Plant Physiol. (1987) 84, 1139-1142
0032-0889/87/84/1139/04/$01.00/0
Interaction of Indoleacetic Acid with Its Inositol and Glucoside
Conjugates in Avena Coleoptile Curvature1
Received for publication July 8, 1986 and in revised form March 30, 1987
TOMASZ J. WODZICKI2, RICHARD P. PHARIS*, AND ALINA B. WODZICKI2
Biology Department, University of Calgary, Calgary, Alberta, Canada, T2N IN4
ABSTRACT
APena coleoptile curvature is promoted by indoleacetic acid (IAA)
IAA-glucoside, and IAA-inositol when these substances are applied in
agar to the decapitated apical end of deseeded plantlets. Absorption of
IHILAA-inositol over a wide range of concentrations during the 20 hour
period of incubation is only 20 to 50% of the applied amount, compared
with 85 to 92% of uptake of the applied iHIIAA at equimolar concentrations. The absorption of IAA-glucoside could not be readily measured.
The stimulation by both IAA-conjugates is very similar to that of free
IAA at low concentrations (0.2 and 0.4 micromolar), but much less at
higher concentrations. The interaction of free UIA with IAA-glucoside is
additive or synergistic (depending on concentration). The interaction of
free IAA with IAA-inositol is an inhibition (i.e. less than additive). The
simultaneous application of equimolar concentrations of free IAA does
not change the chromatographic pattern of the metabolic products of [HI
IAA-inositol. One of the more polar metabolites of IHIIAA-inositol has
chromatographic characteristics similar to the major polar metabolite of
free PHIIAA on an isocratically eluted reversed phase Cis high performance liquid chromatography system that separates a number of IAA sugar
and amino acid conjugates from each other, and from free IAA.
IAA-glucoside, respectively, in aqueous media. The Avena curvature method, however, does allow one to make conclusions
regarding polar transport of IAA, which should not be disregarded (6, 9, 1 1) in any study or hypothesis on morphogenetic
or growth effects of auxin.
MATERUILS AND METHODS
Bioassay. Went's Avena (cv Seger I) coleoptile curvature bioassay for auxin was modified as recommended by Funke (4). Exact
details are given in Wodzicki et al. (12) and references therein.
In its present version the coleoptiles were detached from kernels
24 h before decapitation, which in turn preceded by only 15 min
application of the agar blocks. So modified, this procedure of
cutting the coleoptiles assured high reproducibility of the test
(checked during several years of its use) and the response sensitivity approaching 8 to 10 pg IAA per coleoptile in an 8 ,ul cube
of agar. Technically, the bioassay was done as described by
Kentzer and Rowicka (8). The substances to be tested were
placed in the very bottom of 10 ml conical Pyrex centrifuge
tubes and dried under reduced pressure. They were then dissolved
with 100 ul of hot (800 C) 1% aqueous Difco agar with a Vortex,
and immediately placed on small (6 x 8 mm) rectangular plates
of thick Al foil and covered with a second plate of the Al foil (of
the same size) with a gentle pressing to obtain the desired
(approximately 2.0 mm) thickness of the agar plate. The agar
In studying the interaction of the Avena curvature assay of plates were cooled briefly at +3° C to solidify the agar and after
approximately
natural growth substances collected in agar strips by basipetal careful removal of the upper Al foil cut to 12 cubeswere
exposed to
Coleoptiles
efflux from the cambial region of the pine stem (12), it was found 8 mg each withina razor blade cutter.
h
at
near
100%
20
blocks
for
the
substances
(overnight)
agar
that the presence of natural IAA (in a purified HPLC fraction)
at 24° C, and in complete darkness.
suppressed the expression of curvature by a more polar natural RHExtraction
and Chromatography. In the case of the experisubstance(s). Tentatively, this more polar substance(s) was idenblocks and coleoptified as an IAA-conjugate (i.e. it co-chromatographed with IAA- ments with radioactive substances both agar
determination
of residfor
direct
tiles
were
saved
after
bioassays
IAA
after alkaline hydrolysis)
inositol, and released bioactive
and for extraction. Crushed coleoptiles were
(12). These results implied a possible interdependence of the ual radioactivity,
1 ml ofice cold 80% methanol
processes of IAA release and synthesis of IAA-conjugates (2). extracted in glass vials 3 times with
x
h
2
h at 4° C). The combined
2
0.5
(1
plus
coleoptiles
per
The possible interaction of IAA with two IAA-sugar conjugates
were
and
to dryness with excess
extracts
filtered
evaporated
with
were
thus
tested
herein
and
IAA-glucoside)
(IAA-inositol
the Avena coleoptile curvature assay, on which this apparent absolute methanol under vacuum. They were subsequently disinteraction was originally observed with natural IAA and the solved in a small volume of 28% methanol and subjected to
fractionation on a reversed phase C18 analytical u-Bondputative IAA conjugate from Pinus. According to our knowledge HPLCcolumn
using a Waters Liquid Chromatograph with solvent
neither of the two IAA conjugates has been tested before with apak
this highly sensitive (8-10 pg IAA) and definitive curvature assay, delivery systems 6000A and AGC680. Two HPLC programs
although Nicholls (10) and Keglevic and Pokorny (7) studied were used in this study. Program A was triphasic: (a) isocratic
Avena coleoptile straight growth stimulation by IAA-inositol and 28% aqueous methanol:0.8% acetic acid ( 1-16 min); (b) gradient
28% aqueous methanol:0.8% acetic acid (16-22 min): (c) isocratic absolute methanol (22-34 min): program B was isocratic
' Supported through Natural Sciences and Engineering Research
10% aqueous methanol: 1% acetic acid for 30 min. Both solvents
Council of Canada International Scientific Exchange Award IS-0185, efficiently separated IAA-inositol, IAA-glucoside, IAA-aspartate,
a contract from the Canadian Forestry and IAA-glutamate from IAA (Table I in Ref. 12).
Substances. IAA was purchased from Sigma, and 3-[5n3H]
Service P.R.U.F. program.
2Present address: Department of Forest Botany, Faculty of Forestry, IAA, 16.7 Ci/mmol, was purchased from Amersham. IAAUniversity of Agriculture, 26/30 Rakowicka Street, 02-528 Warsaw, inositol was from R. S. Bandurski. IAA-glucoside was from J.
Poland.
(27 Ci/mmol) was from W. Pengelly.
5-[3H]IAA-inositol
Downloaded from on June 14, 2017Cohen,
- Published
by www.plantphysiol.org
Copyright © 1987 American Society
1139of Plant Biologists. All rights reserved.
Operating grant A-2585, and
1140
WODZICKI ET AL.
1Plant
Physiol. Vol. 84, 1987
RESULTS
IAA and IAA-Inositol Interaction. Preliminary experiments
revealed that stimulation of Avena coleoptile curvature by IAAinositol increased appreciably at concentrations from 0.1 to 0.4
gM, but was relatively stable at concentrations above 0.4 ,M. The
stimulation in all cases was less than that occasioned by equimolar concentrations of IAA. However, the curvature caused by
IAA-inositol alone was not fully expressed when it was tested in
the presence of equimolar amounts of IAA. Instead, a slight
reduction of the IAA stimulation occurred.
A wider range ofconcentrations was then tested in the presence
of [3H]IAA-inositol of high specific activity (Fig. 1, Table I).
Table II summanzes the uptake of radioactivity from agar blocks
by coleoptiles (collected at h 20). The coleoptiles absorbed only
21 to 25% of the IAA-inositol at concentrations in agar of 0.2 to
0.4 Mm, irrespective of whether free IAA was present in the agar
block. At the 0.8 FM concentration (IAA-inositol only) a similar
uptake (32%) occurred. However, at 0.8 Mm when IAA was given
simultaneously, the uptake of IAA-inositol incrased to 59%. A
similar uptake (53%) occurred for IAA-inositol alone when applied at a 1.6 gM concentration (Table II). Taking into account
the actual uptake, the curvature stimulation that was measured
when IAA-inositol was applied alone at concentrations of 0.2 to
0.4 FM could be totally ascribed to the effect of free AA that
might be released from these conjugates (assuming complete
hydrolysis) (Fig. 1) However, at concentrations of 0.8 and 1.6
,uM the stimulation was considerably less than expected had all
the absorbed IAA-inositol been hydrolyzed to yield free IAA. In
fact, there was a negative interaction between the two substances
(Fig. 1). This confirmed the previously noticed suppression of
IAA-inositol stimulated curvature by the presence of equimolar
amounts of free IAA (in agar) at concentrations of0.2 to 0.8 MuM.
At 1.6 uM, IAA-inositol given in the presence of free IAA caused
a significant inhibition of the curvature stimulation expected
from free IAA alone.
Both IAA and IAA-inositol are metabolized in Avena coleoptiles (Figs. 2 and 3). HPLC fractionation was accomplished for
the 80% methanolic extracts from the coleoptiles after their 20
jP 40 0
30
/t a:.o-t'-
40
.~~~~~~~~~0
30
0.
0/
20
/...
816
[3H]LAA-Inositol
cubes
Coleoptile
Uptake
FI.1
h neato
ffe A
n
IAA-inositolintesmua
10
0 0.2
0.8
1.6
CONCENTRATION IN
FIG.
1. The interaction of
fr-ee
8Ml
Agar
3.2.MM
AGAR BLOCK
IAA and IAA-inositol in the stimula-
tion of Avena coleoptile curvature. In the actual curve of IAA + IAAinositol each substance was applied in 8 gl agar cubes in 0.2, 0.4, 0.8,
and 1.6 Mm concentrations. A, Theoretical curve if all of the absorbed
[3HJIAA-inositol had released all of its IAA moiety to be active in
curvature stimulation. B, Theoretical curve if the effect of two substances
had been active at the additive dose, based on amounts absorbed (see
Table II), and assuming release of all of LkA moiety from the absorbed
LAA-inositol. The curvature response (degrees) was calculated for both
theoretical curves from actual log dose response curves of IAA standards.
[3H]IAA
IAA plus [3H]IAA-Inositola
cubes
.^
0
Table II. Uptake of[3HJIAA and [3IJIAA-Inositolfrom Agar Cubes byAvena Coleoptiles during the Went
Avena Curvature Test
Means of 4 replicates ± 1 SE.
Agar
plus
-
Table I. Stimulation ofAvena Coleoptile Curvature (Degrees) by IAA and IAA-Inositol
Means of two replicate series, 8 coleoptiles each, ± 1 SE of the mean.
Concentration (Mm) in 8 ul Agar
Substances Tested
0.1
0.2
0.4
0.8
±
IAA
17.7 0.5
28.4 ± 0.5
38.1 ± 0.4
43.3 ± 0.5
9.6 ± 0.5
IAA-inositol
13.2 ± 0.3
20.0 ± 0.2
20.1 ± 0.3
IAA plus IAA-inositol
17.5 ± 0.5a
27.1 ± 0.4a
37.2 ± 0.5a
a
Equimolar concentrations (0.1, 0.2, and 0.4 Mm) of each substance dissolved in agar.
Concentration
in 8.ul
Agar
- -IAA-
/~~~~~~~~
I-
w
--
,,.'IAA-inositol
*
Coleoptile
Uptake
Agar
cubes
Coleoptile
%
dpm
%
dpm
dpm
dpm
dpm
dpm
JM
317 ± 33
84 ± 23
63 ± 17 25
21
0.2
189 ± 28
25 108±25 602±35
611±47 262±21 21
626±66 213±7
0.4
65 ± 7 701 ± 44
1089 ± 168 538 ± 58 32 627 ± 112 899 ± 104 59
0.8
1.6
1749 ± 310 1867 ± 35 52 1515 ± 114 1712 ± 313 53
31 ± 5 573 ± 65
3.2
a Equimolar concentrations (0.2, 0.4, 0.8, and 1.6 uM) of each substance dissolved in agar.
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 1987 American Society of Plant Biologists. All rights reserved.
Uptake
%
85
92
92
INTERACTION OF IAA WITH IAA-INOSITOL AND IAA-GLUCOSIDE
1141
20 -A
~ ~ A~~
20 ,-~
10-A
-----------
101__
20
1020
-
B
20
10 F
20
o F
,,,,,-
,
10
0
C--
[
t 10
=-..
c
f
F
0
D
0
30r-
10
0
I,
20
0
-
F
..
I,
-----
0
-
o
30
-
E
cL
22 0
20
E
-
lo
o ..
40
4
0
ax 30
20
n
4101-
F
-
30
20 [
-,
-
G
1
,, '
101,t
10
5
0
1
5
10
15
HPLC FRACTIONS
2
5
3
HPLC FRACTIONS (Rt minutes)
FIG. 2. Products of free [3H]IAA and [3H]IAA-inositol metabolism
in Avena coleoptiles over a 20 h period. The histogram denotes percentages of total radioactivity recovered in the successive 1 min fractions of
the C18 HPLC using a three-phasic program: (a) 1 to 16 min isocratic in
aqueous 28% methanol:0.8% acetic acid; (b) 16 to 22 min gradient from
absolute methanol; (c) 22 to
aqueous 28% methanol:0.8% acetic acid
34 min isocratic in absolute methanol. The two replicates refer to extracts
of two separate groups (2 coleoptiles each). A and B, Replicate extracts
from coleoptiles to which [3H]IAA-inositol was applied in 8 1I agar cubes
apically at a concentration of 0.8 uM. C and D, Coleoptiles treated with
[3H]IAA-inositol and ['H]IAA, respectively in equimolar concentrations
of 0.8,gM for each substance. E and F, Extracts of coleoptiles treated with
[3H]IAA at doses of 0.8 Mm and 0.4 gM of IAA respectively. G, Results
of C,8 reversed phase HPLC of the [3H]IAA-inositol used to prepare the
equimolar stock solution (of IAA and IAA-inositol) for the curvature
tests. The unshaded peak (12-13 min) is the Rt of authentic [3H]IAA.
--
h of apical treatment with agar blocks containing [3H]IAA or
[3H]IAA-inositol (the IAA-inositol is probably a mixture of two
or more enantiomorphic forms). The gradient-eluted HPLC
separation shown in Figure 2, although useful to note degree of
metabolism of [3H]IAA to more polar compounds, and [3H]
IAA-inositol to less polar compounds, did not adequately resolve
the very polar metabolites. Hence, an isocratic HPLC in 10%
MeOH was used (Fig. 3). This fractionation revealed several
more polar derivatives (Fig. 3). Free IAA was metabolized mainly
to substances with their RTmax at 4 min and 5 min (Fig. 3), well
to the polar side of [3H]IAA-inositol on the HPLC elution profile
(Fig. 3). A lesser 3H peak (Fig. 3) corresponded to a broad RTm.
20
25
30
(Rt minutes)
FIG. 3. Products of free [3H]IAA and [3H]IAA-inositol metabolism
in Avena coleoptiles over a 20 h period. Histograms denote percentages
of total radioactivity recovered in the successive 1 min fractions of an
isocratic C18 reversed phase HPLC using 10% aqueous methanol:l%
acetic acid for 30 min. The two replicates refer to extracts of two
separately treated coleoptile groups (2 coleoptiles each). A and B, Extracts
from the coleoptiles to which [3H]IAA-inositol was apically applied in 8
,u agar cubes at a concentration of 1.6 uM. C and D, Coleoptiles treated
with [3H]IAA-inositol and ['H]IAA in equimolar concentrations of 1.6
Mm for each substance. E and F, Extracts of the coleoptiles treated with
free [3H]IAA at a concentration of 3.2 gM. G, Chromatographic profile
of the [3H]IAA-inositol used to prepare stock solutions of IAA-inositol
used in the curvature tests. The Rt of free [3H]IAA with this solvent was
42.2 min (elution peak not shown).
(10-12 min) of [3H]IAA-inositol (Fig. 3). The HPLC separation
with 10% aqueous methanol, 1% acetic acid of extracts from the
coleoptiles treated with [3H]IAA-inositol thus yielded four distinct peaks (Fig. 3), two of which coincided with authentic [3H]
IAA-inositol at Rtm. 12 and 16 min. Another (the largest) peak
from [3H]IAA-inositol metabolism occurred at Rtm,, 5 min. This
min 5 peak corresponded to the Rt of the very polar metabolite
of free [3H]IAA, but it contained only half the radioactivity that
was found when only free [3H]IAA was applied to the coleoptiles.
Additionally, a radioactive substance chromatographing at Rtmax
8 min was found in extracts of those coleoptiles treated only with
[3H]IAA-inositol. The isocratic HPLC (28% aqueous methanol,
0.8% acetic acid) did not allow complete separation of all of
these polar substances. However, it did confirm that practically
no [3H]IAA remained in the coleoptiles by h 20. Addition of
['H]IAA to [3H]IAA-inositol in agar at equimolar concentrations
did not result in any significant change of the chromatographic
pattern of the extracts. Hence free IAA did not interfere with
[3H]IAA-inositol metabolism. Thus, the possibility that the neg-
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 1987 American Society of Plant Biologists. All rights reserved.
1142
WODZICKI ET AL.
Plant Physiol. Vol. 84, 1987
tive form. If, however absorption is less than that of free IAA,
the difference between our results and those of Keglevic and
Pokomy (7) could be readily explained. The stimulation by the
two substances combined (in equimolar concentrations) equals
the stimulation which would be expected if both promoters were
50 >
free IAA. Thus, the possibility exists that IAA-glucoside is absorbed as well as IAA but remains partly unhydrolysed unless
IAA is applied simultaneously. This implies that IAA-glucoside
hydrolysis may be stimulated by the presence of free IAA.
1While such a situation may seem unlikely (i.e. induction or
activation of a hydrolytic enzyme by free IAA), both our results
la
and those of Keglevic and Pokorny (7) do point toward an
4
unusually high bioactivity for IAA-glucoside, relative to IAAJ
inositol. Resolution, however, will come only when radioactive
IAA-glucoside becomes available.
Stimulation of Avena coleoptile curvature by IAA-inositol at
the lowest concentrations corresponded to the effect produced
by an equimolar concentration of free IAA, again acting as if all
20O
the absorbed amount was hydrolyzed. Simultaneous application
of IAA and IAA-inositol in equimolar concentrations, however,
I
( l
resulted in less stimulation than would be expected if all the
0
0.4 0.8
1.6
3.2pM
absorbed IAA-inositol was hydrolysed to release free IAA. This
CONCENTRATION IN 8IL AGAR BLOCK
suggests that free IAA may suppress hydrolysis of IAA-inositol
FIG. 4. The interaction of IAA and IAA-glucoside in the stimulation
at higher concentrations. The more general hypothesis of Cohen
of Avena coleoptile curvature. In the curve for IAA + IAA-glucoside
and Bandurski (2, 3) on hormonal homeostasis vis IAA=IAA
each substance was applied in 0.2, 0.4, 0.8, 1.6, and 3.2 ,M concentraconjugates thus tends to be corroborated by at least part of our
tions.
results. However, a natural hydrolytic system of IAA-sugar conjugates analogous to that found in Zea mays (5) has yet to be
ative interaction of IAA and IAA-inositoi on curvature was demonstrated in Avena coleoptiles, nor do we know what the
endogenous IAA conjugates are in the Avena coleoptile in addiaccomplished by limiting IAA-inositol hydrolysis (e.g. limiting tion
to the peptidyl IAA reported by Bandurski and Schulze (1).
the release of free IAA from its conjugate) is not supported by
Analysis by HPLC-RC did not indicate any differences in the
these results. Another logical possibility, of course, is that IAA
metabolism of [3H]IAA-inositol in the presence of added
and IAA-inositol are competing for a site of action.
of free IAA. Thus the inhibition of curvature by IAALIA and IAA-Glucoside Interaction. Stimulation of Avena amounts
via
inositol
for a site of action is indicated, rather
coleoptile curvature by IAA-glucoside applied apically in agar than via anycompetition
effect
on hydrolysis of free IAA from the IAAto
the
of
IAA
effect
free
at
the
concentration
appeared equal
only
of 0.2 uM (Fig. 4). At all greater concentrations (up to 3.2 ,uM) inositol conjugate. The opposite response ofthe Avena coleoptiles
stimulation be free IAA was greater. However, in combination to IAA-glucoside (relative to IAA-inositol) is indicative of a
the two substances produced a synergistic effect on curvature in separate mechanism operating in respect to the coleoptile's ability
all other equimolar concentrations, except the 3.2 uM one, where to deal with various IAA-sugar conjugates. As noted above,
however, resolution of these differing mechanisms awaits the
a reduced curvature resulted (Fig. 4). The synergistic effects of
IAA + IAA-glucoside are as great as if the curvature of the availability of isotope labeled IAA-glucoside.
combination was due to the addition of an equimolar amount
LITERATURE CITED
of free IAA only. However, the effect of IAA-glucoside (alone)
was considerably less than that of IAA alone. Thus, applying
1. BANDURSKI RS, A SCHULZE 1977 The concentration of indole-3-acetic acid
IAA in the presence of IAA glucoside yielded a growth response
and its derivatives in plants. Plant Physiol 60: 211-213
equivalent to doubling the IAA amount. This implies, perhaps,
2. COHEN JD, RS BANDURSKI 1978 The bound auxins: protection of indole-3that significant hydrolysis of IAA from high concentrations
acetic acid from peroxidase-catalysed oxidation. Planta 139: 203-208
3. COHEN JD, RS BANDURSKI 1982 Chemistry and physiology of the bound
(greater than 0.2 uM) of IAA-glucoside may occur only in the
auxins. Annu Rev Plant Physiol 33: 403-430.
presence of appreciable amounts of free IAA.
4. FUNKE H 1939 Uber den Nachweis kleiner Wuchsstoffmengen. Jahrb Wiss
6O,
2 SE
0
0
0.O
40
0
Bot 88: 375-388
5. HALL PJ, RS BANDURSKI 1981 Hydrolysis of [3H]IAA myo-inositol by extracts
of Zea mays. Plant Physiol 67: S-2
6. JACOBS WP 1967 Comparison of the movement and vascular differentiation
effects of the endogenous auxin and of phenoxyacetic acid weedkillers in
stems and petioles of Coleus and Phaseolus. Ann NY Acad Sci 144: 102117
7. KEGLEVit D, M POKORNY 1969 The chemical synthesis of l-O-indol3'ylacetyl)jS-D-glucopyranose. Biochem J 114: 827-832.
8. KENTZER T, K ROWICKA 1963 Przydanosc roznych admian owsa jako materialu testowego do oznaczen regulatorow wzrostu roslin. Acta Agrobot 13:
117-130
9. LEOPOLD AC 1961 The transport of auxin. In W Ruhland, ed, Encyclopedia
of Plant Physiol, Vol 14. Springer-Verlag, Heidelberg, pp 671-682
10. NICHOLLS PB 1967 The isolation of indole-3-acetyl 2-O-myo-inositol from Zea
mays. Planta 72: 258-264
11. THIMANN KV 1972 The natural plant hormones. In FC Steward, ed, Plant
Physiology: A Treatise, Vol 6B. Academic Press, New York, pp 3-365
12. WODZICKI TJ, H ABE, AB WODZICKI, RP PHARIS, JD COHEN 1987 Investigations on the nature of the auxin-wave in the cambial region of pine stems.
Validation of IAA as the auxin component by the Avena coleoptile curvature
amounts of apically applied IAA-glucoside in our experiment
assay and by gas chromatography-mass spectrometry-selected ion monitorDownloaded
from
on June in
14,radioac2017 - Publisheding.
by www.plantphysiol.org
are not known since the conjugate
Plant Physiol 84: 135-143
was not
available
Copyright © 1987 American Society of Plant Biologists. All rights reserved.
DISCUSSION
Stimulation of unilateral growth upon apical application of
IAA-inositol or IAA-glucoside to one side of the cut surface of
Avena coleoptiles appeared similar to the effect obtained with
free IAA only at low concentrations (0.2 and 0.4 ,M) of the
conjugates. At all higher concentrations the stimulation of coleoptile curvature by the IAA conjugates was less than by free
IAA. In this respect only the effect of IAA-inositol at the higher
concentrations agrees with earlier results obtained by Nicholls
(10) with the Avena coleoptile straight growth test. For IAA
glucoside alone, the equal or lesser stimulation of Avena coleoptile curvature (relative to free IAA) measured herein does not
confirm the results of Keglevic and Pokomy (7). They found a
greater (greater than free IAA) promotion of elongation of oat
coleoptile sections in the straight growth test (7). The absorbed