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

Home , Polar auxin transport

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-naph- Schierle 1992), induction of morphological transformation

thylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid in the shoot apex (Okada et al. 1991), and establishment of Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 (TIBA), have various effects on physiological and develop- body axes during embryogenesis (Schiavone and Cooke mental events, such as the elongation and tropism of roots 1987, Liu et al. 1993). It is also known that auxin transport and stems, in higher plants. We isolated NPA-resistant inhibitors inhibit the polar transport of auxin whereby aux- mutants of Arabidopsis thaliana, with mutations designat- in is transported along the body axis of a plant from the ed pirl and pir2, that were also resistant to TIBA. The shoot apex to the root apex (Goldsmith 1977, Scott and mutations specifically affected the root-elongation process, Wilkins 1968). These inhibitors promote the accumulation and they were shown ultimately to be allelic to auxl and of auxin in cultured cells (Rubery and Sheldrake 1974), in ein2, respectively, which are known as mutations that stem segments (Davies and Rubery 1978), and in membrane affect responses to phytohormones. The mechanism of ac- vesicles (Hertel et al. 1983). The polarity of auxin transport tion of auxin transport inhibitors was investigated with has been proposed to result from the basal localization of these mutants, in relation to the effects of ethylene, auxin, auxin-efflux carriers on the plasma membrane. Various and the polar transport of auxin. With respect to the inhibi- effects of auxin transport inhibitors have been proposed to tion of root elongation in A. thaliana, we demonstrated be caused, at the molecular level, by interference with the that (1) the background level of ethylene intensifies the auxin-efflux carrier. However, it remains unclear whether effects of auxin transport inhibitors, (2) auxin transport in- or how blockage of the efflux of auxin by the inhibitors is inhibitors might act also via an inhibitory pathway that does volved in the effects of these inhibitors on physiological not involve ethylene, auxin, or the polar transport of aux- and developmental processes in plants. in, (3) the hypothesis that the inhibitory effect of NPA on Auxin transport inhibitors seem somehow to be relat- root elongation is due to high-level accumulation of auxin ed to phytohormones in terms of their effects. Gravitropism as a result of blockage of auxin transport is not applicable mutants, namely, rgrl of A. thaliana and diageotropica of to A. thaliana, and (4) in contrast to NPA, TIBA itself has tomato, are resistant to NPA as well as to auxin (Simmons a weak auxin-like inhibitory effect. et al. 1995, Muday et al. 1995). Transgenic tobacco plants that overproduce cytokinins also display resistance both to Key words: Arabidopsis thaliana — auxl — Auxin trans- auxin and to auxin transport inhibitors (Li et al. 1994). port inhibitors — ein2 — TV-1-Naphthylphthalamic acid Some effects of ethylene are similar to those of NPA, for ex- (NPA) — Root growth. ample, 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 In higher plants, the class of compounds known as aux- transport of auxin (Burg and Burg 1967, Osborne and in transport inhibitors, including 7V-l-naphthyrphthalamic Mullins 1969, Suttle 1988). The diageotropica mutant is re- acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), has sistant to ethylene, as well as to auxin and to auxin trans- various effects on physiological and developmental pro- port inhibitors (Muday et al. 1995). Resembling auxins, cesses, such as the elongation of stems and roots (Li et al. TIBA is itself transported basipetally through stem seg- 1994, Gaither and Abeles 1975, Muday and Haworth ments (Thomson et al. 1973). 1994), root swelling (Gaither and Abeles 1975, Gaither The pin-formed and pinoid mutants of Arabidopsis 1975), tropism (Khan 1967, Okada and Shimura 1992, thaliana have dramatic abnormalities in their inflores- Takahashi and Suge 1991), formation of root hairs cences, which resemble the structures formed after treat- (Gaither 1975), formation of apical hooks (Schwark and ment 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 re- Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; AVG, aminoethoxyvinylglycine; BA, benzoic acid; EMS, duced, but the relationship between this reduction and the ethyl methanesulfonate; IBA, iodobenzoic acid; NPA, N-l-naph- morphological abnormalities remains unclear. thylphthalamic acid; TIBA, 2,3,5-triiodobenzoic acid. Auxin transport inhibitors have frequently been used

1094 Analysis of effects of auxin transport inhibitors 1095 in studies of polar auxin transport since they have been compound. The vials were sealed and incubated for five days in strongly implicated in the inhibition of such transport. the light. Then a 1-ml sample of air was removed from each vial, However, other effects of these inhibitors have been ig- and the amount of ethylene produced was determined with a gas chromatograph (GC-9A; Shimadzu Corp., Kyoto, Japan), equip- nored, and little attention has been paid to the way in ped with a Porapack T column. which these inhibitors affect physiological and develop- Assay of auxin transport—The method for the determination mental processes in plants. The various effects of auxin of auxin transport was a modified version of that described by transport inhibitors have mainly been explained in terms of Okada et al. (1991). Seedlings were grown for ten days on basal alterations in the concentration and/or distribution of aux- medium solidified with Gellan gum and supplemented with \% su- in as a consequence of the inhibition of the movement of crose, with plates in the vertical position, under illumination at about 5 /YE, and the resultant hypocotyls were used for assays. Ex- auxin. Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 cised hypocotyls of about 15 mm in length were normally placed Isolation and analysis of mutants frequently provide with the apical end pointing downwards in 2.0-ml Eppendorf insights into intrinsic phenomena in biological systems. In plastic tubes that contained ten microliters of a solution of 0.5 x 3 order to characterize details of the mechanism of action of inorganic medium, 370 Bq of 3-[5(n)- H]IAA (Amersham Interna- auxin transport inhibitors, we attempted to isolate mutants tional pic, Buckinghamshire, England), and \% sucrose. TIBA or an analog of TIBA was added to the solution. After incubation at that were resistant to NPA and to dissect the mode of ac- room temperature for 18 h, a segment of about 2 mm was cut off tion of these inhibitors using these mutants, focusing on from the upper end of the segment of hypocotyl and radioactivity phenomena related to the elongation of roots. in this small segment was determined in a liquid scintillation counter. Materials and Methods

Plant materials and growth conditions—Ethyl methanesulfo- Results nate (EMS)-mutagenized M seeds of the Columbia ecotype of 2 Isolation of NPA-resistant mutants—Since 10 fiM A. thaliana and both the auxl-7and ein2-J mutants were obtained from the Lehle Seeds Co. (Tucson, AZ, U.S.A.) and the Arabi- NPA effectively inhibited root elongation and induced the dopsis Biological Resource Center (Ohio State University, Colum- formation of root hairs in A. thaliana, seedlings from bus, OH, U.S.A.), respectively. Plants were grown under continu- EMS-mutagenized M2 seeds were subjected to screening for ous illumination at about 30^E at about 24°C. In general, seeds NPA-resistant mutants at this concentration of NPA. We were sown in pots and supplied with a solution of inorganic com- isolated five NPA-resistant strains. The mutations were all pounds (1 x) that contained 5 mM KNO , 2 mM MgSO , 2 mM 3 4 recessive and could be assigned to two genetic loci, designat- Ca(NO3)2, 2.5 mM potassium phosphate adjusted to pH 5.5, 50 fiU Fe-EDTA, 70^M H3BO3, HytiM MnCl2, 0.5 fiM CuSO4, 1 ed pirl and pir2 (polar auxin transport inhibitor resistant). /iM ZnSO4, 0.2//M NaMoO4, 10/^M NaCl and 0.01 fiM CoCl2. The mutant plants were similar to the wild type in terms of After storage at 4°C for four or five days to synchronize germina- shape, height and fertility. Both pirl-1 and pir2-J were 24 tion, sterile seeds were sown in a sterile container that contained times more resistant to NPA than the wild type at a concen- basal medium (1 x inorganic solution, 100 mg liter"1 myo-in- ositol, 20 mg liter"1 thiamine hydrochloride, 1 mg liter"1 pyridox- tration of NPA that caused 50% inhibition of root growth ine hydrochloride, 1 mg liter"1 nicotinic acid, and 1 mg liter"' d- (Fig. 1A). biotin) that had been solidified by addition of 0.8% agar or 0A% One specific effect of auxin transport inhibitors is the Gellan gum (Wako Pure Chemical Industries, Ltd., Osaka, abolition of gravitropism. NPA at 0.3 to 3^M disrupted Japan), with or without sucrose. the root gravitropism of wild-type seedlings, and at 3 fiM it Determination of sensitivity to auxin transport inhibitors and completely eliminated the gravitropic response. Neither pir phytohormones—Sterilized seeds of the wild type and the mutants mutant displayed altered sensitivity to NPA with respect to were sown on agar-solidified basal medium, as described above, that was maintained in the vertical position so that the roots root gravitropism. However, the background level of root would grow along the surface of the solid medium. After incuba- gravitropism in the absence of NPA in the pirl-1 mutant tion at room temperature for four days, the seedlings were trans- was different from that in the wild type (Fig. IB). There- ferred to new plates prepared with various concentrations of test fore, the lesions in both the pirl and pir2 mutants were spe- compounds, and the positions of root tips were marked on the cifically associated with the effect of NPA on the root-elon- plate. The extent of new root growth was measured after incubation for a further five days. gation process among various possible effects of NPA. Examination of root gravitropism—Sterile seeds were incu- Moreover, the sensitivity to TIBA, another inhibitor of aux- bated on the agar-solidified basal medium, plus \% sucrose with in transport, of pirl-1 and pir2-l was also about 5 and plates in the vertical position. After four days in darkness at about 4 times lower, respectively, than that of the wild type 25°C, the direction from the base of the root to the root tip was (Fig. 2A), indicating that the acquired resistance was not determined relative to the gravity vector. Thus, positively specific to NPA but was common to both auxin transport gravitropic roots were oriented at 0° and negatively gravitropic roots were oriented at 180°. inhibitors. Quantitation of ethylene—About 100 sterilized wild-type Several phytohormone-resistant mutants exhibit alter- seeds were sown in 20-ml vials that contained 10 ml of agar- ed responses to more than one phytohormone, and changes solidified basal medium plus \% sucrose, with or without a test in the sensitivity to auxin transport inhibitors and to phy- 1096 Analysis of effects of auxin transport inhibitors

III • • g 100 X - a A o V 80 BO - a 60 \ 9-V o 40 V s - —o—•— wilpirl-1d type Is,

—a— pirZ-1 Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 1 20 • i i 0.01 0.1 10 100 10 100 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= 14-17 (wild type), n=5-9 (pirl-1), and n= 11-14 (pir2-l); and (B) n=46-52 (wild type), n=31-36 (pirl-1), and n = 24-39 (pir2-l). tohormones often coincide. Thus, it seemed likely that wild-type A. thaliana (Fig. 3). The presence of either the our NPA-resistant mutants might also have acquired pir2 (ein2) mutation or of AgNO3 allowed the partial recov- resistance to other phytohormones. We examined their sen- ery of root growth that would otherwise have been in- sitivity to IAA and 1-aminocyclopropane-l-carboxylic acid hibited by NPA. The roots of wild-type seedlings treated (ACC), a naturally occurring auxin and a precursor of with 10 /xM NPA in the presence and in the absence of ethylene, respectively. The pirl-1 mutant was about 25- AgNO3 were 23% and 73%, respectively, as long as the con- fold more resistant to IAA than the wild type for 50% inhi- trol roots. In the case of pir2-2 seedlings, the roots at 10 bition of root growth. The extent of the inhibition of root j«M NPA were 79% as long as the controls. By contrast, in growth produced by 10yuM ACC was only 20% in pirl-1, the presence of 1 fiM IAA, the addition of AgNO3 to the while it was 51% in the wild type. The pir2-l mutant was medium was associated with no more than a slight increase more insensitive to ACC than the wild type but it was equal- in root length in the wild type (Fig. 3). Similar results were ly sensitive to IAA (Fig. 2B, C). obtained after the application of aminoethoxyvinylglycine In addition to the changes in the sensitivity to phyto- (AVG), a potent inhibitor of ethylene biosynthesis (Yang hormones, pirl exhibited apparent agravitropism even in and Hoffman 1984), instead of AgNO3 (data not shown). the absence of NPA (Fig. IB). These characteristics were Since these results suggested that the action of NPA was very similar to those of the phytohormone-resistant mu- partly exerted via ethylene, we examined whether auxin tants characterized to date. Genetic analysis revealed that transport inhibitors could stimulate ethylene production. pirl and pir2 were allelic to auxl and ein2, respectively. The level of ethylene generated by wild-type seedlings in The auxl and ein2 mutants are known as an auxin-resist- the presence of 10 fiM ACC was about 26 times higher than ant mutant and an ethylene-insensitive mutant, respective- that in the absence of ACC but, contrary to our expecta- ly. Thus, it appeared that both auxin and ethylene were tions, the application of NPA or of TIBA failed to induce associated with the inhibitory effect of auxin transport inhibitors on root growth. Therefore, we attempted to de- termine how the effects of auxin, ethylene and the polar Table 1 Production of ethylene by wild-type seedlings in transport of auxin might interact with the actions of auxin the presence of various agents transport inhibitors. Effects of ethylene—In order to examine the involve- Treatment Ethylene production (nl) ment of ethylene in the effects of NPA on root growth, we performed dose-response analysis using ethylene inhibitors Control 2.2±0.5 and both wild type and pir2 (ein2) mutant seedlings. The ex- IOJUM ACC 52.1±0.1 periment was performed under airtight conditions to accen- 10 /JM NPA 2.6±0.2 tuate the effect of NPA. Silver ions are often used as a strong inhibitor of ethylene action (Beyer 1976), and 10//MTIBA 2.0±0.4 AgNO3, added to the medium at 2fiM, effectively over- Each value represents the mean of results from three sam- came the inhibition by ACC of root growth in seedlings of ples ± standard error. Analysis of effects of auxin transport inhibitors 1097

AgNO3 120 Control

1 1-- 10 uM NPA +

D wild type + • pirZ-Z

10 iiM ACC + Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative root elongation 0.01 10 100 Fig. 3 Effects of NPA, IAA, and ACC on root elongation in the TIBA presence and absence of 2 j/M AgNO3. Seeds were grown on agar- solidified basal medium with 0.1% sucrose in the light, with plates 120 in a vertical position, under airtight conditions. After incubation for 5 days, roots of the wild type and pir2-2 were measured. Elon- gation 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= 11-18 (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 par- 0.001 0.01 0.1 tially sensitive to NPA in terms of root growth (Fig. 3). IAA 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 neg- ative effect on root gravitropism, while the various ana- 0.001 0.01 10 100 logs of TIBA that we tested had little or no effect on either parameter (Fig. 4A, B). The extent of the defect in root ACC gravitropism paralleled that of the defect in the polar transport of auxin. However, root elongation was most strongly 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 ex- inhibited by 3-iodobenzoic acid (3-IBA) among the analogs pressed relative to the mean elongation of the root in the absence of TIBA, followed by TIBA, and no relationship was recog- of test compounds. Bars represent standard errors. Mean values nized between the polar transport of auxin and root length for 100% root growth were 22.7 ±0.7 mm (A), 26.0 ±0.9 mm (B), (Fig. 4A, C). This strong inhibition by 3-IBA was also ob- and 22.1±0.6mm (C) for the wild type; 16.2±1.2mm (A), served with the auxl-7 and pir2-l mutants and with the 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. pir2-l auxl-7 double mutant (Fig. 5). It appears from these (A) n= 14-19 (wild type), n = 8-17 (pirl-1), and n = 7-13 (pir2-l); results that there might exist a pathway that does not in- (B) n= 13-19 (wild type), n=9-l 1 (pirl-1), and n= 10-12 (pir2-l); volve auxin and the polar transport of auxin, as well as a and (C) n=14-16 (wild type), n=9-ll (pirl-1), and n = 9-12 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 1098 Analysis of effects of auxin transport inhibitors

[3H]IAA transported (cpm) effects of auxin transport inhibitors, using the auxl and 0 1000 2000 3000 4000 5000 pir2 (ein2) mutants. Since the auxl mutant was resistant to Acropetal both ethylene and auxin simultaneously, it was unclear Baslpetal whether the phenotypes of the auxl mutant were essentially BA 2-IBA attributable to the resistance to auxin or to the resistance to 3-IBA 4-IBA ethylene. To define whether it was the resistance to auxin TIBA 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 back- Root gravltroplsm (degrees)

ground of the pir2-l mutation, \npir2-l seedlings ethylene Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 10 20 30 40 50 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 Root elongation (mm) obtained with two analogs of TIBA, namely, benzoic acid 10 (BA) and 3-IBA. By contrast, the auxl-7 mutation conferred no significant resistance to NPA on the pir2-l mutant (Fig. 5).

Discussion 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, It has been proposed that auxin transport inhibitors, with an acropetal control (A), on root gravitropism (B), and on such as NPA and TIBA, specifically interfere with the efflux root elongation (C) in wild-type seedlings. Each compound was of auxin from cells, and these compounds have frequently added at 30//M (A) or at 10^M (B, C). Bars represent standard er- been used in the analysis of polar auxin transport. By con- rors. n = 5 (A), n=27-32 (B), and n= 17-19 (C). trast, 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 auxin- Control like activity or by alterations in the concentration and/or distribution of auxin that arise from inhibition of auxin 10 /JM NPA efflux from cells (Katekar and Geissler 1980, Muday and lOpMBA Haworth 1994). To characterize the mechanism of action

30 /JM BA of auxin transport inhibitors in greater detail, we isolated two mutants that were resistant to NPA, with the pirl and 10 uM 3-IBA • wild type DD auxl-7 pir2 mutations, which we eventually found to be allelic to 30 pM 3-IBA • Pir2-1 auxl and ein2, respectively. • pirZ-l auxl-7 10 t>U TIBA The auxl mutant is resistant to both auxin and 30 tiM TIBA ethylene (Pickett et al. 1990) while the ein2 mutant is insensitive to ethylene because of a lesion in the ethylene signal- transduction pathway (Ecker 1995). The auxl mutant is 20 40 60 80 100 120 also known as a root gravitropism mutant, and some lines Relative root elongation (%) of auxl mutants exhibit no gravitropism at all (Okada and Shimura 1992). However, the sensitivity to NPA with re- 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 spect to root gravitropism was found to be wild type both were grown on agar-solidified basal medium with 0.1% sucrose in in pirl (auxl) and in pir2 (ein2) seedlings and, therefore, the light, with plates in a vertical position. After incubation for 5 the two mutations seems specifically to affect the process of days, roots were measured. Elongation is expressed relative to the inhibition of root growth among the various effects of NPA mean elongation of the root in the absence of test compounds. and the mutations might be associated with a defect in hor- Bars represent standard errors. Mean values for control root growth were 15.0±0.5mm for the wild type, 17.3±O.4mm for mone signal transduction rather than in polar auxin trans- auxl-7, 16.8±0.8mm for pir2-l, and 17.8+0.6 mm for pir2-l port itself. auxl-7. n= 15-20 (wild type), n= 13-20 (auxl-7), n= 13-19 (pir2- Since pir mutants that we isolated as NPA-resistant 1) and n = 15-20 (pir2-l auxl-7). mutants were allelic to auxl or ein2, it seemed likely that Analysis of effects of auxin transport inhibitors 1099

pir1 (aux7) pir2 (ein2)

root elongation Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021

polar auxin transport | \\-*- IAA f B

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 insensi- tivity of these mutants to ethylene.

the phytohormones auxin and ethylene might be involved the defect in polar auxin transport reflected the extent of in the effects of auxin transport inhibitors. In fact, there the defect in root gravitropism but not the effect on are many similarities between the effects of ethylene and root elongation (Fig. 4). In particular, 3-IBA markedly those of auxin transport inhibitors, as described above. In prevented root growth but it had no significant effect on addition, in the course of our experiments, we found that polar auxin transport (Fig. 4A, C). This strong inhibition NPA had a greater effect on the root growth of wild-type by 3-IBA was also seen in the auxl-7, pir2-l, and pir2-l seedlings under airtight conditions than under normal auxl-7mutants (Fig. 5). Thus, it seems clear that a pathway growth conditions. This inhibition by NPA was reversed to that is not related to polar auxin transport, to the effects of a considerable extent by the pir2 (ein2) mutation and also auxin, or to the actions of ethylene is a major contributor by the application of ethylene inhibitors such as silver ions to the strong effect of 3-IBA on root growth. To some and AVG (Fig. 3). This result indicated that NPA exerted extent, TIBA might also exert its effect via such a path- its effect in part via ethylene. Resembling root elongation, way (Fig. 6E). BA did not inhibit polar auxin transport the formation of root hairs that was accelerated by NPA (Fig.4A). This result is compatible with the previously was reduced by the pir2 (ein2) mutation and by the inhibi- reported results that BA itself is not transported in a tors of ethylene (data not shown). Thus, ethylene seems basipetal manner in corn coleoptile sections (Hertel et al. likely to be associated with these processes that are stimulat- 1969) and that BA does not affect auxin uptake by seg- ed by NPA. However, neither NPA nor TIBA induced the ments of zucchini hypocotyl (Depta and Rubery 1984). evolution of ethylene (Table 1). These data are consistent The hypothesis is widely accepted that NPA inhibits with those reported previously by Gaither and Abeles the transport of auxin and causes the high-level accumula- (1975), who showed that auxin transport inhibitors, in- tion of auxin, with subsequent inhibition of root growth. cluding NPA and TIBA, did not stimulate ethylene evolu- However, the auxl-7 mutation failed to confer resistance tion by pea seedlings. It appears that the background level to NPA on the background of the pir2-l mutation, in of ethylene is sufficient to intensify the effects of NPA on which ethylene had no effect but IAA inhibited root growth both the growth of primary roots and the formation of in the normal manner. This result leads to the conclusion root hairs (Fig. 6F, G). that the hypothesis is not valid for the inhibition of root Some effects of auxin transport inhibitors have been at- elongation in A. thaliana (Fig. 6C). Unlike the resistance to tributed to the inhibition of auxin transport. However, our NPA, the partial resistance to TIBA caused by the auxl-7 analysis with analogs of TIBA showed that the extent of mutation (Fig. 5) might have arisen from the weak auxin- 1100 Analysis of effects of auxin transport inhibitors like activity of TIBA itself. The difference between the of a variety of additional mutants that reflect diverse modes of action of NPA and TIBA have been discussed in aspects of the action of NPA. some detail: NPA and TIBA seem to have different binding sites (Thomson et al. 1973, Katekar and Geissler 1980, Dep- The authors thank Dr. Hirokazu Tsukaya for helpful advice ta and Rubery 1984, Michalke et al. 1992); moreover, on treatment of seedlings of Arabidopsis thaliana and Drs. Toru TIBA is transported in a polar basipetal manner while NPA Shigematsu and Takeshi Uozumi for quantitation of ethylene. is not (Thomson et al. 1973). These differences between NPA and TIBA at the molecular level might be responsible References for the differences between their effects on root growth. Allan, A.C. and Rubery, P.H. (1991) Calcium deficiency and auxin trans- Since resistance to NPA was not conferred by the port in Cucurbita pepo L. seedlings. Planta 183: 604-612. Downloaded from https://academic.oup.com/pcp/article/37/8/1094/1897776 by guest on 27 September 2021 auxl-7 mutation on the ethylene-insensitive background Bennett, S.R.M., Alvarez, J.A., Bossinger, O. and Smyth, D.R. (1995) (Fig. 5), it is possible that/?//-/ (auxl) mutants might be iso- Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J. 8: lated during the selection for NPA-resistant mutants large- 505-520. Beyer, E.M. (1976) A potent inhibitor of ethylene action in plants. Plant ly because of their resistance to ethylene rather than to aux- Physiol. 58: 268-271. in (Fig. 6F). Burg, S.P. and Burg, E.A. (1967) Inhibition of polar auxin transport by Excised segments of inflorescences of A. thaliana have ethylene. Plant Physiol. 42: 1224-1228. Davenport, T.L., Morgan, P.W. and Jordan, W.R. (1980) Reduction of the capacity for the basipetal transport of auxin (Okada et auxin transport capacity with age and internal water deficits in cotton al. 1991, Bennett et al. 1995). We found that hypocotyls of petioles. Plant Physiol. 65: 1023-1025. A. thaliana also have this capacity. The polar transport of Davies, P.J. and Rubery, P.H. (1978) Components of auxin transport in stem segments of Pisum sativum L. Planta 142: 211-219. auxin in stems is known to be influenced to a considerable Depta, H. and Rubery, P.H. (1984) A comparative study of carrier par- extent by a variety of external and internal factors, such as ticipation in the transport of 2,3,5-triiodobenzoic acid, indole-3-acetic nutrient conditions (Tang and de la Fuente 1986, Allan and acid, and 2,4-dichlorophenoxyacetic acid by Cucurbita pepo L. hypo- Rubery 1991), the age of the seedling (Bennett et al. 1995), cotyl segments. J. Plant Physiol. 115: 371-387. Ecker, J.R. (1995) The ethylene signal transduction pathway in plants. and the position of the stem (Davenport et al. 1980, Morris Science 268: 667-675. and Johnson 1990, Suttle 1991). However, our present tech- Gaither, D.H. (1975) Auxin and the response of pea roots to auxin trans- nique using hypocotyls allows preparation of uniform port inhibitors: morphactin. Planta 55: 1082-1086. Gaither, D.H. and Abeles, F.B. (1975) Sites of auxin action. Plant Physiol. materials in large quantities, and may be valuable for the 56: 404-409. analysis of polar auxin transport in A. thaliana. Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Genetic analysis with mutants can serve as a powerful Plant Physiol. 28: 439-478. Hertel, R., Evans, M.L., Leopold, A.C. and Sell, H.M. (1969) The specific- tool for the dissection of biological phenomena. We at- ity of the auxin transport system. Planta 85: 238-249v tempted to evaluate the effects of ethylene, auxin, and Hertel, R., Lomax, T.L. and Briggs, W.R. (1983) Auxin transport in mem- polar auxin transport on the action of NPA using mutants brane vesicles from Cucurbita pepo L. Planta 157: 193-201. Katekar, G.F. and Geissler, A.E. 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Plant Sci. that the inhibitory effect of NPA on root elongation is due 100: 9-14. to the accumulation of auxin at a high level as a result of Liu, C.-m., Xu, Z.-h. and Chua, N.-H. (1993) Auxin polar transport is blockage of auxin transport is not applicable to A. thaliana essential for the establishment of bilateral symmetry during early embryogenesis. Plant Cell 5: 621-630. (Fig. 6C); and (4) TIBA itself has weak auxin-like activity. Michalke, W., Katekar, G.F. and Geissler, A.E. (1992) Phytotropin-bind- The inhibition of root growth by auxin transport inhibitors ing sites and auxin transport in Cucurbita pepo: evidence for two recogni- appears to be a complex phenomenon in which various fac- tion sites. Planta 187: 254-260. Morris, D.A. and Johnson, C.F. (1990) The role of auxin efflux carriers in tors, including phytohormones, seem to participate and in- the reversible loss of polar auxin transport in the pea {Pisum sativum L.) teract synergistically. To date, the actions of NPA have stem. Planta 181: 117-124. been explained for the most part in terms of inhibition of Muday, G.K. and Haworth, P. (1994) Tomato root growth, gravitropism, auxin efflux. However, in root growth, at least, such is not and lateral development: correlation with auxin transport. Plant Physiol. Biochem. 312: 193-203. the case. The other effects of auxin transport inhibitors re- Muday, G.K., Lomax, T.L. and Rayle, D.L. (1995) Characterization of quire our attention and further examination of these effects the growth and auxin physiology of roots of the tomato mutant, is required for a better understanding of the detailed mech- diageotropica. Planta 195: 548-553. Okada, K. and Shimura, Y. (1992) Mutational analysis of root anisms of action of these inhibitors and the polar transport gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust. of auxin. Further screening might lead to the identification J. Plant Physiol. 19: 439-448. 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