grown in 100 µM ‘purple NPA’ and, following separation by HPLC, the peaks were analyzed by mass spectrometry

Table I. Azonaphthylene (ANA + AONA) and α-naphthylene (αNA) recovery from purified fractions quantitated by integration of the HPLC absorbance peaks. The percent recovery (% recovery) after processing was determined by dividing the amount (i.e. integrated value of peaks normalized to an internal standard) of αNA or ANA + AONA measured in the processed sample by the amount measured in an equal quantity of unprocessed ‘purple NPA’ reagent (× 100). The normalized recovery value represents the amount of αNA or ANA + AONA measured in processed samples corrected for the losses due to processing (see Methods). Recovery values > 1 reflect formation of product. The samples processed were ‘purple NPA’ reagent, growth medium containing ‘purple NPA’ (without seedlings) after 72 h, or extracts from NPA-treated seedlings. The values, represent the mean ± SD from two separate experiments. As determined by ES/MS+ standardized to anthracene and calibrated with CsI (see Methods), ‘purple NPA’ was determined to contain 1.23 % αNA and 0.016 % ANA. Sample (% recovery) ‘purple NPA’ (from 1.17 µg or 1.17 mg sample) Normalized recovery Growth medium – no seedlings (from 40 mL of 100 nM or 100 µM ‘purple NPA’/¼ MS medium) TZ extract (200 seedlings grown in 100 nM or 100 µM NPA)

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100 nM NPA (1.17 µg reagent)

100 µM NPA (1.17 mg reagent)

αNA

ANA + AONA

αNA

ANA + AONA

83 ± 16

86 ± 12

92 ± 9

81 ± 9

1.34 ± 0.79

1.13 ± 0.24

1.86 ± 0.98

1.63 ± 0.20

3.19 ± 1.36

20.01 ± 6.92

4.66 ± 2.03

26.07 ± 4.35

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Figure 4. Electrospray (+) mass spectrum analyses. A, ‘Purple NPA’ reagent in 95 % acetonitrile. B, ‘Purple NPA’ after purification on C18 SepPak cartridges and C18 normal phase/reverse phase HPLC (see Methods). C, C18 SepPak/HPLC-purified RTZ extract from 200 seedlings. Abs peaks, spectroscopic absorbance maxima. Identities of compounds are indicated in parentheses next to molecular mass peaks. ANA, Azonaphthylene; AONA, azoxynaphthylene; NPA, N,1-naphthylphthalamic acid; NPI, N,1-naphthylphthalamide.

(see Methods). As a control, 1.17 mg ‘purple NPA’ reagent and the same volume of incubation medium containing 1.17 mg ‘purple NPA’ (40 mL/100 µM) without seedlings were separately subjected to the identical extraction procedure and analysis. The mass spectral data from the HPLC fractions, representing the second major absorbance peak obtained from the medium control and tissue extract samples, are shown in figure 4 B and C and summarized in table I. The amount of ANA and its oxidative coupling intermediate, 1,1’-azoxynaphthylene (AONA), extracted from the stained transition zones of the seedlings and quantitated by integration of HPLC absorbance peaks exceeded the amount of these compounds present in the original incubation medium by 26-fold (table I). Similar ANA + AONA concentrations were calculated when the second HPLC peak was quantitated by comparison of mass spectral intensities (figure 4 B, C) to internal standards (data not shown). At the 72 h time point, the αNA content (contained primarily in HPLC fractions corresponding to the first major absorbance peak) of both stained tissue extracts and 100 µM

‘purple NPA’ media also significantly exceeded the αNA content of both 1.17 mg of ‘purple NPA’ and ‘purple NPA’ medium incubated without seedlings (table I). Therefore, the vast majority of the ANA in the tissue must be formed in situ rather than being bound from the external medium. The only conceivable source of the ANA in the tissue is the αNA released by the breakdown of NPA by an amidase. This rapid NPA hydrolysis requires living tissue, since it occurred very slowly when seedlings were absent from the medium (see table I). To further test the hypothesis that ANA is formed when αNA is released from NPA, we attempted to reconstitute the reaction using fresh, undegraded NPA and a trace amount (5 µM) of αNA to nucleate the aggregation of the dye. We found that 100 µM undegraded (fresh) NPA did cause some staining, but to a lesser extent than the ‘purple NPA’, consistent with a slower reaction rate (figure 1 C). Figure 1 D shows that 5 µM αNA alone caused neither staining nor marked growth inhibition. However, when 5 µM αNA was added to 100 µM fresh NPA, the effect on staining vol. 37 (6) 1999

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was synergistic (figure 1 E). This suggests that a small amount of αNA in the medium, present either as a contaminant, supplement or product of NPA hydrolysis, is needed to nucleate the reaction forming ANA and to immobilize the product in situ. To determine the lowest NPA concentration at which staining can be detected, Arabidopsis seedlings were treated with NPA concentrations ranging from 10–100 µM in the presence of 5 µM αNA. The lowest concentration of NPA causing visible staining of the RTZ is 30 µM (figure 1 F). When the tissue extraction experiments described above were repeated with media containing only 100 nM (1.17 lg) ‘purple NPA’, hydrolysis products were recovered in proportions similar to those recovered from 100 µM samples (table I), indicating that the putative amidase can hydrolyze NPA at both high and low concentrations.

2.3. Fast Blue B staining and other histochemical assays Based on the foregoing, the enzyme that releases αNA from NPA is most likely an aryl amidase, as diagrammed in figure 3 A. A widely used method for the histochemical localization of aminopeptidases (amidases) involves immobilization of the cleavage product by the formation of a covalent bond to an azo dye such as Fast Blue B [6, 31]. This azo dye coupling reaction is illustrated in figure 3 B. Unlike the slow ‘purple NPA’ staining reaction described above, rapid azo dye coupling involves diazonium-naphthyl (C) coupling (figure 3 B) to form large, insoluble molecules rather than the oxidative (N) coupling of the amino groups of two naphthylamines to form ANA (figure 3 A). The result, however is the same: enzymatically hydrolyzed naphthylamine products are immobilized by covalent incorporation into insoluble compounds that aggregate in situ. To confirm that an amidase is involved in the hydrolysis of NPA at the root-hypocotyl transition zone, we subjected 5-d seedlings to instantaneous Fast Blue B histochemical staining with NPA as the substrate (see Methods). Fast Blue B alone did not stain the seedlings red or purple in any specific region (data not shown). However, as shown in figure 1 G, the cleavage of NPA at the epicotyl-hypocotyl transition zone (HTZ) and the hypocotyl-root transition zone (RTZ) could be readily detected by azo dye coupling with Fast Blue B, independently confirming that enzymatic amido bond cleavage of NPA diagrammed in figure 3 A takes place in vivo at the RTZ and HTZ. (The bright yellow color of the root is a photographic artifact.) Plant Physiol. Biochem.

To determine whether other related enzyme activities were also localized at the RTZ and HTZ, we also carried out a number of other histochemical assays (see Methods), all with negative results (data not shown). No co-localization of polyphenol oxidase, general esterase or myrosinase activity was found. Peroxidase activity, as indicated by staining with 3,3-diaminobenzidine and peroxide was found throughout the root, but not in the RTZ or HTZ regions. Staining of phenolic compounds, lipids, tyramine conjugates, and carbohydrates yielded dissimilar staining patterns in each case. Thus, none of the various staining reactions can account for the specific staining pattern observed with ‘purple NPA’.

2.4. Staining of transparent testa mutants Although preliminary absorption spectra of stained transition zone extracts suggested that the majority of the pigment was not due to anthocyanins, we also examined the NPA staining reaction in the anthocyanin deficient mutants, transparent testa (tt) 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, and 7-1 [18]. To allow for slightly slower rates of germination of some of the tt mutants, the seedlings were allowed to germinate for 60 h rather than 48 h before being placed on NPA/¼ MS medium. As shown in figure 5, the staining patterns of all the mutants tested were similar to that of wild type. Thus, the staining reaction is not due to anthocyanin accumulation.

2.5. [3H]-NPA whole tissue localization studies It is possible that the sites of NPA staining in Arabidopsis seedlings do not correspond to the sites of NPA cleavage, but rather, to sites where the hydrolysis product αNA combines oxidatively to form ANA. In other words, NPA might be cleaved throughout the seedling, but ANA forms only at the HTZ, RTZ and DEZ. If the latter is correct, [3H]-NPA would not be expected to co-localize with the staining reaction in binding studies. To determine whether the stained regions were sites of NPA accumulation, Arabidopsis seedlings were incubated in the presence of 10 µM cold NPA containing 37 Bq⋅mL–1 [3H]-NPA which was labeled on the phthalic acid moiety. Calcium acetate-rinsed seedlings of uniform length were selected at three time intervals, 0.5 mM transverse serial sections were prepared, and the radioactivity in each section was determined (see Methods). As shown in table II, after 24 h, the level of radioactivity in the RTZ was about 20-fold higher than in the other regions. The HTZ was also a site of

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Figure 5. Comparison of staining of wild type (WT) and transparent testa mutants (tt1–7) in the presence of 100 µM ‘purple NPA’.

[3H]-NPA accumulation. These results indicate that the sites of NPA staining correspond to sites of NPA binding and cleavage. The decrease of measured disintegrations in the RTZ and HTZ after 24 h of NPA treatment may indicate the gradual cleavage of the bound NPA (accompanied by the slow diffusion of the released [3H]-labeled phthalic acid into the medium) as well as a decrease in NPA binding activity, perhaps as a result of ANA accumulation at binding sites. Presumably, radioactivity initially accumulates at the sites of staining because binding of [3H]-NPA is relatively rapid compared to cleavage.

2.6. Light regulation of NPA staining The effects of different light treatments on the staining reactions and morphologies of seedlings treated with 50 µM NPA plus 2.5 µM αNA are shown in figure 6. All the seedlings were germinated for 2 d in white light on control medium and transferred to 95 µM NPA plus 5 µM αNA and subjected to various light treatments for an additional 3 d. Control seedlings for each light treatment are shown in figure 6 A–F. Note that the irradiance (60 µE⋅m–2⋅s–1) used in this set of experiments is about half that used in

figure 1 (110 µE⋅m–2⋅s–1), and that all of the light treatments were preceded by 2 d in white light. Hence, the morphologies of the control seedlings differ from those grown continuously under the specific light treatments. This is particularly true in the case of the blue treatment, in which the hypocotyl of the wild type elongated nearly to the same extent as the hy4 mutant. In white light, NPA stained the epicotyl-hypocotyl transition zone (HTZ) (upper arrow), the hypocotylroot transition zone (RTZ) (middle arrow), and the root distal elongation zone (DEZ) (lower arrow) (figure 6 G). Growth was also severely inhibited, as evidenced by the swollen, truncated hypocotyl and the short, hairless root. No NPA staining was observed in the dark, and the seedlings resembled the minus NPA controls, except for the absence of root hairs (figure 6 H). The effects of red, far-red, and blue light are shown in figure 6 I–K. Growth in red (figure 6 I) resulted in little or no staining by NPA, and NPA-induced growth inhibition of the root was also less severe compared to white light. However, NPA induced curvature of the hypocotyl in red light and prevented hypocotyl greening, despite the absence of staining. In far-red

Table II. Net mean (background subtracted) disintegrations normalized to fresh weight of 0.5-mm sections of [3H]-NPA-treated Arabidopsis seedlings. Measurements that were not significantly different from background are indicated as NS. Background measurements ranged from 14–18 DPM⋅mg–1. Tissue section Cotyledon tip HTZ Upper hypocotyl Lower hypocotyl RTZ Root tip

24H (DPM⋅mg–1 fw)

36H (DPM⋅mg–1 fw)

48H (DPM⋅mg–1 fw)

57 ± 11 292 ± 12.6 NS NS 333 ± 15.8 NS

NS 248 ± 3.1 NS NS 306 ± 48.1 NS

18 ± 7.3 NS NS NS NS NS

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Figure 6. Effects of light on controls (A–F) and after NPA staining (G–L). A/G, White light; B/H, dark; C/I, red light; D/J, far-red light; E/K, blue light; F/L, blue light with hy4 mutant.

(figure 6 J), the RTZ (inset) as well as linear files of cells in the vascular tissue (arrows) became stained. Although NPA staining was apparent in far-red light, growth inhibition was less severe than in white light. Blue light caused strong NPA staining of the HTZ, RTZ and DEZ, as well as faint staining in the cortex of the hypocotyl (figure 6 K). The hypocotyl was also truncated and swollen, and the root was strongly inhibited. Blue light thus appears to be the most effective wavelength for promoting the staining reaction as well as growth inhibition in the presence of NPA. To determine whether the promotive effect of blue light on staining is due to physiological regulation or to dye photochemistry, the hy4 mutant was tested. The gene responsible for the hy4 mutation, CRY1, encodes for the putative blue light photoreceptor involved in the growth inhibitory effects of blue light in Arabidopsis [1]. As shown in figure 6 L, the hy4 mutant was neither stained nor strongly inhibited by NPA in blue Plant Physiol. Biochem.

light. Thus, blue light-stimulated NPA staining requires a functional CRY1 gene.

2.7. Inhibitors of NPA staining Various compounds capable of displacing NPA from plasma membranes were tested for their ability to inhibit the NPA staining reaction. A NPA-treated control seedling is shown in figure 7 A. As shown in figure 7 B, curcumin, which inhibited root growth in all treatments, prevented the staining reaction, while both genestein (figure 7 C) and quercitin (figure 7 D) inhibited the staining reaction and partially reversed growth inhibition. In contrast, the non-phytotropin auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), failed to block NPA staining (figure 7 E). Since some arylamidases are aminopeptidases (APs) requiring zinc, calcium or both for activity [37], we tested the effects of EDTA on the NPA staining reaction. Figure 8 A and B show control seedlings treated with 120 µM EDTA and 100 µM fresh NPA

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Figure 7. Effects of NPA binding inhibitors on NPA staining. All treatments are 100 µM undegraded NPA/5 µM αNA plus the treatment indicated. A, NPA control; B, 5 µM curcumin; C, 100 µM genestein; D, 1 µM quercitin; E, 20 µM TIBA.

plus 5 µM αNA, respectively. When EDTA was added to the NPA solution, the staining was reduced (figure 8 C). Addition of 150 µM ZnCl2 to the EDTA solution partially restored the staining at the RTZ, although the color was somewhat altered (figure 8 D). Similar results were obtained with 150 µM MnCl2 (data not shown). Thus, at least some of the staining at the RTZ by NPA is mediated by a metalloenzyme.

of the plasma membrane fraction, as indicated by marker enzyme analysis, was comparable to those published previously for Arabidopsis tissues (table III).

2.8. Direct measurement of NPA amidase activity

To distinguish between integral and peripheral membrane proteins, the plasma membranes were subjected to phase separation using Triton X-114 (see Methods), and the aqueous phase (containing peripheral proteins) was analyzed (table III).

Although the histochemical assays are highly suggestive, direct biochemical measurements are necessary to confirm the presence of the NPA amidase and AP activities in Arabidopsis tissues. As a first step toward a biochemical characterization of these activities, we prepared microsomal membranes from homogenates of light-grown Arabidopsis seedlings and obtained purified plasma membrane fractions using the aqueous polymer two-phase system. The enrichment

NPA amidase activity was measured spectrophotometrically using an azo coupling method for detecting the release of α-naphthylamine (see Methods). The soluble, microsomal, plasma membrane, and Triton X-114 aqueous fractions were assayed at 20 °C. Hydrolysis was not detectable when samples were assayed after a 30-min incubation at 4 °C (data not shown). Boiled samples also exhibited no measurable NPA hydrolyzing activity.

Figure 8. Effects of EDTA plus or minus Zn on the staining activity of NPA. A, 100 µM NPA/5 µM αNA; B, 100 µM NPA/5 µM αNA plus 120 µM EDTA; C, 100 µM NPA/5 µM αNA plus 120 µM EDTA plus 150 µM Zn. Seedlings were germinated for 2 d on ¼ MS and transferred for 3 d to the staining medium.

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Table III. Marker enzyme assays of membrane fractions. Membrane fractions (microsomal, plasma membrane and Triton X-114 aqueous) were assayed for marker enzyme activities (1–5), chlorophyll content (6), and total protein (7). The enzymatic assays utilized and the values obtained for the microsomal fraction (in parentheses) are: 1, VO4-sensitive ATPase (127 µmol Pi released⋅min–1⋅mg–1 protein); 2, NO3-sensitive ATPase (389 µmol Pi released⋅min–1⋅mg–1 protein); 3, DCCD-sensitive pyrophosphatase (309 µmol Pi released⋅min–1⋅mg–1 protein); 4, NADH-cytochrome c reductase (272 µmol cytc reduced⋅min–1⋅mg–1 protein); 5, latent IDPase (894 µmol Pi released⋅min–1⋅mg–1 protein); 6, chlorophyll (271 µg⋅mg–1 protein); 7, total protein (576 mg⋅100 g–1 fw). Calculated average values for assays of three different preparations are given. Assay 1 2 3 4 5 6 7

Plasma membrane (% microsomal)

Triton X-114 aqueous (% microsomal)

402.4 0.7 17.0 38.9 25.7 0.0 2.6

88.0 1.0 9.0 25.0 0.01 1.0 0.1

Plasma membrane ATPase Tonoplast ATPase Tonoplast PPase ER NADH-cytochrome c reductase Golgi latent IDPase Thylakoid chlorophyll Total protein

Figure 9 A shows a pH curve for the NPA amidase activity of the purified plasma membrane fraction. There is a sharp optimum at about 6.4, but the curve is highly asymmetrical, with a broad shoulder of activity on the alkaline side, and a possible small shoulder at around pH 6.0. The complexity of the pH curve suggests that NPA is being cleaved by more than one enzyme. When measured at pH 6.4, NPA amidase activity was detected in the microsomal, plasma membrane and Triton X-114 aqueous phase fractions, but not in the soluble fraction (figure 9 B). Low levels of NPA hydrolysis could be detected in the soluble fraction at pH > 7.6 (data not shown). The specific activity of the Triton X-114 aqueous phase was four to eight times

greater than that of the microsomal and total plasma membrane fractions. In two separate experiments, the NPA amidase activity of the Triton X-114 aqueous (peripheral membrane protein) fraction represented an average of 77 % of the total recovered activity of the reconstituted combined hydrophobic and hydrophilic Triton X-114 phases of the plasma membrane fraction (figure 9 B). However, such results must be interpreted with caution, since detergent activation of the hydrophilic phase or detergent inhibition of the hydrophobic phase may be occurring. The effects of some modulators of amidase or aminopeptidase activity are summarized in table IV. Consistent with the results of histochemical assays, NPA hydrolysis was reduced by both 5 mM EDTA and

Figure 9. NPA hydrolysis and binding. A, The effect of pH on the NPA amidase specific activity of purified plasma membranes. B, Kinetics of NPA hydrolysis by membrane-bound and soluble fractions measured by spectroscopic absorbance of αNA-sulfosomidine conjugates. (·), Soluble fraction; (d), microsomal fraction; (M), total plasma membrane; (´), plasma membrane Triton X-114 aqueous phase (at 20 min time point, aqueous phase represented 77.5 % of total recovered activity – see Methods). C, Specific binding of [3H]-NPA to plasma membrane-enriched fractions at pH 5.3, 6.5 and 7.5 following a temperature shift. (´), pH 5.3; (d), pH 6.4; (_), pH 7.5.

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Table IV. Effects of aminopeptidase modulators on NPA amidase activity in plasma membrane enriched fractions from Arabidopsis seedlings. NPA hydrolysis was monitored by formation of sulfisomidine-αNA conjugates monitored at 540 nm. Net values (i.e. minus A540 boiled controls) ± cumulative SD are normalized to total protein. Significance of pairwise comparisons by Student’s t-test are indicated in t-statistic and probability (P) columns. Inhibition of triiodobenzoic acid and curcumin could not be determined due to interference of the inhibitors with the αNA/sulfisomidine assay. Treatment

Specific activity (nmol αNA⋅mg–1 protein)

% Control

t

P

244 ± 23 66 ± 31 53 ± 19 481 ± 41 235 ± 17 57 ± 26 209 ± 31 154 ± 28 39 ± 40 34 ± 52

100.0 27.2 21.7 197.1 96.2 23.4 85.5 63.2 16.1 13.8

7.839 10.923 – 8.701 0.562 11.184 1.569 4.230 7.564 13.457

0.001 < 0.001 < 0.001 0.604 < 0.001 0.192 0.013 0.002 < 0.001

Control 5 mM EDTA 2 mM EGTA 100 µM MnCl2 100 µM PMSF 1 µM bestatin 5 mM DTT 100 µM genestein 100 nM quercitin 100 nM phloretin

2 mM EGTA; 1 µM bestatin, a specific inhibitor of metallo-APs, also inhibited hydrolysis, while PMSF and DTT did not (table IV). In the presence of 1 mM MnCl2, an activator of metallo-APs, the specific activity increased nearly 2-fold. Phloretin, quercitin and, to a lesser extent, genestein also inhibited NPA hydrolysis. The effects of curcumin and TIBA on NPA hydrolysis could not be determined due to interference with the sulfosomidine assay.

2.9. Hydrolysis vs. binding The presence of NPA amidase activity in Arabidopsis extracts is a potential complicating factor in [3H]NPA binding assays. If hydrolysis occurs, the labeled phthalic acid moiety – which is highly water soluble – would diffuse away, leading to an underestimate of the amount of binding. If so, NPA binding activity might be inversely correlated with NPA amidase activity. To test this hypothesis, NPA binding to plasma membranes was measured at two different temperatures (4 °C and room temperature) and at three different pHs: 5.3, 6.4 and 7.5. The experiments were conducted as follows. The first binding measurements were made after a 1-h incubation at 4 °C; the samples were then shifted to room temperature and additional binding measurements were made at 30, 60 and 90 min. As shown in figure 9 C, NPA binding activity by the plasma membrane was highest at pH 5.3 at 4 °C, the conditions at which the NPA binding assay is normally carried out. These conditions also correspond to those at which NPA amidase activity is negligible (see figure 9 A). At pH 6.4, the pH optimum of NPA cleavage, the binding at 4 °C was reduced by about

one third. However, at room temperature and pH 6.4 – the conditions at which amidase activity is maximal – the amount of NPA binding decreased by ∼90 % during the first 30 min. The effect of temperature on binding is less drastic at the other pHs. Thus, there is an inverse relationship between NPA binding and NPA amidase activity in the purified plasma membrane fraction.

3. DISCUSSION 3.1. The NPA staining reaction We made the serendipitous discovery that solutions of old, partially degraded NPA stain Arabidopsis seedlings at the hypocotyl-root transition zone and other regions, including the epicotyl-hypocotyl transition zone, the distal elongation zone, the bases of lateral root primordia and the vascular bundles of the inflorescence. Using absorbance and mass spectrometry, we detected trace amounts of αNA and ANA in the solid reagent as well as in the stained tissue. Subsequent quantitative analysis indicated that the amount of ANA accumulating in the tissue farexceeded the initial amount of ANA in the incubation medium, indicating that ANA was being formed in situ by the hydrolysis of NPA. Under the mild conditions utilized in these studies, abiotic formation of ANA from NPA is a very slow process and clearly did not take place to any significant degree in the control (minus seedling) experiments. After 72 h, the combined amount of ANA and AONA formed abiotically in solution under conditions vol. 37 (6) 1999

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identical to those used for incubating seedlings was less than two times the amount found in the starting material (table I). In vivo, αNA, one of the products of the apparent enzymatic hydrolysis of the NPA amide bond, steadily accumulates (data not shown) and subsequently conjugates in an oxidative amino coupling reaction to form ANA, which finally aggregates to produce discrete, localized staining. Although the coupling reaction may result directly from the interaction of free naphthylamines with nitrene intermediates formed during peptide (amide) bond hydrolysis [9], it may also involve additional enzymatic activities. In bacteria, peroxidases have been shown to conjugate aromatic amines to form azo linkages [15], but oxidases, nitrilases or aromatic aldoxime-synthetic enzymes could also conceivably perform that function in plants. Further studies on the chemical mechanism of coupling are needed for a complete understanding of the NPA staining reaction. We postulated that the ability of ‘purple NPA’ to stain the tissue was due to the ability of trace amounts of ANA in the solution to promote the aggregation (immobilization) of the ANA produced in the tissue. Hydrophobic azonaphthylene dyes are known to form highly associated aggregates in aqueous environments. For example, a sulfonated diphenyl bis azonaphthylene, Congo Red (MW = 697 Da), yields an apparent molecular weight of 8 000 Da when analyzed by gel chromatography [32]. Naphthylamine-conjugated substrates have been widely used in histochemical coupling reactions to detect the activities of a variety of hydrolytic enzymes, including peptidases, glucosidases and phosphatases [11]. The effectiveness of these staining reactions depends on the ability of a coupling compound to rapidly immobilize, and thus localize, the cleaved substrate. Because the staining reaction depends on aggregation of the product, this would explain the requirement for relatively high NPA concentrations (> 30 µM), the initially slow rate of staining, and the enhancement of staining found when ANA or αNA was included with undegraded NPA in the reconstitution studies (see figure 1 E). Since ‘purple NPA’ concentrations ≤ 30 µM did not result in any staining, NPA might be cleaved nonspecifically as part of a detoxification mechanism. However, the results of LC-MS analysis of the reaction products at 100 nM NPA (see table I) demonstrated that cleavage also occurs at nanomolar NPA concentrations. Although NPA is cleaved at low concentrations, the reaction is difficult to detect histochemically, presumably because the ANA fails to aggregate and diffuses into the medium. Consistent with this interPlant Physiol. Biochem.

pretation, labeling studies with [3H]-NPA at 20 nM, a concentration too low to cause staining, demonstrated that NPA localizes at the sites of staining, but the label is gradually lost due to diffusion out of the tissue. When localized to the catalytic site of an enzyme, ANA may itself act as an inhibitor of amidases or aminopeptidases. In vivo, ANA may eventually become sulfonated as indicated by unidentified minor MS molecular mass peaks at 368 and 451 Da that were found to contain both naphthyl- and oxygencontaining groups (data not shown). Sulfonated bisazonaphthylene dyes such as the selective protease inhibitor Congo Red have carbohydrate- and glycoprotein-binding characteristics [26] and some sulfonated cyclic compounds have been found to inhibit NPA activity [34]. Unfortunately, the spectroscopic interference of ANA and other bis-azo dyes at 540 nm precludes spectroscopic studies of their effects on NPA hydrolysis kinetics. We conclude that ‘purple NPA’ staining is due to the ability of the ANA contaminant in solutions of partially degraded NPA to facilitate the immobilization (by hydrophobic aggregation) of the newly synthesized ANA at the sites where it is produced by amidase action [32]. Since staining is inhibited by EDTA, activated by Mn2+, and restored by low concentrations of Zn2+, at least part of the activity appears to involve metalloenzymes. The NPA staining reaction was found to be lightdependent. In agreement with the results of Jensen et al. [17], blue light was required for optimal growthinhibition by NPA. Blue light wavelengths were also the most effective for the NPA staining reaction. Since the hy4 mutant was relatively insensitive to NPA in blue light, and was equally refractory in the NPA staining assay, the light dependence of the staining reaction cannot be due to simple dye photochemistry, but requires a functional blue light signal transduction pathway. NPA staining was also blocked by several compounds – curcumin, genestein, and phloretin – which have been shown to displace NPA from its binding site on plasma membranes [3, 16]. Genestein and quercitin also partially reversed the effects of NPA on growth at concentrations consistent with those found to inhibit NPA binding in zucchini hypocotyls [3, 16]. As discussed below, one possible interpretation of these results is that the amidase responsible for the NPA staining reaction either binds NPA, or regulates NPA binding.

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3.2. Biochemical assays In addition to the histochemical tests, we also performed enzymatic assays with membrane and soluble fractions from Arabidopsis seedlings. NPA amidase activity was detected in both the microsomal and purified plasma membrane fractions, but not in the soluble fraction. The specific activity was particularly high in a Triton X-114 aqueous phase of purified plasma membranes, which corresponds to peripheral membrane proteins. However, low levels of activity were also detected in the detergent-soluble fraction, considered to be integral plasma membrane proteins (data not shown). Since a NPA-hydrolyzing enzyme is, by definition, a NPA-binding protein, these findings may be significant in view of the recent controversy concerning the identity of the putative NPA-binding protein as an integral [4] vs. peripheral [8] plasma membrane protein. To examine the relationship between NPA binding to the plasma membrane and NPA amidase activity, binding assays were carried out at three different pHs and at two different temperatures. NPA binding was shown to be inversely correlated with NPA amidase activity, i.e. under conditions giving high amidase activity, NPA binding was low, and vice versa. This suggests that NPA hydrolysis may be a hitherto unrecognized complicating factor in NPA binding studies. Aryl amidase activity has been identified in herbicide metabolism studies of a number of plants [19], but, to our knowledge, it has only been assayed in crude extracts. Although attention has focused on soluble APs ( [2, 35] and references therein). APs have also been identified in microsomal membranes [29]. The sensitivity of NPA hydrolysis to EGTA, bestatin, and MnCl2 and insensitivity to DTT and PMSF are consistent with metallo-aminopeptidase activity [35]. In the accompanying paper [23], the properties of several aminopeptidases associated with Arabidopsis plasma membranes are described. Finally, the finding that αNA is released from NPA in plant tissues has potential implications for the use of certain pesticides on agricultural crops. Since αNA, aniline, azobenzene and other substituted aromatic amines that could potentially be produced by the aryl amidase/AP activity described herein have previously been identified as carcinogens [28], the safety of some applications of widely used arylamide pesticides may need to be re-evaluated.

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4. METHODS 4.1. Materials Arabidopsis thaliana Columbia (Col-0) wild type and transparent testa mutant seeds were obtained from the Arabidopsis Biological Resources Center at Ohio State. All reagents were obtained from Sigma (St. Louis, MO) with the exception of the following: N,1-naphthylphthalamic acid, which was originally obtained from Frinton Laboratories (Vineland, NJ) (purple or partially degraded NPA) and subsequently from Chemservices (West Chester, PA) (fresh NPA); and 2,3,4,5-[3H]-N,1-naphthylphthalamic acid (labeled on the phthalic acid moiety), which was a gift from Dr Paul Bernasconi and Novartis Crop Protection, Inc. (Palo Alto, CA). 1,1'-Azonaphthylene, 1,2'azonaphthylene, and 2,2'-azonaphthylene were synthesized according to the method of Naito et al. [24]. 4.2. Staining of seedlings with NPA Arabidopsis seeds were surface sterilized with 10 % Clorox for 5 min, rinsed with sterile water, and dried with ethanol. With the exception of the initial experiments in which seeds were germinated directly on NPA-amended media, seeds were first planted on ¼ Murashige Skoog basal salts (MS), 10 mM MES, pH 6.2 (¼ MS) in 0.15 % Phytagel, plus 0.05 % DMSO. After 48-h incubation (60 h in the case of transparent testa mutant staining) in continuous light (Sylvania Gro-Lux bulbs, 110 µE⋅m–2⋅s–1 at surface level) at 22 °C, seedlings were transferred to identical media augmented with the treatment noted and incubated for 72 h at the same light and temperature conditions. Seedlings for membrane preparations were grown for 6 d on vertical mesh transfer plates with ¼ MS as the growth medium as previously described [22] For light treatments, plants were grown for 2 d on control media in white light, and transferred to Petri dishes containing ¼ MS + 100 µM NPA in 1 m3 light boxes. The light sources consisted of two Sylvania broad spectrum fluorescent tubes (for white and blue light) or three 100-W incandescent bulbs (for red and far-red) equipped with heat diffusers and cutoff filters that supplied white (open window), red (2423 acrylic – Rohm and Haas, Philadelphia, PA), blue (2045 acrylic – Rohm and Haas), or far-red (Roscolene #874 + #863 + Roscolux #94, Rosco Labs, Stamford, CT) light at 60 µE⋅m–2⋅s–1. 4.3. Other histochemical assays Instantaneous amidase azo dye coupling assays were carried out using the methods described by vol. 37 (6) 1999

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Frank [10] and Chayen et al. [6]. First, 5-d-old Ws Arabidopsis seedlings were incubated for 1 h at 30 °C in a buffer of ¼ MS, 20 mM MES, 1 mM NaN2, 5 % DMSO, 0.05 % Fast Blue B salt (pH 6.1), and 1 mM fresh NPA. Seedlings were washed in 50 mM CuSO4 for 2 min to stop the reaction [6] and photographed immediately through a dissecting microscope. All photographs of histochemical assays were taken with Kodak Ektachrome 200 daylight film using a dissecting microscope-mounted camera fitted with a polarizing filter. This method resulted in better visualization of staining than was found with tungsten film but, because a daylight filter was not used, also caused a yellow cast that could only be partially removed in subsequent image processing. Control histochemical enzyme assays for polyphenol oxidase (EC 1.14.18.1), general esterase, and myrosinase (EC 3.2.3.1) activities were performed as described by Gahan [11] utilizing diethyldithiocarbamate, α-naphthol AS D acetate dye coupling, and sinigrin assays, respectively. Peroxidase (EC 1.11.1.7) activity was localized using the method of ThordalChristensen et al. [33] with 3,3-diaminobenzidine and peroxide as substrates. Nonspecific staining of phenolic compounds was localized with ferric chloride [11], lipids were stained with Oil Red O and Sudan Red B [5], carbohydrates were localized with both benzidine and phthalic acid [7], and hydroxycinnamoyl transferase (EC 2.3.1.110) activity was localized with 2 mM tyramine [25].

4.4. [3H]-NPA localization and binding Seedlings were grown for 72 h as above, then transferred to media containing 20 nM unlabeled NPA and 37 Bq⋅mL–1 [3H]-NPA. Twenty seedlings were selected at each time point, rinsed twice in ice cold 10 mM calcium acetate, then fresh sectioned transversely in 0.5-mm segments using a razor blade. Sections were selected from the cotyledon tips, cotyledon node/apical tip region, upper hypocotyl, lower hypocotyl, transition zone and root tip of each seedling, pooled accordingly, blotted dry for 5 min, weighed, and counted in a Beckman LS5801 scintillation counter after 24 h to allow dissipation of any chemiluminescence. The experiment was repeated three times and values reported are the means and standard deviations. NPA binding experiments were conducted as described by Bernasconi et al. [3, 4], except that 25 mM MES-BTP buffers of constant ionic strength at pH 5.3, 6.4 and 7.5 were utilized in place of sodium citrate and 1 mM NaMoO4 was omitted. Samples were incubated Plant Physiol. Biochem.

in 5 nM [3H]-NPA at 4 °C for 1 h, then incubated at room temperature for 30, 60 and 90 min. Membrane proteins were collected on 0.1 % polyethyleneiminetreated GF-B (Whatman, Maidstone, Kent, England) glass fiber filters as described by Bernasconi et al. [3, 4], washed with 5 mL cold buffer, and assayed for bound [3H]-NPA in a Beckman LS-5801 scintillation counter. [3H]-NPA specific binding activity was determined by subtracting the activity of samples incubated with 5 nM [3H]-NPA + 5 µM cold NPA. In place of dialysis, Triton X-114 aqueous fractions were washed extensively on microconcentrating spin filters as described above before use in NPA-binding assays. The binding assays of Triton X-114 fractions were then performed within the same microconcentrating filters. Unbound [3H]-NPA was removed by two cycles of centrifugation and washing with 400 mL binding assay buffer, followed by centrifugation to near dryness. The microconcentrating filters were then placed directly in scintillation fluid and counted in a Beckman LS5801 scintillation counter. Background activity was determined by repeating the assay with microconcentrating filters only.

4.5. Extraction of azo dyes and analysis by absorbance and mass spectroscopy Arabidopsis thaliana Wassilewskija seedlings were germinated under light as above for 54 h, then transferred to Petri dishes containing 40 mL ¼ MS in 0.15 % Phytagel supplemented with 100 µM ‘purple NPA’ for 72 h. The green tissue above the purplestained transition zone from 200 seedlings grown on the same plate was excised and the remaining tissue was rinsed in ice cold 12 Mohm water, blotted dry for 5 min, and weighed. The seedlings were then incubated in 5 mL dimethylformamide with shaking for 30 min to extract the dye, then centrifuged for 5 min at 16 000 × g. The supernatant was collected into a fresh tube and the volume was brought up to 100 mL with 12 Mohm water, and applied in 20-mL fractions to 1 mL C-18 Sep Pak cartridges (Waters, Milford, MA). Each column was washed with 20 mL water, and eluted with acetonitrile. The eluate was reduced under vacuum to 500 µL, yielding a dark magenta color. The extract was separated by combined reverse and normal phase HPLC using a 5-µ 4.2 × 25 cm C-18 column (Supelco). Solvent flow was 0.5 mL⋅min–1 with water and acetonitrile containing 0.2 % formic acid delivered in the following program: initial – 80 % acetonitrile: 20 % water; 15 min ramp to 96 % acetonitrile:4 % water; 5 min hold at 96 % acetonitrile:4 % water;

A. Murphy, L. Taiz

15 min ramp to 80 % acetonitrile:20 % methanol. Fractions (0.5 mL) were collected corresponding to the absorbance peaks detected at 254 nm, brought to a pH of 7.2 with 0.1 N NaOH, lyophilized, and resuspended in 100 mL 80 % acetonitrile. For purposes of comparison, 1.17 mg ‘purple NPA’ was dissolved in acetonitrile and assayed directly by ES/MS+ and absorbance spectroscopy. In addition, 1.17 mg ‘purple NPA’ in 5 mL dimethylformamide was also processed and assayed by the same method used with the tissue extracts described above. Finally, 40 mL ¼ MS + 100 µM NPA medium exposed to the same light conditions as seedlings for 72 h was extracted with 5 mL dimethylformamide, then processed and analyzed utilizing the same method as was used with tissue extracts. Each experiment was repeated twice. Aliquots (20 lL) from the collected HPLC fractions were utilized for each absorbance or mass spectroscopy measurement. Quantitation of compounds was accomplished by integration of HPLC absorbance peaks at 254 nm using 1 µM anthracene as an internal standard and confirmed by comparison of electrospray MS intensities from different samples to internal anthracene standards after calibration with CsI. For analysis of UV absorbance profiles, a sample from each fraction was added to 0.5 mL cyclohexane or separately to 0.5 mL ethanol and scanned from 220–390 nm in a Spectronic Genesys5 spectrophotometer (Spectronic, Rochester, NY). For visible absorbance measurements, each sample was dissolved both in cyclohexane and, to maximize the absorbance of primary amines, separately in cyclohexane/0.1 N HCl then scanned from 400–700 nm. Molecular masses and argon collision daughter constituents of the HPLC fractions were measured with a Quattro II quadrupole electrospray mass spectrometer and analyzed with MassLynx software (Micromass/Waters, Manchester, UK). Spectra were compared to published databases. After determining that the molecular mass peak (298 Da) of the second HPLC peak contained 1,1’azoxynaphthylene (AONA), pure ANA was processed as described above. Preliminary experiments established that the majority product (87 %) detected by MS was AONA, indicating that the purification process converted most of the ANA in extracts to AONA. Some naphthylphthalimide (NPI), which is in equilibrium with NPA in aqueous solution, was also detected in the NPA solutions. To determine whether ANA is formed in tissues in the concentration range at which NPA acts as a phytotropin rather than an herbicide (e.g. ∼100 nM

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NPA), all of the above analytical procedures were repeated with a 1/1 000 dilution of ‘purple NPA’ (100 nM NPA media or 1.17 lg solid reagent).

4.6. Preparation of plasma membrane and soluble protein fractions The preparation of microsomal and soluble membrane fractions was based on the methods described by Hall and Moore [14], and Shimogawara and Usuda [30]. Although the results presented are for seedlings, similar results were also obtained using young inflorescences. First, 35 g of tissue from 6-d-old seedlings or early inflorescences without opened flowers were collected and immediately ground in a mortar and pestle on ice in 100 mL 0.3 M sucrose, 25 mM Hepes (pH 8.5), 50 mM EDTA, 0.5 % BSA, 3.5 mM DTT, 0.5 g PVP, 200 ng⋅mL–1 leupeptin, then centrifuged at 8 000 × g, 4 °C for 15 min. To obtain a microsomal pellet, the supernatant from the 8 000 × g spin was collected and centrifuged at 100 000 × g, 4 °C for 45 min. Microsomal pellets were resuspended in a buffer consisting of 10 mM BTP-Mes (pH 7.8), 0.1 % Brij-35, 250 mM sucrose, 20 % glycerol, 0.5 % BSA, 1 mM DTT (resuspension buffer) and again centrifuged at 100 000 × g for 30 min, after which the supernatant was discarded. Enriched plasma membrane fractions were obtained directly from microsomal pellets using the phase separation method of Shimogawara and Usuda [30]. After addition of glycerol to 15 % (v/v), the purified plasma membrane fractions were frozen in liquid nitrogen until use. For the soluble fraction, the supernatant from the 100 000 × g spin was collected, incubated on ice for 2 h with stirring after addition of solid (NH4)2SO4 to 75 % saturation, then centrifuged at 8 000 × g, 4 °C for 30 min. The pellets were collected, dialyzed against resuspension buffer overnight at 4 °C, then frozen in liquid nitrogen until use. Integral and peripheral membrane proteins of the plasma membranes were further fractionated by Triton X-114 phase separation as described by Bernasconi et al. [4]. Both aqueous and detergent phases obtained in this manner were washed with 400 lL resuspension buffer on Microcon 10 (Amicon, Beverly, MA) spin filter columns and centrifuged to near dryness. The washing procedure was repeated six times, the retentate was eluted with resuspension buffer and then frozen in liquid nitrogen until further use. As specific NPA binding activity has been found to decrease after Triton X-114 phase separation [4], total recoverable activity for all amidase activity investivol. 37 (6) 1999

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gated was determined by combining the phases obtained by Triton X-114 phase separation in a Microcon 10 spin filter, centrifuging to near dryness at 4 °C, and washing four times with 400 lL AP assay buffer (as above) to remove detergent. Samples were then resuspended in 100 lL assay buffer before protein quantitation and amidase enzyme assays.

4.7. NPA amidase assay A colorimetric NPA amidase assay was developed based on a method for quantitation of micromolar concentrations of free αNA in aqueous solution described by Younis and Bashir [39]. Aqueous αNA concentrations from 2–20 µM can be determined by coupling αNA with freshly diazotized sulfisomidine and measuring the absorbance of the azo-coupled product at 540 nm with a relative SD of 0.35–4.2 %. Unlike the Younis assay, which requires acidic conditions and the activation of the primary amino group of free αNA by treatment with HONO, the NPA amidase assay measures the well-documented rapid coupling of diazotized sulfosomidine with activated naphthylamines released when a naphthylamide conjugate, such as NPA, is hydrolyzed [6, 10]. This NPA amidase assay yields reproducible results in a pH range of 5.2–8.0 (data not shown). Under the conditions utilized, there was no measurable sulfosomidine coupling product when free αNA was incubated with boiled plasma membrane or soluble fractions for 30 min at room temperature (data not shown), after which time the diazotized sulfisomidine is no longer stable [39]. All assays were restricted to 20 min, since some of the inhibitors tested (bestatin, curcumin, genestein and quercitin) caused an increase in background absorbance at 540 nm in the boiled controls after this time period. Diazotized sulfosomidine solution (100 mL) was added to samples containing ∼75 ng protein in 700 mL aryl amidase (AA) assay buffer consisting of ¼ MS/0.1 % Brij 35/25 mM BTP-Mes pH 6.4 (unless otherwise indicated) and the reaction was initiated with 2 mL 100 mM NPA (final concentration = 250 µM) in DMSO. Release of αNA was monitored continuously at 540 nm. Absorbances of boiled extract blanks were simultaneously subtracted at each timepoint. Concentrations of αNA solutions, prepared in boiled sample fractions diluted in AP assay buffer augmented with 3 mM HONO, were calibrated using a αNA standard range of 2–20 µM. 4 °C assays were conducted by incubating samples at 4 °C and measuring at room temperature (approximately 5 s) at 5-min intervals. The results of the continuous NPA hydrolysis Plant Physiol. Biochem.

assay were confirmed by the quantitation of αNA formation by post coupling with Fast Garnet GBC.

4.8. Other enzyme and protein assays Membrane marker enzymes were assayed as follows: chlorophyll (thylakoid), NADH-cytochrome c reductase (endoplasmic reticulum – EC 1.6.99.3), and latent IDPase (Golgi apparatus – EC 3.6.1.6) were assayed as described in Hall and Moore [14]. DCCDsensitive pyrophosphatase (tonoplast – EC 3.6.1.1) activity was assayed according to the method of Zhen et al. [40] using 500 µM DCCD. Vanadate sensitive (plasma membrane – EC 3.6.1.35) and nitrate sensitive (tonoplast – EC 3.6.1.3) ATPase activities were assayed using the method described by Müller et al. [21] utilizing 100 µM vanadate and 100 mM nitrate. Total protein was quantitated with the Bio-Rad protein quantitation reagent (Bio-Rad, Hercules, CA). To reduce detergent interference with protein determination, it was necessary to dilute samples 1:100 in water, precipitate with trichloroacetic acid, and wash the pellets twice with acetone before quantitation. In some cases, total protein was confirmed by quantitation with Nano-Orange reagent (Molecular Probes, Eugene, OR) or amido black [27].

4.9. Cautionary note Naphthylamines and their derivatives are extremely hazardous and carcinogenic substances that should be handled with extreme caution. Even short-term exposure of small dosage poses a considerable health risk [28].

Acknowledgments We thank Drs Paul Bernasconi and Mathias Müller for valuable discussions during the course of this work. We also thank Drs Jack Okamura and Diane Jofuko for the generous use of their microscope. This research was supported by Grant No. 94-37100-0755 from the United States Department of Agriculture.

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