Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

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1 Plant Cell Physiol. 37(8): (1996) JSPP 1996 Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana Hironori Fujita and Kunihiko Syono Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo, 153 Japan Polar auxin transport inhibitors, including TV-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have various effects on physiological and developmental events, such as the elongation and tropism of roots and stems, in higher plants. We isolated NPA-resistant mutants of Arabidopsis thaliana, with mutations designated pirl and pir2, that were also resistant to TIBA. The mutations specifically affected the root-elongation process, and they were shown ultimately to be allelic to auxl and ein2, respectively, which are known as mutations that affect responses to phytohormones. The mechanism of action of auxin transport inhibitors was investigated with these mutants, in relation to the effects of ethylene, auxin, and the polar transport of auxin. With respect to the inhibition of root elongation in A. thaliana, we demonstrated that (1) the background level of ethylene intensifies the effects of auxin transport inhibitors, (2) auxin transport inhibitors might act also via an inhibitory pathway that does not involve ethylene, auxin, or the polar transport of auxin, (3) the hypothesis that the inhibitory effect of NPA on root elongation is due to high-level accumulation of auxin as a result of blockage of auxin transport is not applicable to A. thaliana, and (4) in contrast to NPA, TIBA itself has a weak auxin-like inhibitory effect. Key words: Arabidopsis thaliana auxl Auxin transport inhibitors ein2 TV-1-Naphthylphthalamic acid (NPA) Root growth. In higher plants, the class of compounds known as auxin transport inhibitors, including 7V-l-naphthyrphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), has various effects on physiological and developmental processes, such as the elongation of stems and roots (Li et al. 1994, Gaither and Abeles 1975, Muday and Haworth 1994), root swelling (Gaither and Abeles 1975, Gaither 1975), tropism (Khan 1967, Okada and Shimura 1992, Takahashi and Suge 1991), formation of root hairs (Gaither 1975), formation of apical hooks (Schwark and Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; AVG, aminoethoxyvinylglycine; BA, benzoic acid; EMS, ethyl methanesulfonate; IBA, iodobenzoic acid; NPA, N-l-naphthylphthalamic acid; TIBA, 2,3,5-triiodobenzoic acid. Schierle 1992), induction of morphological transformation in the shoot apex (Okada et al. 1991), and establishment of body axes during embryogenesis (Schiavone and Cooke 1987, Liu et al. 1993). It is also known that auxin transport inhibitors inhibit the polar transport of auxin whereby auxin is transported along the body axis of a plant from the shoot apex to the root apex (Goldsmith 1977, Scott and Wilkins 1968). These inhibitors promote the accumulation of auxin in cultured cells (Rubery and Sheldrake 1974), in stem segments (Davies and Rubery 1978), and in membrane vesicles (Hertel et al. 1983). The polarity of auxin transport has been proposed to result from the basal localization of auxin-efflux carriers on the plasma membrane. Various effects of auxin transport inhibitors have been proposed to be caused, at the molecular level, by interference with the auxin-efflux carrier. However, it remains unclear whether or how blockage of the efflux of auxin by the inhibitors is involved in the effects of these inhibitors on physiological and developmental processes in plants. Auxin transport inhibitors seem somehow to be related to phytohormones in terms of their effects. Gravitropism mutants, namely, rgrl of A. thaliana and diageotropica of tomato, are resistant to NPA as well as to auxin (Simmons et al. 1995, Muday et al. 1995). Transgenic tobacco plants that overproduce cytokinins also display resistance both to auxin and to auxin transport inhibitors (Li et al. 1994). Some effects of ethylene are similar to those of NPA, for example, inhibition of stem and root elongation (Knight et al. 1910, Ecker 1995), disruption of gravitropism (Knight et al. 1910), induction of the formation of root hairs (Tanimoto et al. 1995), and inhibitory effects on the polar transport of auxin (Burg and Burg 1967, Osborne and Mullins 1969, Suttle 1988). The diageotropica mutant is resistant to ethylene, as well as to auxin and to auxin transport inhibitors (Muday et al. 1995). Resembling auxins, TIBA is itself transported basipetally through stem segments (Thomson et al. 1973). The pin-formed and pinoid mutants of Arabidopsis thaliana have dramatic abnormalities in their inflorescences, which resemble the structures formed after treatment of seedlings with auxin transport inhibitors (Okada and Shimura 1992, Bennett et al. 1995). In these two mutants, the rate of the polar transport of auxin is reduced, but the relationship between this reduction and the morphological abnormalities remains unclear. Auxin transport inhibitors have frequently been used 1094

2 Analysis of effects of auxin transport inhibitors 1095 in studies of polar auxin transport since they have been strongly implicated in the inhibition of such transport. However, other effects of these inhibitors have been ignored, and little attention has been paid to the way in which these inhibitors affect physiological and developmental processes in plants. The various effects of auxin transport inhibitors have mainly been explained in terms of alterations in the concentration and/or distribution of auxin as a consequence of the inhibition of the movement of auxin. Isolation and analysis of mutants frequently provide insights into intrinsic phenomena in biological systems. In order to characterize details of the mechanism of action of auxin transport inhibitors, we attempted to isolate mutants that were resistant to NPA and to dissect the mode of action of these inhibitors using these mutants, focusing on phenomena related to the elongation of roots. Materials and Methods Plant materials and growth conditions Ethyl methanesulfonate (EMS)-mutagenized M 2 seeds of the Columbia ecotype of A. thaliana and both the auxl-7and ein2-j mutants were obtained from the Lehle Seeds Co. (Tucson, AZ, U.S.A.) and the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, U.S.A.), respectively. Plants were grown under continuous illumination at about 30^E at about 24 C. In general, seeds were sown in pots and supplied with a solution of inorganic compounds (1 x) that contained 5 mm KNO 3, 2 mm MgSO 4, 2 mm Ca(NO 3 ) 2, 2.5 mm potassium phosphate adjusted to ph 5.5, 50 fiu Fe-EDTA, 70^M H 3 BO 3, HytiM MnCl 2, 0.5 fim CuSO 4, 1 /im ZnSO 4, 0.2//M NaMoO 4, 10/^M NaCl and 0.01 fim CoCl 2. After storage at 4 C for four or five days to synchronize germination, sterile seeds were sown in a sterile container that contained basal medium (1 x inorganic solution, 100 mg liter" 1 myo-inositol, 20 mg liter" 1 thiamine hydrochloride, 1 mg liter" 1 pyridoxine hydrochloride, 1 mg liter" 1 nicotinic acid, and 1 mg liter"' d- biotin) that had been solidified by addition of 0.8% agar or 0A% Gellan gum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), with or without sucrose. Determination of sensitivity to auxin transport inhibitors and phytohormones Sterilized seeds of the wild type and the mutants were sown on agar-solidified basal medium, as described above, that was maintained in the vertical position so that the roots would grow along the surface of the solid medium. After incubation at room temperature for four days, the seedlings were transferred to new plates prepared with various concentrations of test compounds, and the positions of root tips were marked on the plate. The extent of new root growth was measured after incubation for a further five days. Examination of root gravitropism Sterile seeds were incubated on the agar-solidified basal medium, plus \% sucrose with plates in the vertical position. After four days in darkness at about 25 C, the direction from the base of the root to the root tip was determined relative to the gravity vector. Thus, positively gravitropic roots were oriented at 0 and negatively gravitropic roots were oriented at 180. Quantitation of ethylene About 100 sterilized wild-type seeds were sown in 20-ml vials that contained 10 ml of agarsolidified basal medium plus \% sucrose, with or without a test compound. The vials were sealed and incubated for five days in the light. Then a 1-ml sample of air was removed from each vial, and the amount of ethylene produced was determined with a gas chromatograph (GC-9A; Shimadzu Corp., Kyoto, Japan), equipped with a Porapack T column. Assay of auxin transport The method for the determination of auxin transport was a modified version of that described by Okada et al. (1991). Seedlings were grown for ten days on basal medium solidified with Gellan gum and supplemented with \% sucrose, with plates in the vertical position, under illumination at about 5 /YE, and the resultant hypocotyls were used for assays. Excised hypocotyls of about 15 mm in length were normally placed with the apical end pointing downwards in 2.0-ml Eppendorf plastic tubes that contained ten microliters of a solution of 0.5 x inorganic medium, 370 Bq of 3-[5(n)- 3 H]IAA (Amersham International pic, Buckinghamshire, England), and \% sucrose. TIBA or an analog of TIBA was added to the solution. After incubation at room temperature for 18 h, a segment of about 2 mm was cut off from the upper end of the segment of hypocotyl and radioactivity in this small segment was determined in a liquid scintillation counter. Results Isolation of NPA-resistant mutants Since 10 fim NPA effectively inhibited root elongation and induced the formation of root hairs in A. thaliana, seedlings from EMS-mutagenized M 2 seeds were subjected to screening for NPA-resistant mutants at this concentration of NPA. We isolated five NPA-resistant strains. The mutations were all recessive and could be assigned to two genetic loci, designated pirl and pir2 (polar auxin transport inhibitor resistant). The mutant plants were similar to the wild type in terms of shape, height and fertility. Both pirl-1 and pir2-j were 24 times more resistant to NPA than the wild type at a concentration of NPA that caused 50% inhibition of root growth (Fig. 1A). One specific effect of auxin transport inhibitors is the abolition of gravitropism. NPA at 0.3 to 3^M disrupted the root gravitropism of wild-type seedlings, and at 3 fim it completely eliminated the gravitropic response. Neither pir mutant displayed altered sensitivity to NPA with respect to root gravitropism. However, the background level of root gravitropism in the absence of NPA in the pirl-1 mutant was different from that in the wild type (Fig. IB). Therefore, the lesions in both the pirl and pir2 mutants were specifically associated with the effect of NPA on the root-elongation process among various possible effects of NPA. Moreover, the sensitivity to TIBA, another inhibitor of auxin transport, of pirl-1 and pir2-l was also about 5 and 4 times lower, respectively, than that of the wild type (Fig. 2A), indicating that the acquired resistance was not specific to NPA but was common to both auxin transport inhibitors. Several phytohormone-resistant mutants exhibit altered responses to more than one phytohormone, and changes in the sensitivity to auxin transport inhibitors and to phy-

3 1096 Analysis of effects of auxin transport inhibitors g ao V BO a o s I I I X \ 9- V - o wild pirl-1type Is, a pirz-1 i i V A NPA (/im) Fig. 1 Effects of NPA on root elongation (A) and root gravitropism (B) in wild-type, pirl-1, and pir2-l seedlings. Elongation is expressed relative to the mean elongation of the root in the absence of NPA. Bars represent standard errors. Mean values for 100% root growth were 22.7±0.7 mm for the wild type, 25.8±0.7 mm for pirl-1, and 23.9±0.8 mm iorpir2-l. (A) n= (wild type), n=5-9 (pirl-1), and n= (pir2-l); and (B) n=46-52 (wild type), n=31-36 (pirl-1), and n = (pir2-l). tohormones often coincide. Thus, it seemed likely that our NPA-resistant mutants might also have acquired resistance to other phytohormones. We examined their sensitivity to IAA and 1-aminocyclopropane-l-carboxylic acid (ACC), a naturally occurring auxin and a precursor of ethylene, respectively. The pirl-1 mutant was about 25- fold more resistant to IAA than the wild type for 50% inhibition of root growth. The extent of the inhibition of root growth produced by 10yuM ACC was only 20% in pirl-1, while it was 51% in the wild type. The pir2-l mutant was more insensitive to ACC than the wild type but it was equally sensitive to IAA (Fig. 2B, C). In addition to the changes in the sensitivity to phytohormones, pirl exhibited apparent agravitropism even in the absence of NPA (Fig. IB). These characteristics were very similar to those of the phytohormone-resistant mutants characterized to date. Genetic analysis revealed that pirl and pir2 were allelic to auxl and ein2, respectively. The auxl and ein2 mutants are known as an auxin-resistant mutant and an ethylene-insensitive mutant, respectively. Thus, it appeared that both auxin and ethylene were associated with the inhibitory effect of auxin transport inhibitors on root growth. Therefore, we attempted to determine how the effects of auxin, ethylene and the polar transport of auxin might interact with the actions of auxin transport inhibitors. Effects of ethylene In order to examine the involvement of ethylene in the effects of NPA on root growth, we performed dose-response analysis using ethylene inhibitors and both wild type and pir2 (ein2) mutant seedlings. The experiment was performed under airtight conditions to accentuate the effect of NPA. Silver ions are often used as a strong inhibitor of ethylene action (Beyer 1976), and AgNO 3, added to the medium at 2fiM, effectively overcame the inhibition by ACC of root growth in seedlings of wild-type A. thaliana (Fig. 3). The presence of either the pir2 (ein2) mutation or of AgNO 3 allowed the partial recovery of root growth that would otherwise have been inhibited by NPA. The roots of wild-type seedlings treated with 10 /xm NPA in the presence and in the absence of AgNO 3 were 23% and 73%, respectively, as long as the control roots. In the case of pir2-2 seedlings, the roots at 10 j«m NPA were 79% as long as the controls. By contrast, in the presence of 1 fim IAA, the addition of AgNO 3 to the medium was associated with no more than a slight increase in root length in the wild type (Fig. 3). Similar results were obtained after the application of aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene biosynthesis (Yang and Hoffman 1984), instead of AgNO 3 (data not shown). Since these results suggested that the action of NPA was partly exerted via ethylene, we examined whether auxin transport inhibitors could stimulate ethylene production. The level of ethylene generated by wild-type seedlings in the presence of 10 fim ACC was about 26 times higher than that in the absence of ACC but, contrary to our expectations, the application of NPA or of TIBA failed to induce Table 1 Production of ethylene by wild-type seedlings in the presence of various agents Treatment Control IOJUM ACC 10 /JM NPA 10//MTIBA Ethylene production (nl) 2.2± ± ± ±0.4 Each value represents the mean of results from three samples ± standard error.

4 Analysis of effects of auxin transport inhibitors 1097 AgNO Control 10 um NPA D wild type pirz-z TIBA IAA ACC Fig. 2 Effects of TIBA (A), IAA (B), and ACC (C) on root elongation in wild-type, pirl-1, and pir2-l seedlings. Elongation is expressed relative to the mean elongation of the root in the absence of test compounds. Bars represent standard errors. Mean values for 100% root growth were 22.7 ±0.7 mm (A), 26.0 ±0.9 mm (B), and 22.1±0.6mm (C) for the wild type; 16.2±1.2mm (A), 21.3±1.2mm (B), and 16.7±0.9mm (C) for pirl-1; and 20.3± 0.8 mm (A), 26.5±0.8 mm (B), and 19.2±0.6 mm (C) for pir2-l. (A) n= (wild type), n = 8-17 (pirl-1), and n = 7-13 (pir2-l); (B) n= (wild type), n=9-l 1 (pirl-1), and n= (pir2-l); and (C) n=14-16 (wild type), n=9-ll (pirl-1), and n = iim ACC Relative root elongation Fig. 3 Effects of NPA, IAA, and ACC on root elongation in the presence and absence of 2 j/m AgNO 3. Seeds were grown on agarsolidified basal medium with 0.1% sucrose in the light, with plates in a vertical position, under airtight conditions. After incubation for 5 days, roots of the wild type and pir2-2 were measured. Elongation is expressed relative to the mean elongation of the root in the absence of test compounds. Bars represent standard errors. Mean values for control root growth were 13.1 ±0.6 mm for the wild type and 18.4±1.5mm forpir2-2; n= (wild type), and n = ll-15 (pir2-2). ethylene production (Table 1). Effects of the polar transport of auxin The pir2 mutant was completely insensitive to ethylene but it was partially sensitive to NPA in terms of root growth (Fig. 3). Thus, there seemed that NPA could act via pathways that did not involve ethylene. To investigate whether inhibition of the polar transport of auxin is important for the physiological action of auxin transport inhibitors, we examined the relationships between the effects of TIBA and its analogs on root elongation, root gravitropism, and the basipetal transport of auxin. We found that the hypocotyl of A. thaliana has the capacity for the polar basipetal transport of auxin (Fig. 4A) as does the inflorescence (Okada et al. 1991, Bennett et al. 1995). TIBA completely prevented the polar transport of auxin and had a considerable negative effect on root gravitropism, while the various analogs of TIBA that we tested had little or no effect on either parameter (Fig. 4A, B). The extent of the defect in root gravitropism paralleled that of the defect in the polar transport of auxin. However, root elongation was most strongly inhibited by 3-iodobenzoic acid (3-IBA) among the analogs of TIBA, followed by TIBA, and no relationship was recognized between the polar transport of auxin and root length (Fig. 4A, C). This strong inhibition by 3-IBA was also observed with the auxl-7 and pir2-l mutants and with the pir2-l auxl-7 double mutant (Fig. 5). It appears from these results that there might exist a pathway that does not involve auxin and the polar transport of auxin, as well as a pathway that does not involve ethylene. Effects of auxin In the last set of experiments, we evaluated the extent to which auxin contributes to the 1.2

5 1098 Analysis of effects of auxin transport inhibitors [ 3 H]IAA transported (cpm) Acropetal Baslpetal BA 2-IBA 3-IBA 4-IBA TIBA Root gravltroplsm (degrees) Root elongation (mm) 10 Fig. 4 Effects of TIBA and its analogs, BA, 2-IBA, 3-IBA, and 4-IBA, on the basipetal transport of auxin in hypocotyl segments, with an acropetal control (A), on root gravitropism (B), and on root elongation (C) in wild-type seedlings. Each compound was added at 30//M (A) or at 10^M (B, C). Bars represent standard errors. n = 5 (A), n=27-32 (B), and n= (C). Control 10 /JM NPA lopmba 30 /JM BA 10 um 3-IBA 30 pm 3-IBA 10 t>u TIBA 30 tim TIBA wild type DD auxl-7 Pir2-1 pirz-l auxl Relative root elongation (%) Fig. 5 Effects of NPA, BA, 3-IBA, and TIBA on root elongation in the wild type, auxl-7, pir2-l, andpir2-i auxl-7. Seedlings were grown on agar-solidified basal medium with 0.1% sucrose in the light, with plates in a vertical position. After incubation for 5 days, roots were measured. Elongation is expressed relative to the mean elongation of the root in the absence of test compounds. Bars represent standard errors. Mean values for control root growth were 15.0±0.5mm for the wild type, 17.3±O.4mm for auxl-7, 16.8±0.8mm for pir2-l, and mm for pir2-l auxl-7. n = (wild type), n= (auxl-7), n= (pir2-1) and n = (pir2-l auxl-7). 120 effects of auxin transport inhibitors, using the auxl and pir2 (ein2) mutants. Since the auxl mutant was resistant to both ethylene and auxin simultaneously, it was unclear whether the phenotypes of the auxl mutant were essentially attributable to the resistance to auxin or to the resistance to ethylene. To define whether it was the resistance to auxin and not to ethylene of the auxl mutant that participated in the effect of auxin transport inhibitors on root growth, we introduced the auxl-7 mutation into seedlings on a background of the pir2-l mutation, \npir2-l seedlings ethylene had no effect but the inhibitory effect of IAA was normal. The extent of inhibition of root growth by TIBA at 10 /im inpir2-l andpir2-l auxl-7seedlings was 44% and 19%, respectively, as compared in the control without TIBA. Thus, the pir2-l auxl-7 double mutant was more resistant to TIBA than the pir2-l single mutant. Similar results were obtained with two analogs of TIBA, namely, benzoic acid (BA) and 3-IBA. By contrast, the auxl-7 mutation conferred no significant resistance to NPA on the pir2-l mutant (Fig. 5). Discussion It has been proposed that auxin transport inhibitors, such as NPA and TIBA, specifically interfere with the efflux of auxin from cells, and these compounds have frequently been used in the analysis of polar auxin transport. By contrast, little attention has been paid to the mode of action of these inhibitors, and the various effects of these inhibitors have been explained only in terms of their own weak auxinlike activity or by alterations in the concentration and/or distribution of auxin that arise from inhibition of auxin efflux from cells (Katekar and Geissler 1980, Muday and Haworth 1994). To characterize the mechanism of action of auxin transport inhibitors in greater detail, we isolated two mutants that were resistant to NPA, with the pirl and pir2 mutations, which we eventually found to be allelic to auxl and ein2, respectively. The auxl mutant is resistant to both auxin and ethylene (Pickett et al. 1990) while the ein2 mutant is insensitive to ethylene because of a lesion in the ethylene signaltransduction pathway (Ecker 1995). The auxl mutant is also known as a root gravitropism mutant, and some lines of auxl mutants exhibit no gravitropism at all (Okada and Shimura 1992). However, the sensitivity to NPA with respect to root gravitropism was found to be wild type both in pirl (auxl) and in pir2 (ein2) seedlings and, therefore, the two mutations seems specifically to affect the process of inhibition of root growth among the various effects of NPA and the mutations might be associated with a defect in hormone signal transduction rather than in polar auxin transport itself. Since pir mutants that we isolated as NPA-resistant mutants were allelic to auxl or ein2, it seemed likely that

6 Analysis of effects of auxin transport inhibitors 1099 pir1 (aux7) pir2 (ein2) polar auxin transport \\-*- IAA f B root elongation Fig. 6 Schematic representation of the action of auxin transport inhibitors on root elongation in A. lhaliana. The inhibitors interfere with the polar transport of auxin (A) and, as a consequence, they might change the level and the distribution of IAA in cells (B), which in turn might be responsible for the disruption of gravitropism. By contrast, the hypothesis that the inhibitory effect of NPA on root elongation is due to the high-level accumulation of auxin as a result of blockage of auxin transport (B) is not applicable to A. thaliana (C). There might be pathways in which the polar transport of auxin is involved (D) and in which it is not involved (E). Neither NPA nor TIBA induces ethylene production (F). However, the background level of ethylene intensifies the inhibitory effect of NPA on root elongation (G). The resistance to auxin transport inhibitors of thepirl (auxl) and pir2 (einl) mutants appears to be due predominantly to the insensitivity of these mutants to ethylene. the phytohormones auxin and ethylene might be involved in the effects of auxin transport inhibitors. In fact, there are many similarities between the effects of ethylene and those of auxin transport inhibitors, as described above. In addition, in the course of our experiments, we found that NPA had a greater effect on the root growth of wild-type seedlings under airtight conditions than under normal growth conditions. This inhibition by NPA was reversed to a considerable extent by the pir2 (ein2) mutation and also by the application of ethylene inhibitors such as silver ions and AVG (Fig. 3). This result indicated that NPA exerted its effect in part via ethylene. Resembling root elongation, the formation of root hairs that was accelerated by NPA was reduced by the pir2 (ein2) mutation and by the inhibitors of ethylene (data not shown). Thus, ethylene seems likely to be associated with these processes that are stimulated by NPA. However, neither NPA nor TIBA induced the evolution of ethylene (Table 1). These data are consistent with those reported previously by Gaither and Abeles (1975), who showed that auxin transport inhibitors, including NPA and TIBA, did not stimulate ethylene evolution by pea seedlings. It appears that the background level of ethylene is sufficient to intensify the effects of NPA on both the growth of primary roots and the formation of root hairs (Fig. 6F, G). Some effects of auxin transport inhibitors have been attributed to the inhibition of auxin transport. However, our analysis with analogs of TIBA showed that the extent of the defect in polar auxin transport reflected the extent of the defect in root gravitropism but not the effect on root elongation (Fig. 4). In particular, 3-IBA markedly prevented root growth but it had no significant effect on polar auxin transport (Fig. 4A, C). This strong inhibition by 3-IBA was also seen in the auxl-7, pir2-l, and pir2-l auxl-7mutants (Fig. 5). Thus, it seems clear that a pathway that is not related to polar auxin transport, to the effects of auxin, or to the actions of ethylene is a major contributor to the strong effect of 3-IBA on root growth. To some extent, TIBA might also exert its effect via such a pathway (Fig. 6E). BA did not inhibit polar auxin transport (Fig.4A). This result is compatible with the previously reported results that BA itself is not transported in a basipetal manner in corn coleoptile sections (Hertel et al. 1969) and that BA does not affect auxin uptake by segments of zucchini hypocotyl (Depta and Rubery 1984). The hypothesis is widely accepted that NPA inhibits the transport of auxin and causes the high-level accumulation of auxin, with subsequent inhibition of root growth. However, the auxl-7 mutation failed to confer resistance to NPA on the background of the pir2-l mutation, in which ethylene had no effect but IAA inhibited root growth in the normal manner. This result leads to the conclusion that the hypothesis is not valid for the inhibition of root elongation in A. thaliana (Fig. 6C). Unlike the resistance to NPA, the partial resistance to TIBA caused by the auxl-7 mutation (Fig. 5) might have arisen from the weak auxin-

7 1100 Analysis of effects of auxin transport inhibitors like activity of TIBA itself. The difference between the modes of action of NPA and TIBA have been discussed in some detail: NPA and TIBA seem to have different binding sites (Thomson et al. 1973, Katekar and Geissler 1980, Depta and Rubery 1984, Michalke et al. 1992); moreover, TIBA is transported in a polar basipetal manner while NPA is not (Thomson et al. 1973). These differences between NPA and TIBA at the molecular level might be responsible for the differences between their effects on root growth. Since resistance to NPA was not conferred by the auxl-7 mutation on the ethylene-insensitive background (Fig. 5), it is possible that/?//-/ (auxl) mutants might be isolated during the selection for NPA-resistant mutants largely because of their resistance to ethylene rather than to auxin (Fig. 6F). Excised segments of inflorescences of A. thaliana have the capacity for the basipetal transport of auxin (Okada et al. 1991, Bennett et al. 1995). We found that hypocotyls of A. thaliana also have this capacity. The polar transport of auxin in stems is known to be influenced to a considerable extent by a variety of external and internal factors, such as nutrient conditions (Tang and de la Fuente 1986, Allan and Rubery 1991), the age of the seedling (Bennett et al. 1995), and the position of the stem (Davenport et al. 1980, Morris and Johnson 1990, Suttle 1991). However, our present technique using hypocotyls allows preparation of uniform materials in large quantities, and may be valuable for the analysis of polar auxin transport in A. thaliana. Genetic analysis with mutants can serve as a powerful tool for the dissection of biological phenomena. We attempted to evaluate the effects of ethylene, auxin, and polar auxin transport on the action of NPA using mutants with defects in the responses to hormones. We focused here on the mode of action of auxin transport inhibitors. With respect to root elongation in A. thaliana, we can conclude that (1) the background level of ethylene is sufficient to intensify the effect of NPA (Fig. 6F, G); (2) there might be a pathway in which ethylene, auxin, and the polar transport of auxin are all not involved (Fig. 6E); (3) the hypothesis that the inhibitory effect of NPA on root elongation is due to the accumulation of auxin at a high level as a result of blockage of auxin transport is not applicable to A. thaliana (Fig. 6C); and (4) TIBA itself has weak auxin-like activity. The inhibition of root growth by auxin transport inhibitors appears to be a complex phenomenon in which various factors, including phytohormones, seem to participate and interact synergistically. To date, the actions of NPA have been explained for the most part in terms of inhibition of auxin efflux. However, in root growth, at least, such is not the case. The other effects of auxin transport inhibitors require our attention and further examination of these effects is required for a better understanding of the detailed mechanisms of action of these inhibitors and the polar transport of auxin. Further screening might lead to the identification of a variety of additional mutants that reflect diverse aspects of the action of NPA. The authors thank Dr. Hirokazu Tsukaya for helpful advice on treatment of seedlings of Arabidopsis thaliana and Drs. Toru Shigematsu and Takeshi Uozumi for quantitation of ethylene. References Allan, A.C. and Rubery, P.H. (1991) Calcium deficiency and auxin transport in Cucurbita pepo L. seedlings. Planta 183: Bennett, S.R.M., Alvarez, J.A., Bossinger, O. and Smyth, D.R. (1995) Morphogenesis in pinoid mutants of Arabidopsis thaliana. 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