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Produced by the NASA Center for Aerospace Information (CASI)
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 biosynthesis, 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 concentrations < 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. ,, ,. _ .. -- - ------_.. _..^
v

I,,

a

^i 

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

x

d 

5.5

5,0

60 110 180 240

TIME (min)

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 direction 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 associacaused 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
-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 concentrations 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 differential 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

Z

0

i

Zz

0JW
A

B

C

ORIGINAL PAGE 15

OF POOR QUALITY

65 AbA

6 4 ---------

63

P

H 62

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.
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 gravitrapism. 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.
REFERENCES

Abou-Mandour, A. and Hartung, W. (1980). Z. PfZanzenphysioZ.

+ 100, 25-33.

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.

Feldman, L.J. (1981). PZanta 153, 471-475.

Gaither, D.H., Lutz, D.H. and Forrence, L.E. (1975). PZ.

PhysioZ. 55, 948-949.

Hartung, W. (1976). PZanta 128, 59-62.

Keeble, F., Nelson, M.G. and Snow, R. (1931). Loo. L. Soc.,

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Filet, P.E. (1974). In "Plant Growth Substances 1973, Proc.

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Pilet, P.E. and Chanson, A. (1981). PZ. Foi. Lett. 21,

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Thimann, K.V. (1937). Am. J. Bot. 24, 407-412.

Trewavas, A. (1981). Pl. CeZZ and D2vir. 4, 204 -228.

Went, F.W. and Thimann, K.V. 1937). "Phytohormones".

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N

i C711

Appendix ^^^+^

Copies of "in press" publications describing in more detail work

accomplished during the Tirst half of support.

I