In order to test the idea that auxin action on root growth may be mediated by H(+) movement, the correlation of auxin action on growth and H(+) movement in roots was examined along with changes in H(+) efflux patterns associated with the asymmetric growth which occurs during gravitropism. The effects of indoleacetic acid (IAA) and abscisic acid (AbA) on growth, H(+) secretion, and gravitropism in roots were compared. Results show a close correlation existent between H(+) efflux and growth in maize roots. In intact roots there is strong H(+) efflux from the elongation zone. Growth-promoting concentrations of IAA stimulate H(+) efflux. During gravitropism the H(+) efflux from the elongation zone becomes asymmetric; the evidence indicates that auxin redistribution contributes to the development of acid efflux asymmetry. That AbA stimulates root growth is reflected in its ability to stimulate H(+) efflux from apical root segments

Evans, M. L.

Mulkey, T. J.

English

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COMPARATIVE EFFECTS OF AUXIN AND
ABSCISIC ACID ON GROWTH, HYDROGEN
ION EFFLUX AND GRAVITROPISM IN
PRIMARY ROOTS OF MAIZE
Michael L. Evans and Timothy J. Mulkey
Department of Botany, Ohio State
University, Columbus, Ohio
43210, U.S.A.
1. INTRODUCTION
According to the acid growth hypothesis of auxin action,
promotion of cell elongation by auxin is dne, at least in
part, to stimulation of hydrogen ion (ti+) efflux from the
cytoplasm into the cell wall. Acidification of the wall is
thought to enhance wall loosening and allow rapid growth
(Rayle and Cleland, 1977). There is now considerable
evidence that gcid efflux plays a role in auxin action on
cell eiongation in stems and coleoptiles. However, rela—
tively little study has been made of the role of H+ movement
in auxin action on roots. This is in spite of the fact that
auxin can either promote (at low concentrations) or inhibit
(at higher concentrations) elongation in both shoot and root
cells. Based on this bimodal concentration dependence,
Thimann (1937) proposed that the molecular basis of auxin
action is fundamentally the same in shoots and roots. This
proposal is strengthened by the finding that root growth is
strongly stimulated by acid pH just as is shoot growth
(Edwards and Scott, 1974; Evans, 1976).
In order to test the idea that auxin action on root
growth may be mediated by H+ movement, we have examined the
correlation of auxin action on growth and H+ movement in
roots and looked for changes in H+ efflux patterns assoc -
iated with the asymmetric growth which occurs during
gravitropism. We have also compared the effects of IAA and
AbA on growth, H+ secretion, and gravitropism in roots.
'l. MATERIALS AND METHODS
The experiments were done using the following cultivars
of maize (Zea mays- L.): Bear hybrid WF9 x 38MS, Orla 231,
and LO 11. Unless otherwise indicated, the experiments were
done with WF9 x 38MS seedlings. Dark grown seedlings were
raised with a brief red light treatment as described by
(NASA—CR-174355) COMPARATIVE EFFECTS OF
	 N85-18444
AUXIN AND ADSCISIC ACID ON GlCNTH, HYDROGEN
.ION EFFLUX AND GRAVIT50PISM IN PRIMARY ROOTS
OF MAIZE 40hio State Univ_, Columbus.) 11 P
	 Unclas
HC A02/MF
 A01	 CSCL 02C G3/43 17519
Mulkey et al. ,(1981) or raises in total darkness. The
seedlings were used when they were 2.5 to 3.5 d old, when
the primary root was about 1.5 cm long.
Root elongation was measured using the root auxanometer
described by Evans (1976). The time course of gravitropism
was determined from time-lapse Super-8 mm films of seedlings
mounted horizontally in a Plexiglas chamber. The chamber
was maintained at 100% relative humidity and 22 +/- 2C.
The pattern of acid efflux from roots of intact seedlings
was determined using the modification of the Weisenseel
method described by Mulkey and Evans (1981). Seedlings are
placed on a plate of agar containing the pH indicator dye,
bromocresol purple. Acid efflux from the root causes the
indicator to become yellow. In regions of apparent acid
entry into the root, the pH of the medium adjacent to the
root increases and the dye in that region turns red.
To obtain a quantitative measurement of hormone effects
on H+ efflux from maize roots, 1-cm apical root segments
were used, and acid efflux was measured as a decrease in the
pH of 3.5 ml of 1 mM KPO 4 buffer containing 60 segments
(Mulkey et al., 1982a). Since treatment of apical root
segments with IAA or AbA enhances their ethylene biosynthe-
sis, the effects of IAA and AbA on H+ efflux in root
segments were measured using segments pretreated with a
solution containing 1 pM aminoethoxyvinylglycine plus 0.1 mM
cobalt nitrate (AVG/Co 2+) to minimize the enhancement of
ethylene formation (Mulkey at al., 1982a).
Abscisic acid (+/- cis,trans) was purchased from Sigma
Chemical Co., St. Louis, M0, USA and from Calbiochem-Behring
Corp., La Jolla, CA, USA. AVG was provided by Dr. Richard
Gladon, Dept. Horticulture, Iowa State University, Ames, IA,
USA.
3. RESULTS
Auxin Action on Growth and Ht Movement in Roots
Using intact seedlings of maize, we obtain stimulation of
root elongation by low (e.g. < 1 nM) concentrations of IAA
only occasionally. In most experiments, IAA in concentrat-
ions < 1 nM has no effect on root elongtion. However, if
the roots are pretreated with AVG/Co (Mulkey et al.,
1982b), we obtain consistent promotion of elongation using
IAA in concentrations from 0.1 nM to 10 nM (Fig. 1).
The ability to obtain consistent promotion of root growth
with low concentrations of auxin and inhibition with high
concentrations, has allowed us to compare auxin action on H+
efflux and growth over both concentration ranges. When a
growth promoting concentration of auxin is added to isolated
.ii«• ' amt. ,,
	
,.	 _	
.. -- - ------_.. _..^
vI,,
a
^i ^i
4'
corn root tips, there is strong enhancement of H+ efflux
from the segments. This parallels the promotion of growth
in intact roots using the same concentration of auxin (Fig:
2). Conversely, treatment of isolated root tips with a
growth inhibiting concentration of IAA causes an apparent
uptake of H+ from the medium, and this is paralleled by
inhibition of growth (Fig. 2).
Fig. 2. Concentration dependence of IAA action on elongation
of maize roots pretreated with AVG/CO 2+ . Solid lines: roots
pretreated with , AVG/Co 2+ 2 h and 'ther treated with the
indicated concentration of IAA (plus AVG/CO 2+) .
 The growth
rate rats measured 2 h after transfer to the IAA containing
solutions. Dashed line: control roots held in 'buffer 2 h and
then treated with IAA. Means, +,/- S.E. are shorn.
VisuaZiaation of H+
 EffZux Patterns During Straight Growth
and Gravitropism
The above results indicate a close relationship between
H+ movement and growth rate in maize roots. However, since
the H+ efflux experiments were done on apical root segments
and the growth experiments were done using intact roots, we
also wanted to look at H+ efflux in intact, relatively
undisturbed roots. This was done by placing whole seedlings
on a plate of agar containing the pH indicator dye,
bromocresol purple. With the seedling oriented vertically,
the pH pattern shown is Figure 3A develops around the root
within 8 min and intensifies thereafter. The medium
adjacent to the elongation zone and the medium along the
root hair zone become yellow, indicating acid efflux. The
medium adjacent to the non-growing region behind the
elongation zone and the medium around the tip of the root
become red, indicating acid uptake. When 2 uM IAA is
included in the medium (Fig. 3B) the medium along the
elougati.on zone becomes red instead of yellow, indicating H+
f
T'0	 a.1nM IAA
s
1.0	 7YM UA
n•5
6.0
xd 
5.5
5,0
60	 110	 180	 240
TIME (min)
ORIGINAL PAGE'19
OF POOR QUALITY,
Fig. 2. Effect of IAA on growth and H+ movement in maize
roots. Top: Effect of IAA on eZongation. A singZe intact
root was treated with the indicated concentration of IAA.
The root receiving 0.1 nM IAA was pretreated for 1 h with
AVG/Co 2+ prior to exposure to auxin. Bottom: Effect of IAA
on H+ effZux. The indicated concentrations of IAA were
added to sixty 1-om apical root segments in 3,5 mZ of
medium. The segments receiving 0.1 rad I/A were pretreated
for 3 h with AVG/CO 2+ prior to exposure to auxin.
uptake into the elongation zone. These data show that
intact roots secrete acid from the elongation zone and that
growth inhibiting concentrations of IAA reverse the direct-
ion of acid movement in that region.
When a seedling in contact with an agar/dye plate is
oriented horizontally there is, of course, a shift in the
growth pattern of the root as positive gravitropism occurs.
The rapid growth rate on the top relative to that on the
bottom is paralleled by intense acid efflux on the top and
diminished acid efflux on the bottom {Fig. 4).
The correlation between the Ht efflux pattern and
gravitropism suggests the possibility that asymmetric acid
efflux may cause the asymmetric growth which leads to
positive gravitropism. If so, the manner of development of
asymmetric acid efflux becomes important in considering
models of gravitropism. According to the Cholodny/Went
hypothesis (Went and Thimann, 1937), root gravitropism
results from movement of auxin toward the lower side of the
root where it accumulates to growth-inhibiting levels.
Since we find that high concentrations of auxin inhibit e
OGK^"
OF F(; . .
Fig. 3. H+ en-Mix pattern o,f Ma-' Z( J-oors on agar pZates
containing :he pil indicator d^ e, bromccresoZ purple. --he
'i.ght regions represent tjeZZow zones inclica-,irg acid erfZux.
the dark regions represent red zoneR inaicating acid uptake.
„: eoti+ti• Z. B: ig.^ IAA. C:0.2 ra?, AAA. Photos taken 2 h after
lacing Ate.;:	 "r or the
efflux from roots while
efflux, it is possible that
aced with gravitropism is
We find that inhibitors c
root gravitropism (e.g.
prevent the development
^,ravistimulated roots (Fig.
low concentrations promote 1i+
the H+ efflux asymmetry associa-
caused by auxin redistribution.
f auxin transport which prevent
naphthylphthalamic	 acid) also
of	 acid	 efflux 3syametry in
4).
Co ,l: rative E,; feels of AbA and IAA or: Root Growth and
Grav i tro - is^
A second major hypothesis of root gravitropism is the
root cap inhibitor hypothesis. According to this model,
root gravitropism results from the movement of a growth
inhibitor from the root cap to the elongation zone where it
accumulates on the lower side (Keeble et al., 1931; Pilet,
1974). AbA is suggested to he a major component of the cap
inhibitor (Pilet and Rivier, 1980). With this model in
mind, we have examined the effects of AbA on H+ efflux,
growth and gravitropism in several cultivars of maize.
Using the root auxanometer, we find that the initial
effect of a wide concentration range of AbA is to promote
• A .
-t
CRIGMAL p!;'^'_
OF POOR QUALITY
growth (Fig. 5).	 With low concentrations (e.g. 0.1 x1m)
there is a transient stimulation of elongation.	 Using
higher concentrations (e.g. 0.1 mM) there is a period of
stimulatioa followed by long 	 term inhibition of root
elongation, so that the net effect on growth is inhibitory.
Fig. 4. Sequence of chanaes in surface pH patter ,a along the
primary root of a maize seedling exposed to a gravitrcpic
stimulus. Regions of high pH (reddish purple in color)
appear as dark areas. Regions of low pH (geZZow in color)
appear as 'Light areas. FZapsed time after pZacirw the root
in a horizontal p,,sition on the agarldgc plate are (A) 2
min, (B) 20 min, and (C) 120 min. The seedZing ehoum in (D)
rats treated in the same manner but placed on an agar/due
plate containing 4.86 nM. naphthylphthaZamie acid (NPA) and
photographed after 18 h.
However, with both low and high concentrations of AbA, the
initial stimulatory phase lasts several hours. Data very
similar to these have also been reported by Smith and Ho
(198"2).
The initial stimulatory effect of even high concentrat-
ions of AbA is reflected in the inability of AbA to inhibit
or reverse H+ efflux from the elongation zone of intact
roots of maize (Fig. 3C). In fact we find that treatment
of apical root sections with AbA causes stimulation of H+
efflux (Fig. 6), an observation consistent with the initial
growth promoting effect of AbA.
The fact that the initial effect of applied AbA on intact
roots is stimulatory, conflicts with the proposed different-
ial inhibitory action of AbA in root gravi*ropism. We
therefore looked at the ability of unilateral. application of
2.6
20 bA
26 	 10^M AbA
1.0
104 M AbA
0.6
4 0 10 16 20 24 28 32 36 40 44
TIME Mrs)
Fig, 5. Time course of AbA effects on primary root
elongation in intact seedlings of maize. The indicated
concentrations of AbA were added at the arrow. QuaZitatively
similar data were obtained with the three cuZtivars cf maize
tested.
AbA to modify root gravitropism in several cultivars of
maize (Fig. 7). We found that application of an agar
blocklet containing 0.1 mM AbA to the upper surface of
horizontal roots, accelerated gravitropism (Fig. 7, column
1) while application of AbA to the bottom of the root
retarded gravitropism (Fig. 7, column 2). These results are
contradictory to the proposed role of AbA as a growth
inhibitor i,n gravitropism.
4. DISCUSSION
Our results show a close correlation between H + efflux
and growth in maize roots. In intact roots there is strong
H+ efflux from the elongation zone and apparent H+ influx on
both sides of the elongation zone. Concentrations of IAA
which inhibit root growth also inhibit H+ efflux, while
growth promoting concentrations of IAA stimulate H+ efflux.
During gravitropism the H+ efflux from the elongation
zone becomes asymmetric with greater H + efflux on the top
relative to that on the bottom. This parallels the
asymmetric growth which causes gravitropism. The possibili -
ty that auxin redistribution contributes to the development
of acid efflux asymmetry and hence asymmetric growth, is
indicated by the observation that inhibitors of auxin
transport prevent both the development of asymmetric acid
efflux and gravitropism.
The effects of applied AbA on root growth and gravitrop -
ism are inconsistent with its suggested role as an
endogenous growth inhibitor mediating gravitropism. We find
r
E
E
w
Z0
iZz0JW
t
AB
C
fio^
a^
.
ORIGINAL PAGE 15
OF POOR QUALITY
65	 AbA
6 4 ---------
63
P
62H
60
	
^^	 10 7M
59	
104M
J 8
- 
--A -.	 -1
330 360 390 420 450 480 520
Toms in min
Fig. 6. Stzmulaz.::'r. of Elf  effZUX frcr, rriaL:wt_ root	 by
ALA.	 The indicated concentrations of ALA were added to
sixty 1-cm apieaZ root segments in 3.5 mZ of medium.
	
1	 2	 3
Fig. ?. Effect of unilate-rai	 appZication rf AbA on
gravitropism in maize roots. Column 1: AbA (0.1 rW) applied
to the top (bottom block, agar only). Column 2: AbI applied
to the bottom (top block, agar only). Column 3: control with
agar blocks only.	 The roots were oriented horizontally
immediately after applying the agar (fro) blocks.	 Photos
taken 1 h after gravistimulation. Row A: cv WF9 x 3SMS. Rom
B: cv Or1 a 231. Rory C: cv LC 11.
i
I,0,
that the initial,effect of applied AbA is to promote root
growth and that the period of growth promotion is long lived
compared with the time required for completion of gravitrap-
ism. The ability of AbA to stimulate ruot growth is
reflected in its ability to stimulate a efflux from apical
root segments. It is also manifested in the ability of
asymmetric application of AA to enhance gravitropism when
the hormone is applied to the top of a horizontal root and
its ability to inhibit gravitropism when applied to the
bottom.	 This aspect of our results is in agreement with
other reports of the ability of AbA to promote root growth
(Gaither et al., 1975;	 Yamaguchi
	 and Street, 1977;
Abou—Mandour and Hartung, 1980; Smith and Ho, 1982). In
light of these findings, it would seem that, if AbA mediates
gravitropism in roots, its redistribution would need to be
toward the top of the root, not the bottom. Ironically,
Suzuki et al. (1979) reported accumulation of AbA on the
upper side of graviresponding roots, and Hartung et al.
(1976) noted a preferential short term upward movement of
labelled AbA in horizontal maize roots.
Our results confirm other reports of long term inhibition
of root growth by high concentrations of AbA (Pilet and
Chanson, 1981). 'We see long term inhition of elongation
with concentrations of AbA equal to or greater than about
0.1 MM. However, the inhibition is slow to develop and
occurs at such high concentrations that it is unlikely to be
of physiological significance.
Although our data are consistent with a possible role for
IAA in mediating root gravitropism, the results do not
address a number of objections which have been raised to the
Cholodny/Went model of auxin mediation of gravitropism
(Wilkins, 1979). The possibility remains that, root
gravitropism may not be mediated by changes in hormone
concentration (Digby and Firn, 1980; Trewavas, 1981) or that
is may be mediated by redistribution of some, as yet,
unidentified inhibitor from the root cap (Suzuki et al.,
1979; Feldman, 1981).
ACKNOWLEDGEMENTS
We thank Konrad Kuzmanoff for his assistance. Supported
by MSA grant NAGW-297, by NSF grant PCM 8103298, and by a
Sigma Xi grant to T.J.M.
^I
it
wREFERENCES
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1	 Digby, R.D. and Firn, J. (1981) J. Rev. PZ, PhysioZ. 27,y	 131-148.
Edwards, K.L. and Scott, T.K. (1974). PZanta 119, 27-37.
Evans, M.L. (1976). PZ. PhysioZ. 58, 599-601.
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Ni C-
711
Appendix
	 ^^^+^
Copies of	 "in press" publications describing in more detail work
accomplished during the Tirst half of support.
I


Comparative effects of auxin and abscisic acid on growth, hydrogen ion efflux and gravitropism in primary roots of maize

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