Regulation of auxin transport by aminopeptidases and ... - Springer Link

Planta (2000) 211: 315±324

Regulation of auxin transport by aminopeptidases and endogenous ¯avonoids Angus Murphy, Wendy Ann Peer, Lincoln Taiz Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA Received: 2 December 1999 / Accepted: 16 January 2000

Abstract. The 1-N-naphthylphthalamic acid (NPA)binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin eux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophanspeci®c aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, amino acyl-b-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin eux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more eective inhibitors of low-anity speci®c [3H]NPA-binding than were the ¯avonoids quercetin and kaempferol but had no eect on high-anity binding. Since the APs are inhibited by ¯avonoids, we compared the localization of endogenous ¯avonoids and APs in seedling tissue. A correlation between AP and ¯avonoid localization was found in 5- to 6-d-old seedlings. Evidence that these ¯avonoids regulate auxin accumulation in vivo was obtained using the ¯avonoid-de®cient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root.

Abbreviations: aa-NA = amino acyl-b-naphthylamide; AP aminopeptidase; DPBA = diphenylboric acid 2-aminoethyl ester; MS = Murashige & Skoog; NA = naphthylamine; NPA = 1-Nnaphthylphthalamic acid; Pro = proline; Trp = tryptophan; Tyr = tyrosine Correspondence to: L. Taiz; E-mail: [email protected]; Fax: +1-831-459-3139

These results implicate APs and endogenous ¯avonoids in the regulation of auxin eux. Key words: Aminopeptidase ± Arabidopsis(auxin transport) ± Auxin ± Flavonoid ± 1-Nnaphthylphthalamic acid ± Polar transport(auxin)

Introduction A key feature of the plant growth hormone auxin (indole-3-acetic acid or IAA) is its unique ability to be transported in a polar fashion from shoot tip to root tip in a wide range of plant species (reviewed by Goldsmith 1977). There is convincing evidence that the polarity of auxin transport is governed at the cellular level and consists of two primary processes: IAA uptake and IAA eux. Indole-3-acetic acid can be taken up by cells either by diusion across the membrane in the protonated form or by proton co-transport via a carrier protein (reviewed by Lomax et al. 1995; Bennett et al. 1998; Leyser 1999). Eux is mediated by auxin-anion eux carriers. Recently, genes encoding both the auxin in¯ux and eux carriers have been cloned and localized in Arabidopsis (Bennett et al. 1996; Chen et al. 1998; GaÈlweiler et al. 1998; Luschnig et al. 1998; MuÈller et al. 1998; Utsuno et al. 1998; Marchant et al. 1999). Consistent with previous immunological studies (Jacobs and Gilbert 1983), the eux carriers have been localized at the basal ends of parenchyma cells presumed to be involved in polar transport. The preferential localization of the eux carriers at the base of each shoot cell, or at the apical end of root cells, is thought to determine the general polarity of auxin transport in plant tissues (Bennett et al. 1998; Leyser 1999). There is pharmacological evidence that polar auxin transport is regulated. Speci®cally, a group of synthetic auxin transport inhibitors (ATIs) block polar transport (Katekar and Geissler 1977). The herbicide 1,N-naphthylphthalamic acid (NPA) is an example of an ATI that

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A. Murphy et al.: Role of aminopeptidases and endogenous ¯avonoids in auxin transport

can block auxin eux from plant cells. When auxin eux is blocked, continued cellular uptake of the hormone usually results in auxin accumulation. The NPA does not appear to compete with IAA, but instead binds to a plasma-membrane protein that regulates the IAA eux carrier. Since certain naturally occurring ¯avonoids, e.g. quercetin, apigenin, and kaempferol, have been found to displace NPA from its binding site and also block polar auxin eux and stimulate auxin accumulation (Jacobs and Rubery 1988; Faulkner and Rubery 1992; Bernasconi et al. 1996), it has been suggested that polar auxin transport may be regulated in vivo by endogenous ¯avonoids. We recently reported that NPA is enzymatically cleaved to naphthylamine (NA) and phthalic acid in light-grown Arabidopsis thaliana seedlings (Murphy and Taiz 1999a). When NPA was used at concentrations ³30 lM, oxidative coupling of the liberated NAs produced magenta-colored staining. The reaction was localized in three zones: immediately below the cotyledonary node, the hypocotyl-root transition zone (referred to hereinafter as the transition zone), and the root distal elongation zone. In biochemical assays, NPA amidohydrolase activity localized to the plasma membrane with the majority of the activity partitioning with fractions enriched in peripheral membrane proteins. We hypothesized that aminopeptidases (APs) might be involved in hydrolyzing the amide bond of NPA. Using amino acyl b-naphthylamide (aa-NA) conjugates as substrates, tyrosine (Tyr)-, tryptophan (Trp)-, and proline (Pro)speci®c AP activities were found to co-localize with NPA hydrolysis in light-grown seedlings (Murphy and Taiz 1999b). The speci®city of the AP activity was subsequently con®rmed biochemically with tripeptides and amino acyl conjugates of both aminomethylcoumarin and amino¯uoromethylcoumarin. Soluble Pro-AP activity was consistent with that of cytosolic APs (Bartling and Weiler 1992) while Tyr/Trp-AP activities were NPA-activated and exhibited characteristics consistent with previously described IAA-amidohydrolases (Davies et al. 1999). However, plasma-membrane Tyr-, Trp-, Pro-, and Xaa-Pro-speci®c AP activities were inconsistent with previously described plant hydrolase activity and were strongly inhibited by the NPA-binding antagonists quercetin and kaempferol, although poorly inhibited by NPA itself (Murphy and Taiz 1999b). The ®ndings that ¯avonoid-sensitive APs hydrolyze NPA at speci®c regions of Arabidopsis seedlings and that arti®cial AP substrates have NPA-like growth-inhibiting activity raise the possibility that APs may play a role in auxin transport. However, it has not yet been established that the same AP substrates that inhibit growth also inhibit auxin eux. Nor has it been shown that the regions where the APs are localized are sites of auxin transport regulation, that is, auxin accumulation. The current investigation had four main objectives: (i) to test whether the AP substrates (aa-NA conjugates) having ATI-like activity also block auxin eux; (ii) to test whether the AP inhibitor bestatin inhibits auxin eux and/or NPA-binding to microsomal membranes; (iii) to determine whether endogenous ¯avonoids co-localize

with APs in Arabidopsis seedlings; and (iv) to determine whether endogenous ¯avonoids regulate auxin accumulation in vivo. Materials and methods Reagents and seed stocks. All chemical reagents were from Sigma (St. Louis, Mo., USA) with the exception of NPA (Chemservices, West Chester, Pa., USA) and [3H]NPA (donated by Novartis Crop Protection, Palo Alto, Calif., USA). All seed stocks were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Net auxin uptake. Seedlings of Arabidopsis thaliana (L.) Heynh. were grown in Vertical Mesh Transfer (VMT) tanks as previously described (Murphy and Taiz 1995). Net auxin uptake by tissue sections of 5-d-old seedlings was measured by the method of Garbers et al. (1996), except that sections of both light- and darkgrown upper hypocotyls and hypocotyl-root transition zones were assayed. The sectioning method was as described below except that only upper-hypocotyl and transition-zone sections were utilized. The incubation medium contained 1.0 lM [14C]IAA [1.11 e+13 Bq (300 nCi) mL)1]. The concentration of the aa-NA conjugates was 10 lM. Bestatin was used at 1 lM. Tripeptides were used at 100 lM. Control uptake experiments replacing [14C]IAA with [14C]benzoic acid revealed no signi®cant dierence between aa-NA treatments and controls. Counts of radiolabeled IAA were determined in a Beckman LS 5801 scintillation counter after a 24-h equilibration period to allow dissipation of chemiluminescence. Assay of NPA binding. Competition of bestatin and aa-NA with NPA binding was assayed as described by Ruegger et al. (1997) and Muday et al. (1993), except that samples were incubated for 30 min at 1 °C (on ice in 4 °C cold room) to limit the eects of hydrolysis of both NPA and inhibitors. Microsomal vesicles were prepared as described previously (Murphy and Taiz 1999a). Membranes (0.2 mg dry weight) were resuspended in 200-lL aliquots of NPA binding assay buer. Assays of [14C]IAA transport and leakage in whole seedlings. Seedlings were grown in a VMT tank on quarter-strength Murashige and Skoog (1962) nutrient solution (1/4 MS) (pH 6.2) for 5 d, then transferred to 1/4 MS, 1/4 MS + 10 nM naringenin, or 1/4 MS + 10 nM NPA, as noted, for 12 h. The seedlings were then transferred to fresh 1/4 MS, after which 0.2 lL of 10 nM [14C]IAA in ethanol [85 e + 12 Bq (50 nCi) lL)1] was applied with a 1-lL glass syringe to the apical tip of each seedling. The seedlings were then incubated for 4 h in white light. Ten seedlings from each treatment were rinsed brie¯y in 1/4 MS and divided under humid conditions into four 2-mm segments as follows: (i) cotyledons were removed and the apical 2 mm of upper hypocotyl was excised; (ii) roots and remaining hypocotyl tissue were excised from the transition zone, leaving a 2-mm section corresponding to the transition zone plus a small amount of root and shoot tissue ¯anking it on either side; (iii) a 2-mm section was excised from the basal portion of the upper root; (iv) the apical 2 mm of the lower root was excised. Radioactivity in each set of segments was determined as described above. Because of the small amounts of tissue involved, neither dry nor fresh weights of individual sections could be reproducibly measured. To allow comparison, 200 seedlings were sectioned and the total fresh weights of the various tissue segments were determined. When determined in this manner, the fresh weight of light-grown transition zone » light-grown upper hypocotyl, dark-grown transition zone »1.3 dark-grown upper hypocotyl. To study auxin accumulation by root tips, 20 Arabidopsis seedlings were grown as above except that, during the ®nal 4 h incubation, the apical 2 mm of each seedling root rested on a


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0.5 cm ´ 4 cm strip of Whatman 3 MM paper mounted on Saran Wrap. After 4 h, the isolated paper strips were placed in scintillation ¯uid and counted. Statistical treatments. All uptake and transport experiments were repeated three times and the data shown represent the means and SD of three experiments. Statistical signi®cance was calculated in a pairwise fashion using Student's t-test. Analysis of variance of treatments vs. controls was calculated by the method of NeumanKeuls. Flavonoid localization studies. Seedlings were grown in VMT tanks. Where indicated, the medium was supplemented with 10 nM naringenin at the end of 3 d. Seedlings were stained as described by Sheahan and Rechnitz (1993) except that staining was for 5±15 min using saturated (0.25%) diphenylboric acid 2-aminoethyl ester (DPBA) and either 0.02% (whole seedling and plasmolysis photographs) or 0.005% (tissue localization photographs) Triton X-100 (v/v). Where indicated, seedlings were plasmolyzed with 2 M mannitol for 5 min prior to staining. The ¯uorescence was visualized on a ¯uorescence microscope with a ¯uorescein isothiocyanate ®lter (excitation 450±490 nm, suppression LP 515 nm) or on a Leitz DMIRB inverted photoscope equipped with a Leica TCS NT laser confocal imaging system. Flavonoid identities were determined by visualizing standards with and without DPBA. Fluorescence maxima within the ®lter range were obtained for the following standards: naringenin (515 nm, 522 nm, 523 nm, 527 nm), phloretin (518 nm, 520 nm, 523 nm), apigenin (516 nm), taxifolin (dihydroquercetin) (517 nm, 522 nm), kaemperferol (520 nm), quercetin (543 nm), and quercitrin (534 nm); ¯avonols and dihydro¯avonols ¯uoresce 10- to 100-fold more than other ¯avonoids. Con®rmation of ¯avonoid identity. Flavonoid localization was con®rmed microscopically by visualization after treatment with NH3 vapor for 2 min or 5% AlCl3 in 95% ethanol (v/v) for 10 min. Methanolic extracts of the seedling sections described in transport experiments above were further analyzed by TLC ( NH3), UV/ vis spectrophotometry (Harborne et al. 1975; Markham 1982), and HPLC. Flavonoid glycosides and aglycones were prepared for analysis from seedling sections as described by Burbulis et al. (1996) and analyzed by HPLC using a C18 column (Supelco, 5 lm; 250 mm long, 4.2 mm i.d.), with UV absorbance monitoring at 254 nm. Separation was achieved with a gradient of 2% methanol in water (pH 3.0 with H3PO4) and acetonitrile at a ¯ow rate of 1 ml min)1 with a highly concave pro®le (16 min for unhydrolyzed ¯avonoids, 30 min for aglycone products of acid hydrolysis). Spectral characteristics of collected peaks were further analyzed as described by Markham (1982) and Harborne et al. (1975).

Results Aminopeptidase substrates and bestatin inhibit auxin eux. The ability of various aa-NA conjugates to inhibit auxin eux, as re¯ected by an increase in net uptake of [14C]IAA, was tested in both the upper hypocotyls and transition zones of 5-d-old Arabidopsis seedlings. The concentration used (10 lM) is below the threshold for the histochemical staining reaction described previously (Murphy and Taiz 1999b). When net uptake was measured in the upper hypocotyls, NPA increased the net uptake of IAA by 80% and 95%, respectively, in dark-grown and light-grown upper hypocotyls (Fig. 1A). Smaller increases in net IAA uptake were also observed in dark-grown upper hypocotyls with Trp-NA (54 21%), Pro-NA (38 24%), and GlyPro-NA (54 21%). A small

Fig. 1A,B. Net [14C]IAA uptake by light-grown and dark-grown Arabidopsis seedlings in the presence of 10 lM NPA, aa-NA conjugates, or bestatin (error bars indicate SD, n = 3). A Twomillimeter-long upper hypocotyl sections. B Two-millimeter-long sections centered on the transition zone

but signi®cant increase (34 9%) was observed in light-grown upper hypocotyls with Tyr-NA. However, His-NA and Ala-NA (not shown) caused no net increase in auxin uptake in either light-grown or dark-grown upper hypocotyls unless used in 10-fold excess. In preliminary experiments, when 200 seedlings were sectioned as described, the fresh weights of light-grown upper hypocotyls were approx. 2.3-fold those of darkgrown upper hypocotyls. As such, normalized net auxin uptake in control dark-grown upper hypocotyls was approx. 2-fold that of light-grown upper hypocotyls. The ability of AP substrates to mimic NPA in blocking auxin eux suggests two possibilities: either APs form part of the NPA-binding auxin transport regulatory complex, or the aa-NA conjugates are suciently similar to NPA in structure that they bind to the NPA-binding protein. Enhancement of net auxin uptake by bestatin, an inhibitor of most APs, would support the former possibility. As shown in Fig. 1A, treatment with 1 lM bestatin enhanced net auxin uptake approx. 2-fold in dark-grown upper hypocotyls and approx. 4-fold in light-grown upper hypocotyls. Thus bestatin is more active than NPA in light-grown seedlings. When normalized to approximate fresh weights (see Materials and methods) the stimulation of net IAA

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uptake by NPA and the aa-NA conjugates was more pronounced in the transition zone sections than in the upper hypocotyl sections. Although NPA more than doubled net IAA uptake in light-grown seedlings, it had little or no eect in dark-grown seedlings. In light-grown seedlings, Tyr-, Trp-, and Pro-NA all caused an increase in net IAA uptake equal to or greater than that of NPA. Only Trp-NA increased net IAA uptake in transition zones of dark-grown seedlings. In contrast to the upper hypocotyl, net uptake in light-grown transition zone sections increased less than 2-fold after bestatin treatment, while net uptake in dark-grown transition zone sections was not signi®cantly dierent from controls (Fig. 1B). We also tested the eects of various tri-peptides (10 lM) on net auxin uptake in the transition zone. Of these, only Tyr-[Tyr/Pro]-Xaa and Trp-[Trp/Phe/Tyr]Xaa treatments increased net IAA uptake over the controls: 26 6% and 45 5%, respectively. The reduced ability of the tripeptides to inhibit auxin eux when compared to aa-NA conjugates may be related to the apparent higher anity of APs for aa-NA conjugates or, more likely, to the localized aggregation of the oxidatively coupled naphthylamines released by aa-NA conjugates after AP hydrolysis (Murphy and Taiz 1999a,b). Aminopeptidase substrates and bestatin compete with lowanity NPA binding to microsomal membranes. Competition assays using 3H-labeled NPA were conducted to determine whether aa-NA conjugates and bestatin compete with NPA for its binding site in microsomal membranes. Since maximal eects of both the aa-NA conjugates and bestatin on net auxin uptake were observed in light-grown seedlings, tissue from 6-d light-grown seedlings was utilized to prepare membrane vesicles. The results are shown in Fig. 2. Tyrosine-, Trp-, and, to a lesser extent, Pro-NA caused a decrease in NPA binding in a saturable manner, while His-NA had no signi®cant eect in the nanomolar range (Fig. 2A). The maximum NPA displacements by Tyr-, Trp-, and Pro-NA were 25, 23, and 21%, respectively. The halfmaximum displacement concentrations were 38, 114, and 130 nM, respectively. As shown in Fig. 2B, increases in bestatin concentration above 5 nM resulted in decreases in speci®c [3H]NPA binding, with a half-maximum displacement of 16.5% being reached at 31 nM and a maximum displacement of 33% being reached when bestatin was used in 50-fold excess (250 nM). Further increases in bestatin concentration had no eect other than the reduction of non-speci®c binding, as indicated by the slight increase in speci®c binding with bestatin concentrations greater than 250 nM. To compare these results with previously published reports of ¯avonoid displacement of NPA in darkgrown zucchini hypocotyl microsomes (Jacobs and Rubery 1988; Bernasconi 1996), speci®c NPA binding in the presence of quercetin and kaempferol was assayed. Increases in concentrations of both ¯avonoids resulted in a nearly linear decrease of speci®c NPA

Fig. 2A,B. Inhibition of speci®c NPA binding by bestatin, quercetin, and kaempferol. Microsomal vesicles prepared from Arabidopsis seedlings were incubated with 5 nM [3H]NPA and inhibitors at the concentrations indicated for 30 min, bound to glass ®lters containing 0.3% polyethyleneimine, washed and assayed in scintillation ¯uid (see Materials and methods). Speci®c binding was determined by incubating identical samples with 10 lM cold NPA (error bars indicate SD, n = 3). A j, Trp-NA; d, Tyr-NA; s, Pro-NA; n, His-NA. B j, bestatin; d, quercetin; m, kaempferol

binding (Fig. 2B). The NPA binding decreased to 60±65% with inhibitor concentrations in 200-fold excess (1 lM) (Fig. 2B) and was reduced to near background levels by ¯avonoid concentrations greater than 50 lM (data not shown). Flavonoids co-localize with APs in Arabidopsis seedlings. If APs act as components of a regulatory complex that inhibits auxin eux, and if ¯avonoids serve as eectors of the complex in vivo, then endogenous ¯avonoids should co-localize with the APs in Arabidopsis seedlings. We used the ¯uorescent dye diphenylboric acid 2-aminoethyl ester (DPBA) for the dierential localization of various ¯avonoids in Arabidopsis seedling hypocotyls and roots. The procedure not only indicates areas of ¯avonoid localization, but also allows preliminary identi®cation based on color. Flavonoid localization and identities were con®rmed by a series of tests, including AlCl3, NH3 vapor, TLC, HPLC and UV/vis spectrophotometric analysis (see Materials and methods).


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For reference, Fig. 3A shows the histochemical localization of NPA hydrolysis, presumably representing the sum of the three AP activities speci®c for Tyr, Pro, and Trp (Murphy and Taiz 1999b). As reported previously, staining is present below the cotyledonary node, at the transition zone, and at the distal elongation zone. (The faint diagonal lines between the transition zone and distal elongation zone are caused by light refraction from the agar medium.) As a control, untreated wildtype Ler seedlings were examined for auto¯uorescence. Chlorophyll in the cotyledons and hypocotyl exhibited a red auto¯uorescence; a faint blue-green auto¯uorescence due to sinapate esters was distributed throughout the

seedling, but was masked by chlorophyll in the cotyledons and hypocotyls. Figure 3B shows a composite ¯uorescence micrograph of a DPBA-stained 5-d Ler seedling. The cotyledons, upper hypocotyl, and transition zone ¯uoresced orange, characteristic of quercetin. In the lower root (Fig. 3B, inset), chalcone-naringenin (bright yellow ¯uorescence) was observed in the distal elongation zone, and kaempferol (bright green ¯uorescence) in the root cap. This staining pattern was observed in all wild type seedlings. Lower root ¯uorescence is not evident in the whole-seedling composite due to reciprocity failure of the ®lm using the lower-magni®cation objective.

Fig. 3A±F. Co-localization of NPA and ¯avonoid staining in Arabidopsis seedlings. A Staining of NPA in a wild-type Ler seedling treated with 100 lM NPA. Arrows indicate areas of NPA hydrolysis (magenta staining). B Composite of a 5-d Ler seedling stained with saturated (0.25%) DPBA and 0.02% Triton X-100. Arrows indicate areas of ¯avonoid staining. Chlorophyll auto¯uorescence is red. Inset: detail of distal elongation zone and root tip. C In the transition zone of a 4-d Ler seedling, kaempferol (green ¯uorescence) is localized as a single row of cells (arrow), and quercetin (orange ¯uorescence) occurs

as a cone in the root cortex. D A 5-d tt4 (¯avonoid de®cient) seedling has chlorophyll and sinapate ester (light blue-green) auto¯uorescence only. Arrow indicates transition zone. E A 5-d tt4 seedling supplemented with 50 nM naringenin. Kaempferol, quercetin, and chalcone-naringenin selectively accumulate at the transition zone. F Top: confocal image of the midsection of a 5-d Ler transition zone. Bottom: ¯avonoids are localized to the plasma membrane (pm) in the root of a 5-d Ler plasmolyzed seedling treated with 2 M mannitol for 5 min prior to staining. cw, cell wall

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Higher magni®cation of the transition zone revealed a ring of green-¯uorescing, kaempferol-containing cells (observed in >95% of stained seedlings) above the orange-¯uorescing, quercetin-enriched tissue (Fig. 3C, arrow). This is the same ring of cells in which NPA staining initially develops (see Fig. 3A). The quercetincontaining cells formed a cone which extended into the stele of the root immediately below the ring of kaempferol-containing cells (Fig. 3C). This region corresponds to the site of NPA staining after prolonged exposure (Murphy and Taiz 1999a). Consistent with studies of gene expression (Shirley 1995), ¯avonoid ¯uorescence was ®rst detectable at 3 d, reached a maximum at 5 d, and diminished by 7 d. As determined by HPLC and spectrophotometric analysis, the visualized ¯avonoids were primarily aglycone or hydrophobic species. Signi®cant levels of ¯avonoid glycosides were ®rst detected in 7-d seedlings and were accompanied by a dramatic decrease in levels of hydrophobic species. The peak of ¯avonoid production at 5 d corresponds to the peak of AP activity (Murphy and Taiz 1999b). Flavonoid staining in tt4 mutants can be restored by application of the precursor naringenin. If ¯avonoids serve as endogenous eectors of the auxin eux regulatory complex, mutants de®cient in ¯avonoids should exhibit altered patterns of auxin distribution. The tt (transparent testa) mutants of Arabidopsis are blocked at various steps in the biosynthesis of anthocyanins and their ¯avonoid intermediates (Burbulis et al. 1996). Figure 3D shows the staining patterns in the tt4-1 mutant, which is nearly devoid of all ¯avonoids (Shirley et al. 1995). Because tt4 is a chalcone synthase mutant, ¯avonoid levels are drastically reduced, while an excess of sinapate esters accumulates (Li et al. 1993; Sheahan 1996). As shown in Fig. 3D, the only ¯uorescence visible in the stained tissue was from chlorophyll and sinapate esters in all observed seedlings. However, treatment of tt4 with the intermediate naringenin resulted in the production and accumulation of kaempferol and quercetin in the transition zone. Kaempferol accumulated in the epidermal cells of the transition zone, quercetin accumulated in interior cells, and chalcone-naringenin was present in patches of cells within the quercetin-rich zone (Fig. 3E). In 5-d naringenin-treated tt4 seedlings, staining was not detectable in the distal elongation zone, even though HPLC and spectrophotometric analysis of root tip extracts indicated that the ¯avonoid content had increased to approx. 25% of the wild-type level. However, within 24 h the staining of the distal elongation zone in the naringenin-treated tt4 seedlings exceeded that of the untreated wild type (data not shown). Thus, when tt4 was supplied with exogenous naringenin, the rate of ¯avonoid biosynthesis in the distal elongation zone lagged slightly behind that of the transition zone. These patterns were observed in all stained seedlings. Flavonoid-de®cient mutants have altered patterns of auxin distribution. To determine whether the ¯avonoid-de®cient mutant tt4 exhibited abnormal auxin transport

in whole seedlings, 0.2 lL of 10 nM [14C]IAA (50 nCi lL)1) was applied with a syringe to the apical tips of 5-d light-grown seedlings (10 per treatment), and the seedlings were incubated for 4 h in white light. The seedlings were then rinsed brie¯y in 1/4 MS, divided into four uniform sections, and counted for radioactivity. The sections, which included the upper hypocotyl, transition zone, upper root, and lower root, accounted for the entire axis below the cotyledons except for an approx. 1-mm section between the upper hypocotyl and transition zone. The results of the whole-seedling transport studies are shown in Fig. 4. In wild-type Ler seedlings, a steep gradient of [14C]IAA from the upper hypocotyl to the upper root was observed. Although radioactivity measurements could not be reproducibly normalized to either dry or fresh weights of individual upper root and lower root sections (see Materials and methods), from approximate measurements the amounts of [14C]IAA in the upper hypocotyl and lower root were about equal (fresh weight of upper hypocotyl » 1.4 ´ upper root » 7.4 ´ lower root). The pattern of radioactive auxin distribution in the ¯avonoid-de®cient mutant tt4 diered markedly from that of the wild type. The amounts of [14C]IAA in the upper hypocotyl, transition zone, and lower root were strongly reduced, while the amount of radioactivity in the upper root increased. The increase in the radioactivity of the upper root suggests that more auxin had been transported basipetally from the hypocotyl to the root in the tt4 mutant due to the absence of ¯avonoids. On the other hand, the reduced amount of radioactivity in the lower root suggests that [14C]IAA was leaking out of the root tip in the mutant. Naringenin restores the wild-type pattern of auxin transport in the tt4 mutant. If the altered pattern of auxin transport in tt4 is due to the absence of ¯avonoids, it should be possible to restore the normal pattern by supplying the intermediate naringenin to the mutant. As

Fig. 4. Polar transport of [14C]IAA by wild-type (wt) Ler and tt4 seedlings of Arabidopsis in control growth medium (MS) and by tt4 seedlings in medium augmented with 10 nM naringenin or NPA as indicated (error bars indicate SD, n = 3). Inset: Accumulation/eux of [14C]IAA from Ler and tt4 seedling root tips 10 nM naringenin


shown in Fig. 4, auxin distribution in naringenin-treated tt4 seedlings was similar to that of the untreated wild type. The eects of NPA on the distribution of [14C]IAA in the tt4 seedlings were similar to those of naringenin (Fig. 4). To determine whether the low levels of radioactivity in the lower root of tt4 was due to IAA leakage into the external medium, seedlings labeled with [14C]IAA were incubated on moistened ®lter paper and the radioactivity released into the paper surrounding the root was counted. As shown in the inset of Fig. 4, tt4 roots released about 3.5-fold more auxin into the external medium than wild-type roots. Treatment with 10 nM naringenin completely prevented the leakage of auxin by the tt4 roots. Flavonoids are localized to cell membranes. The wholeseedling [14C]IAA transport studies strongly suggest that endogenous ¯avonoids regulate auxin eux from cells during polar transport from the shoot tip to the root tip. However, to regulate auxin transport on the plasma membrane, ¯avonoids must be localized to the plasma membrane. Figure 3F (top) shows a confocal ¯uorescence micrograph of a DPBA-stained transition zone. By adjusting the focal plane it could be determined that the ¯avonoids are present in both epidermal and cortical cells of the transition zone, but appear to be absent from vascular tissue. In the plasmolyzed root cell shown in Fig. 3F (bottom), the ¯uorescence is clearly localized on the plasma membrane, although internal membranes also appeared to contain ¯avonoids. These patterns were observed in all stained seedlings. Discussion One or more NPA-binding proteins are thought to negatively regulate the auxin anion eux carrier involved in polar transport. Studies of NPA binding to plasma-membrane vesicles and solubilized membrane proteins suggest that NPA interacts with either an F-actin-binding peripheral membrane protein (Muday et al. 1993; Dixon et al. 1996; Butler et al. 1998), an integral membrane tyrosine kinase (Bernasconi et al. 1996), or both. In support of the two-binding-sites hypothesis, Michalke et al. (1992) have provided compelling evidence for both strong and weak NPAbinding sites in zucchini and suggest that the weak NPA-binding site is involved in regulating auxin transport. Additional support for this hypothesis comes from studies with membrane-impermeant sulfonated ¯avonoids which suggested that both extracellular and internal sites are present (Faulkner and Rubery 1992). Recent reports that plasma-membrane-localized NPA amidohydrolase activity appears to be at least partially composed of ¯avonoid-sensitive Tyr-, Trp, and Prospeci®c AP activities in Arabidopsis seedlings (Murphy and Taiz 1999a, b) further support the likelihood of multiple plasma-membrane NPA-binding sites, although only one may have auxin transport regulatory activity.

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Could APs, themselves, be involved in the NPAsensitive regulation of auxin eux? In animal cells, cell surface proteases and APs have been implicated in a variety of regulatory processes (Taylor 1996). Thus far, the systemin-processing protease in tomato is the only cell surface protease that has been shown to participate in plant signal transduction (Schaller and Ryan 1994). Interestingly, bestatin produces systemin-like eects in tomato, presumably as a result of an interaction with the systemin protease (Schaller et al. 1995). Two lines of evidence suggest a role for cell surface APs in regulating auxin accumulation. We previously reported that Tyr-, Pro-, and Trp-b-naphthylamide substrates have NPAlike inhibitory eects on growth in Arabidopsis seedlings (Murphy and Taiz 1999b). In this paper we have presented evidence that both bestatin and these same aa-NA substrates block auxin eux from tissues. Furthermore, the eects of both NPA and the aa-NA substrates on auxin eux are greater in the transition zone than in the upper hypocotyl, consistent with the localization of AP activity in the transition zone. Bestatin also inhibited auxin eux in the transition zone almost as much as the aa-NA conjugates. The presence of additional inhibition by bestatin in the upper hypocotyl suggests the presence of another bestatinsensitive AP with distinct auxin-regulatory functions in the upper hypocotyl. The dissociation of NPA from microsomal membranes by aa-NA conjugates and bestatin not only supports a role for APs in the regulation of auxin eux, but also suggests a site of AP activity. Inhibition of NPA binding by both aa-NA conjugates and bestatin is eective at low concentrations but then saturates at approx. 35%, thereby suggesting that both aa-NAs and bestatin compete with weak NPA binding at the site thought to correlate with auxin transport inhibition (Michalke et al. 1992). As early as 1987, Katekar et al. (1987) noted that NPA is 30- to 100-fold less active in bioassays than binding studies indicate, suggesting that NPA binding to non-receptor factors must take place. Katekar et al. (1987) even suggested that amidohydrolases might hydrolyze bound NPA, but did not suggest that amidases themselves might be NPA binding proteins. An alternative explanation to the discrepancy between activity and binding is that some APs act as regulators of auxin eux. Recently, three putative plasma-membrane-associated APs from Arabidopsis have been puri®ed, sequenced, and cloned in our laboratory (data not shown). Molecular genetic studies of these AP genes may provide clues to the speci®c mechanisms involved. The ability of ¯avonoids to displace NPA from its binding site and to block auxin eux from cells was ®rst demonstrated by Jacobs and Rubery (1988), and this observation has since been con®rmed by others (Bernasconi 1996). However, ¯avonoids appear to interact with both strong and weak NPA-binding sites. In the present study, the ¯avonoids quercetin and kaempferol caused a dose-dependent linear inhibition of NPA binding, suggesting either a weaker or, more likely, a

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non-competitive interaction with the proposed NPAbinding site on the aminopeptidase. The inhibitory eects of dihydrochalcones and ¯avonoids on a number of animal APs is well documented (Parellada 1995). We previously showed that Tyr-, Pro-, and Trp-AP activities are strongly inhibited by genestein, quercetin, and phloretin ± ¯avonoids known to displace NPA from its binding site (Murphy and Taiz 1999b). In addition, both the activity of NPA-hydrolyzing APs and seedling ¯avonoid biosynthesis reach maxima at 5 d (Murphy and Taiz 1999b; Pelletier et al. 1999). In blocking auxin eux, it seems unlikely that AP substrates and inhibitors simply mimic the binding of endogenous ¯avonoids to a single site. But, as shown in Fig. 5, there is some structural similarity between all of the compounds involved. For example, Tyr-NA is structurally similar to the highly eective phytotropin 2-naphthylphthalic acid (2-NPA), although Tyr-NA lacks the carbonyl group shown to be critical to phytotropin activity (Katekar et al. 1987). However, as suggested by Katekar et al. (1987), if sulfonated, phosphorylated, or hydrolyzed in vivo in such a way as to produce an acid group, aa-NA conjugates would then conform well to the criteria for interaction with the NPA-binding site. Bestatin also conforms well to the criteria established by Katekar et al. (1987) for phytotropin binding and activity, with the exception that the linkage between its aromatic ring and conjugated carbonyl-containing moiety is ¯exible rather than rigid. Finally, all of the molecules involved have the capacity to disrupt the activity in a catalytic site by coordination or chelation of a divalent cation. It is possible that all of the compounds interact with a single site, but more likely, interact with multiple sites with dierent speci®city or location as demonstrated previously by assays with membrane-impermeant sulfonated ¯avonoids (Faulkner and Rubery 1992). In this paper we have shown that in Arabidopsis seedlings, ¯avonoids co-localize with the APs as follows: (i) kaempferol and quercetin are localized in the regions of the cotyledonary node and transition zone; (ii) chalcone-naringenin accumulates at the distal elongation zone; and (iii) kaempferol is present in the root tip. The co-localization of ¯avonoids and APs in speci®c regions of the seedlings suggests that these tissues may be sites where auxin eux is regulated. Consistent with this idea, Mathesius et al. (1998a, 1998b) showed that ¯avonoids accumulate in cortical cells involved in the formation of clover root nodules

and that both ¯avonoids and NPA induce auxin accumulation in these cells. Aminopeptidase substrates in the form of aa-NA conjugates have a demonstrated NPA-like eect on growth (Murphy and Taiz 1999b), but the eects of endogenous ¯avonoids have been more dicult to document. Both quercetin and kaempferol are toxic to Arabidopsis in low nanomolar concentrations (data not shown) and their biosynthesis is tightly regulated throughout development (Burbulis et al. 1996; Pelletier et al. 1999). However, some morphological dierences, such as increased lateral root formation, decreased in¯orescence growth, decreased seed viability over time, and decreased cell elongation in the lower hypocotyl have been noted in ¯avonoid-de®cient mutants of Arabidopsis (data not shown). In most cases, wild-type phenotypes could be partially restored by exogenous application of naringenin. A detailed report of these results is currently being prepared for publication. The [14C]IAA transport studies described herein support a role for the localized ¯avonoids in auxin accumulation. Flavonoid-de®cient tt4 mutant seedlings exhibited an altered pattern of auxin distribution that could be overcome by the inclusion of the ¯avonoid intermediate naringenin in the growth medium. The auxin distribution pattern in the tt4 mutant suggested that less auxin was being retained by certain tissues along the polar transport pathway, such as at the cotyledonary node, transition zone and root tip. Direct measurements of auxin leakage from the root tip indicated that less auxin was retained in tt4 than in the wild type, while naringenin treatment restored auxin accumulation to wild-type levels. The ability of ¯avonoids to prevent auxin leakage from the tip provides compelling evidence that endogenous ¯avonoids are required for normal auxin accumulation by plant cells. Consistent with this model, when auxin transport was examined in the tt3 mutant, which accumulates higher levels of quercetin and kaempferol than the wild type, [14C]IAA levels were signi®cantly higher in both the transition zone and lower root (data not shown). Other ligands besides ¯avonoids may also be able to bind to and regulate the cell surface APs we have described, thus aecting polar auxin transport. For example, Tyr- and Pro-rich proteins in the cell wall could also act as substrates and/or eectors of cell surface APs. Cell surface APs may process systemin-like signaling peptides (Schaller et al. 1995) as they do in animals (Taylor 1996). Plasma-membrane APs could also poten-

Fig. 5. Chemical structures of a-N-naphthylphthalamic acid (1-NPA), b-N-naphthylphthalamic acid (2-NPA), tyrosineb-naphthylamide (Tyr-bNA), quercetin, and bestatin


tially be involved in hydrolysis of amino acyl conjugates of signaling molecules of local inhibitors. Further studies will be needed to con®rm the involvement of such APs in the regulation of polar auxin transport. This work was funded by US Dept. of Agriculture Grant No. 94-37100-0755.

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