It is characteristic of a great number of biologically active substances that the responses which they elicit are twofold, low concentrations of the material promoting a particular activity, and higher concentrations inhibiting it. This is the case with the auxin-induced growth responses of plants. An active auxin such as indole acetic acid (IAA) brings about and is essential to growth in length of stems, hypocotyls and other plant organs including the Avena coleoptile

Bonner, James

Foster, Robert J.

McRae, D. Harold

English

Caltech Authors - Main

BOTANY: FOSTER ETA!.
A UXIN-INDUCED GROWTH INHIBITION A NATURAL
CONSEQUENCE OF TWO-POINT A TTACHMENT*
By ROBERT J. FOSTER, D. HAROLD MCRAE, AND JAMES BONNER
KERCKHOFF LABORATORIES OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY
Communicated October 9, 1952
Introduction.-It is characteristic of a great number of biologically active
substances that the responses which they elicit are twofold, low concentra-
tions of the material promoting a particular activity, and higher concentra-
tions inhibiting it. This is the case with the auxin-induced growth re-
sponses of plants. An active auxin such as indole acetic acid (IAA) brings
about and is essential to growth in length of stems, hypocotyls and other
plant organs including the Avena coleoptile. Excised sections of this object
grow in length when they are floated in solutions containing low concentra-
tions of IAA and this growth is a simple function of auxin concentration as
> 3.0
= 2.0 I
1.0
E
E
3.0 2 3x 10
33.00
o 2.0
z
F
0
v) 100 200 300 x 10-§
CONCENTRATION IAA: moles/liter
FIGURE 1
Avena coleoptile section growth as a function of IAA
concentration: a (above), over a low concentrations range
0-3 X 10-5M; b, over a greater concentration range
0-300 X 10-5M.
is shown in figure la. With increasing auxin concentration, however,
growth rises to a maximum only to decline as auxin concentration is still
further increased (Fig. lb).
It has been shown in earlier papers' that a molecule in order to be active
as an auxin must satisfy a number of structural requirements among which
is the requirement for two suitably positioned reactive groups. These two
groups, a carboxyl group located in a side chain attached to an unsaturated
cyclic nucleus2 and a substitutable group of critical reactivity in the nucleus
ortho to the side chain3 appear to be the functional groups through which
1014 PROC. N. A. S.
BOTANY: FOSTER ETAL.
the auxin molecule makes attachment to some receptor entity within the
plant. The combined auxin, bound through its two reactive positions to
two functional sites of the receptor entity, appears to be the form which is
active in eliciting growth responses. Although the exact chemical nature
and biochemical function of the auxin-receptor complex is not known, the
existence and character of the complex can be deduced from kinetic con-
siderations4 and from the study of the relationships of chemical structure to
physiological activity of synthetic auxin analogs.'
It is the purpose of this paper to show that the inhibition of growth in-
duced by supra-optimal concentrations of auxin is a natural and indeed an
inescapable consequence of the two-point attachment by which auxin is
bound within the plant to form the functional entity.
Materials and Methods.-All of the experimental work reported here has
been based on measurements of the rate of elongation of excised sections of
Avena coleoptiles floated in solutions containing an active auxin.6 Avena
seedlings were grown in vermiculite contained in stainless steel trays and
maintained in a dark room under low intensity red light at a temperature of
25-26°C. and a relative humidity of 90%. When the seedlings had
attained an age of 82-84 hrs. they were selected for uniformity. Those
plants with a coleoptile length between 2.75 and 3.25 cm. were decapitated
by removal of a 2-3 mm. apical tip and the first leaves removed. Five
mm. apical sections were then cut from each coleoptile with a double-
bladed cutting tool. The sections were pooled and randomly distributed in
lots of 20 to individual Petri dishes containing 20 ml. of the test solution.
A basal medium containing 0.0025 M potassium maleate buffer (pH 4.5)
and 3 per cent sucrose was employed. To this the varied auxins, adjusted
to pH 4.5, were added as desired.
Sections were allowed to grow in the dark at 25-26° for a period of 12
hrs. and their length then measured under a microscope. This growth
measurement yields the initial growth velocity since the growth of Avena
coleoptile sections under the present conditions is linear with time for a
period of 18 hrs. or more.
Kinetic Treatment of Avena Section Growth.-The elongation of excised
Avena coleoptile sections in response to exogenous auxin may be formally
treated by the enzymee kineties Qf Michaelis and Menten.7 For this treat-
-met,tetnhitial;oi velocity of the sections (v) is determined as a func-
tion of substrate (IAA) concentration [S]. Classical enzyme kinetics are
based upon the hypothesis that substrate combines with enzyme (E) to
form an enzyme-substrate complex (ES) which then decomposes to form
the product of the reaction (in this case growth) and to reform E. In this
fornulation which is outlined in equation (1), K8 and k are constants which
express, respectively, the affinity constant for the formation of ES from E
VO-L. 38, 1952 1015
BOTANY: FOSTER ET AL.
KS k
E + S 'ES growth + E (1)
and S and the constant characterizing the rate of decomposition of ES to
end product.
30 .
CQ20
I-
3
w
(n
2.01
cn
0
CM
-I>
I.o'
2 3 4
CONCENTRATION IAA moles/liter
w ~~~~~~~~12x 10
(S) liters/mole
FIGURE 2
Avena coleoptile section growth as a function of IAA concentration:
a (above), growth rate plotted as a function of IAA concentration; b,
reciprocal of growth rate plotted as function of reciprocal of concen-
tration.
Equation (1) may be expressed in terms of reaction velocity (v) which is
dependent, as shown in equation (2) upon [S] and the constants K8 and
Vmax [IS
vK8+[IS] (2)
PROC. N. A. S.1016
BOTAN Y: FOSTER ET AL.
Vmax. The latter is determined as the reaction velocity of the system in
the presence of non-limiting substrate concentration and in which all E
is therefore combined as ES. The relationship between v and [SI for any
system which follows the formulation of equation (2) must be expected to
be a hyperbolic one in which V.ax. represents the value of v which is
asymptotically approached as [S] is increased and in which K8 is the value
of [S] at which one half of the maximum reaction velocity is attained.
Figure 2a shows how the data relating growth rate of Avena coleoptile sec-
tions to IAA concentration fall smoothly on such an hyperbola.
A critical test of the applicability of equation (2) to a particular system
consists in relating graphically the reciprocal of reaction velocity (l/v) to
the reciprocal of substrate concentration (1/ [S]). Any system following
Michaelis-Menten kinetics is expected
to yield a straight line for which the E S
intercept on the ordinate is 1/Vax. [i
and the slope K,! Vma,.. Figure 2b +s
shows that the data for Avena section
growth as a function of IAA concen- E+ E
tration fit this formulation with preci- ESI 2 ES1,2
sion over the lower range of substrate
concentration. The systematic devi- + /
ation of the data for the higher con- I /
centrations, which is an expression of
a depression in growth rate at the ES2
higher auxin levels, may be understood
in the light of an extension of the GROWTH = k [ES ]
Michaelis-Menten treatment as will be 1,2
shown hereafter. FIGURE 3
Kinetic Treatment of Two-Point At- Equilibria relating the formation of
tachment.-The treatment thus far has active receptor-substrate complex
dealt implicitly with systems in which (ES1, 2) to the formation of inactivedea lmplc ty l t l lchreceptor-substrate complex (E5S2S).
attachment of substrate to enzyme is
consummated through a single point. Let us now consider the manner in
which two-point attachment may be expected to alter this picture.8 A
molecule of auxin (S) might be expected to become first associated with
one of the two receptive sites on the enzymatic entity and then, by becom-
ing associated with the second receptive site, to consummate the two-point
attachment essential to enzymatic (growth) activity. This sequence of
reactions will predominate at low auxin concentrations. At higher auxin
concentrations, however, there is an appreciable probability that two auxin
molecules may simultaneously approach the receptor entity and simul-
taneously react with it, each combining through a single reactive group.
Such interaction will yield an inactive auxin-receptor complex in which
1017VOL. 38, 1952
BOTANY: FOSTERETALP
each auxin molecule prevents the other from consummating a two-point
attachment.
The several equilibria which relate the monomolecular and bimolecular
substrate-receptor complexes are illustrated in figure 3. These equilibria
involve the following quantities:
E = Receptive entity of the plant.
S = Auxin.
ES1 = Auxin-receptor entity complex combined through point 1.
ES1,2 = Auxin-receptor entity complex combined through points 1 and 2.
This complex, the enzymatically active one, may be pre-
sumed to be formed from either ES1 or ES2.
ES1S2 = Bimolecular auxin-receptor entity complex in which two auxin
molecules are involved, one attached through point 1, the
other through point 2. This complex is presumed to be
enzymatically inactive.
In the classical enzyme kinetics only the equilibria which result in the
formation of the active complex, ES1,2 are considered. If we include in the
consideration the formation of the complex ES1S2, the classical expression
relating reaction velocity to substrate concentration may be shown to be
altered to the form given in equation (3).
Vex [S]
K8' + [5] + [S]2/C
This expression differs from that of classical kinetics in that it includes a
term [S]2/C which is a measure of the probability that a second molecule of
substrate will become attached to the receptor entity before the first
molecule has consummated its two-point attachment. The reaction
velocity v, which is proportional to ES1,2, is expressed in terms of [S] and the
experimentally determinable constants Vex, K,' and C. Equation (3) pre-
dicts a maximum reaction velocity (growth rate) at a substrate concentra-
tion of \/K8'-C. This maximal rate is decreased below the rate which
would be achieved in a classical system by the amount to which the inactive
complex ES1S2 is formed in the system at the substrate concentration
VK8'-C.
Equation (3) will now be applied to data derived from experimental
determination of the relation of auxin concentration to rate of Avena
coleoptile section growth. We shall first evaluate the constants Vex and K8'
which correspond to the constants Vx;- and K8 of classical enzyme ki-
netics. These two constants may be determined in the low range of auxin
concentration in which the formation of the bimolecular complex ES1S2 is
negligible. This is most readily done on the reciprocal plot of figure 2b.
Vex is the extrapolated intercept on the ordinate while K8' is the slope
1018 PROC. N. A. S.
BOTANY: FOSTER ET AL.
divided by the intercept. The remaining constant C, which is a measure
of the probability that two molecules will react with the two receptor sites
of the reactive entity before either has had a chance to consummate its
two-point attachment, must be evaluated in the region of high concentra-
tion in which the formation of this complex predominates. This-is-done as
5 2.0
E
E
1.0
0
z
2o
2
I
: liters/mole
FIGURE 4
At high auxin concentration, growth of Avena coleoptile
sections is inversely proportional to the substrate concen-
tration. The limiting slope of the lower portion of this
curve = Vexc C.
LOG CONCENTRATION moles/liier
FIGURE 5
Avena section growth as a function of IAA concentra-
tion. The solid line is that for equation (3); the points
represent data from five separate experiments.
shown in figure 4 which presents growth velocity as a function of the recip-
rocal of the substrate concentration. It can be seen that as the auxin con-
centration is increased, the growth velocity becomes inversely proportional
to it and from the limiting slope which is VexC, the value of C may be deter-
mined.
VOL. 38, 1952 1019
BOTANY: FOSTER ETAL.
It appears then that at the extremes of the auxin concentration range
auxin affects growth in the manner predicted by equation (3). This
equation describes accurately the effects of auxin on the growth response of
Avena coleoptile. sections over the entire range of auxin concentrations to
which this organ is sensitive, a range of one hundred thousand fold. That
this is so is shown in figure 5. Since the range of concentration is so large,
; 2.0
~ oE
It
0 .
CD
z
0
n
_t6 -D -4
LOG CONCENTRATION: moles/liter
FIGURE 6
Avena section growth as a function of 2,4-D concentra-
tion. The solid line is that for equation (3); the points
represent data from two separate experiments.
5 2.0
1.0 X0
z .0
0
-7 -6 -5 -4 -3
LOG CONCENTRATION: moles/liter
FIGURE 7
Avena section growth as a function of NAA concentra-
tion. The solid line is that for equation (3); the points
represent experimental data.
the data are presented in the form of initial growth velocity as a function ot
the log of auxin concentration. The curve of figure 5 is that for equation
(3) using the values for the constants determined as outlined above. The
individual points of figure 5 represent data from 5 separate and complete
experiments. These fit the curve within experimental error over the entire
100,000 fold range of concentrations. That the maximum growth rate
occurs at a substrate concentration of x/XKTC is also verified by the data.
1020 PROC. N. A. S.
0
BOTANY: FOSTER ETAL.
The synthetic growth substances such as 2,4-dichlorphenoxy acetic acid
(2,4-D) and naphthalene acetic acid (NAA) are, like the naturally occurring
IAA, active in promoting the growth of Avena coleoptile sections at low
concentrations, active in inhibiting growth at higher concentrations.
That the kinetic relations of 2,4-D and of NAA in their effects on Avena
section growth are also in accord with the two-point attachment concept is
evident from the data of figures 6 and 7. This is true even though the
constants Vex, K8' and C for these substances differ appreciably among
themselves and differ also from those determined for IAA, a fact summa-
rized in table 1.
Discussion.-The concept that auxins are bound within the plant to an
appropriate receptor entity through which auxin-induced growth responses
are mediated was originally based not only on the known binding of auxins
to plant proteins9 but also and more specifically on the fact that the
kinetics of auxin action are in close accord with the expectations of classical
TABLE 1
SUMMARY OF THE PARAMETERS WHICH RELATE GROWTH OF AVENA COLEOPTILE
SECTIONS TO AUXIN CONCENTRATION
vexs IKs',a C,b V/Ks' X C,c
Auxin mm./12 hrs. moles/liter moles/liter moles/liter
IAA 3.1 3.4 X 10-7 4.0 X 1O-4 1.2 X 1O-5
2,4-D 2.3 5.5 X 10-7 1.4 X 10-4 0.9 X 10-5
NAA 2.1 6.4 X 10-7 1.7 X 10-4 1. 1 X 10-6l
a Concentration of auxin at which growth rate is 0.5 Vex and ES1,2 = Efree.
b Concentration of auxin at which growth rate is 0.5 maximal due to formation of the
complex which contains two auxin molecules. ES1,2 = ES1S2.
c Concentration of auxin at which growth rate is maximal.
enzyme kinetics.4 The extension of this concept to include the specifica-
tion that the binding must be consummated through a two-point attach-
ment of the auxin molecule to the receptor entity was first suggested by the
observation of Muir, Hansch and Gallup3 that a free ortho group of critical
reactivity as well as a free carboxyl group are essential to auxin activity.
The hypothesis that both of these groups are in fact essential to auxin
activity and by implication essential to auxin binding has been strengthened
by the finding that auxin-like molecules in which either of the two essential
groupings is blocked or abolished are not ottly inactive as auxins but also
are in fact competitive inhibitors of auxin action. 1O The matters con-
sidered in this paper constitute a critical experimental test of the two-point
attachment concept as applied to auxin action. If two-point attachment
of auxin to its receptor is essential for the formation of the complex which
induces growth it follows that inhibition of growth by the formation of
bimolecular complexes may be expected at higher auxin concentrations.
The fact that auxin-induced growth inhibition not only does occur but also
VOL. 38, 1952 1021
BOTANY: FOSTER ETAL.
that it follows in.elegant detail the kinetics of bimocular complex formation
provides strong support of the whole two-point attachment concept.
Of-what practical use may it be to express and to think about auxin-
induced growth inhibition in terms of the kinetics of two-point attachment?
One evident application may be in the study of the herbicidal action of
synthetic growth substances such as 2,4-D. It is a peculiarity of these
materials that although they are exceedingly deleterious to particular
plants at higher concentrations, they act at lower concentrations in a
manner indistinguishable from that of IAA itself. We might therefore in-
quire in how far the herbicidal action of 2,4-D and related materials may
be owing to the formation of bimolecular complexes of the kind here
described and we might attempt to answer this question by determination
of the extent to which herbicidal activity is a function of the second power
rather than of the first power of the applied growth substance concentra-
tion.
The twofold action of auxin on plant growth appears then to have its
basis in a requirement for two-point attachment of the auxin to its receptor,
a requirement which makes possible the formation of an inactive bimolec-
ular complex at higher auxin concentrations. It will be of evident interest
to discover whether the twofold action of other biologically active sub-
stances may in some instances similarly reside in a requirement for multiple
attachment.
Summary.-The relationship between Avena coleoptile section growth
rate and auxin concentration over a wide range has been demonstrated to
be predictable on the basis of a requirement for two-point attachment of
the auxin molecule to some receptive entity in the plant. In particular,
the inhibition of growth at higher auxin concentrations, which may be the
cause of herbicidal activity of certain chemicals, has been shown to be a
natural and predictable consequence of the two-point attachment of the
auxin molecule to the receptor entity within the plant.
* Report of work supported in part by the Herman Frasch Foundation.
'Summarized in Bonner, J., and Bandurski, R. S., Ann. Rev. Plant Physiol., 3, 59
(1952).
2 Koepfli, J. B., Thimann, K. V., and Went, F. W., J. Biol. Chem., 122, 763 (1938).
3 Muir, R. M., Hansch, C., and Gallup, A. H., Plant Physiol., 24, 359 (1949).
4 McRae, D. H., and Bonner, J., Abstracts, Am. Soc. Planjt Physiol., Western Sec-
tion, 1952, p. 7.
6 Hansch, C., Muir, R. M., and Metzenberg, R., Plant Physiol., 26, 812 (1951). See
also reference 3 above.
6 Bonner, J., J. Gen. Physiol., 17, 63 (1933).
7 Michaelis, L., and Menten, M. L., Biochem. Z., 49, 333 (1913).
8 Foster, R., and Niemann, C., PROC. NATL. ACAD. SCI. (in press).
9 Wildman, S., and Gordon, S., Ibid., 28, 217 (1942).
10 McRae, D. H., and Bonner, J., Plant Physiol. (in press).
1022 PROC. N. A. S.


Auxin-induced growth inhibition a natural consequence of two-point attachment

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Auxin-induced growth inhibition a natural consequence of two-point attachment

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