Plant Cell Physiol. 37(8): 1094-1101 (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
Analysis of effects of auxin transport inhibitors
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 a n d / o r 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.
1095
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)-3H]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.
Materials and Methods
Results
Plant materials and growth conditions—Ethyl methanesulfonate (EMS)-mutagenized M2 seeds of the Columbia ecotype of
Isolation of NPA-resistant mutants—Since 10 fiM
A. thaliana and both the auxl-7and ein2-J mutants were obtained
NPA
effectively inhibited root elongation and induced the
from the Lehle Seeds Co. (Tucson, AZ, U.S.A.) and the Arabiformation of root hairs in A. thaliana, seedlings from
dopsis Biological Resource Center (Ohio State University, Columbus, OH, U.S.A.), respectively. Plants were grown under continuEMS-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 comisolated five NPA-resistant strains. The mutations were all
pounds (1 x ) that contained 5 mM KNO3, 2 mM MgSO4, 2 mM
recessive and could be assigned to two genetic loci, designatCa(NO3)2, 2.5 mM potassium phosphate adjusted to pH 5.5, 50
ed pirl and pir2 (polar auxin transport inhibitor resistant).
fiU Fe-EDTA, 70^M H3BO3, HytiM MnCl2, 0.5 fiM CuSO4, 1
/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 germinashape, 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 concenbasal medium (1 x inorganic solution, 100 mg liter"1 myo-in1
1
ositol, 20 mg liter" thiamine hydrochloride, 1 mg liter" 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,
root gravitropism. However, the background level of root
that was maintained in the vertical position so that the roots
gravitropism in the absence of NPA in the pirl-1 mutant
would grow along the surface of the solid medium. After incubation at room temperature for four days, the seedlings were transwas different from that in the wild type (Fig. IB). Thereferred to new plates prepared with various concentrations of test
fore, the lesions in both the pirl and pir2 mutants were specompounds, and the positions of root tips were marked on the
cifically associated with the effect of NPA on the root-elonplate. The extent of new root growth was measured after incubagation process among various possible effects of NPA.
tion for a further five days.
Moreover, the sensitivity to TIBA, another inhibitor of auxExamination of root gravitropism—Sterile seeds were incuin transport, of pirl-1 and pir2-l was also about 5 and
bated on the agar-solidified basal medium, plus \% sucrose with
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
inhibitors.
roots were oriented at 180°.
Several phytohormone-resistant mutants exhibit alterQuantitation of ethylene—About 100 sterilized wild-type
ed
responses
to more than one phytohormone, and changes
seeds were sown in 20-ml vials that contained 10 ml of agarsolidified 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
I
g
100
o
V
80
I
•
I
•
-
X
A
a
-
BO
a
40
1
20
9-
V
o
s
V
\
60
-
—•— wild
type
—o—
pirl-1
—a— pirZ-1
•
i
0.01
0.1
Is,
i
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
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 25fold 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
AgNO3, 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 AgNO3 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
AgNO3 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 AgNO3 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 AgNO3 (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
Ethylene production (nl)
2.2±0.5
ACC
52.1±0.1
10 /JM NPA
2.6±0.2
10//MTIBA
2.0±0.4
IOJUM
Each value represents the mean of results from three samples ± standard error.
Analysis of effects of auxin transport inhibitors
1097
AgNO3
120
Control
1
10 uM NPA
1--
+
D wild type
• pirZ-Z
+
10 iiM ACC
+
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Relative root elongation
0.01
10
100
TIBA
120
0.001
0.01
0.1
IAA
0.001
10
0.01
100
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= 14-19 (wild type), n = 8-17 (pirl-1), and n = 7-13 (pir2-l);
(B) n= 13-19 (wild type), n=9-l 1 (pirl-1), and n= 10-12 (pir2-l);
and (C) n=14-16 (wild type), n = 9 - l l (pirl-1), and n = 9-12
Fig. 3 Effects of NPA, IAA, and ACC on root elongation in the
presence and absence of 2 j/M AgNO3. 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 = 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 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
Analysis of effects of auxin transport inhibitors
1098
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).
[3H]IAA transported (cpm)
0
1000
2000
3000
4000
5000
Acropetal
Baslpetal
BA
2-IBA
3-IBA
4-IBA
TIBA
Root gravltroplsm (degrees)
10
20
30
40
50
Root elongation (mm)
10
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,
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 = 17-19 (C).
Control
10 /JM NPA
lOpMBA
30
/JM
BA
10 uM 3-IBA
• wild type
DD auxl-7
• Pir2-1
• pirZ-l auxl-7
30 pM 3-IBA
10 t>U TIBA
30 tiM TIBA
20
40
60
80
100
120
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 17.8+0.6 mm for pir2-l
auxl-7. n = 15-20 (wild type), n = 13-20 (auxl-7), n = 13-19 (pir21) and n = 15-20 (pir2-l auxl-7).
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
Analysis of effects of auxin transport inhibitors
1099
pir1 (aux7)
pir2 (ein2)
root elongation
polar auxin transport |
\\-*B
IAA f
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-
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.
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(Received April 12, 1996; Accepted September 2, 1996)