333

Pla,nta 9 Springer-Verlag 1988

Abscisic acid and water transport in sunflowers Marianne Ludewig, Karl Diirffling, and Hans Seifert Institut ffir Allgemeine Botanik und Botanischer Garten, Universit~it Hamburg, Ohnhorststrasse 18, D-2000 H a m b u r g 52, Federal Republic of Germany

Abstract. The role of abscisic acid (ABA) in the transport of water and ions from the root to the shoot of sunflower plants ( H e l i a n t h u s annuus) was investigated by application of A B A either to the root medium or to the apical bud. The exudation at the hypocotyl stump of decapitated seedlings was measured with and without hydrostatic pressure (0-0.3 MPa) applied to the root. All A B A concentrations tested (10-1~ -4 tool-1 1) promoted exudation. Maximal amounts of exudate (200% of control) were obtained with ABA at 10- 6. mol- 1- z and an externally applied pressure of 0.1 MPa. The effect was rapid and long-lasting, and involved promotion of ion release to the xylem (during the first hours) as well as an increase in hydraulic conductivity. Abscisic acid applied to the apical bud had effects similar to those of the rootapplied hormone. Increased rates of exudation were also obtained after osmotic stress was applied to the root; this treatment increased the endogenous level of A B A in the root as well as in the shoot. Water potentials of the hypocotyls of intact plants increased when the roots were treated with ABA at 5~ C, whereas stomatal resistances were lowered. The results are consistent with the view that ABA controls the water status of the plant not only by regulating stomatal transpiration, but also by regulating the hydraulic conductivity of the root. Key words: Abscisic acid and water transport H e l i a n t h u s - Hydraulic conductivity - Ion transport - Osmotic stress - R o o t (water transport) Water transport. Abbreviations and symbols." A B A : abscisic acid ; Tv = volume flow; L p = h y d r a u l i c conductivity; PEG=polyethyleneglycol; gt = water potential; 7t~ = osmotic potential; g = osmotic value; AP = hydrostatic pressure

Introduction Controversial views exist on the effect of abscisic acid (ABA) on the transport of water and ions through roots (for a review see Andersen and Proebsting 1984). Several workers have found, after direct application of A B A to roots, an increased volume flow which may be the result of either increased hydraulic conductivity (Glinka 1977; Andersen and Proebsting 1984) or increased ion transport (Karmoker and Van Steveninck 1978), or both (Glinka 1980; Fiscus 1981). Others, however, found that A B A reduced volume flow in roots (Pitman and Welfare 1978; Markhart et al. 1979), whereas Erlandson et al. (1978) observed no effect at all. Some of the differences in the experimental results may be the consequence of differences in the plant species used, in the methods used for application, and in the time at which the effects were measured. All relevant studies published so far have been performed with exogenous ABA, which was applied to the root systems of decapitated seedlings at only a few concentrations. Dose-response relationships have not been studied in detail. In most studies, only the changes in the osmotic potential of the xylem sap, but not the changes of specific ions have been analyzed. Moreover, nothing is known about the effect of shoot-applied A B A on the water uptake of and flow through the root, nor is evidence available whether stress-induced A B A has an effect on volume flow from the root to the shoot. A promotion of water uptake from the soil by stress-induced endogenous ABA could be as beneficial for the regulation of the water status as the well-known control of stomatal transpiration by ABA. Therefore, the objective of this paper was to

326

M. Ludewig et al. : ABA and water transport in sunflower

r e i n v e s t i g a t e t h e e f f e c t o f A B A o n w a t e r a n d ion transport in roots with emphasis on a possible ecophysiological function.

ABA 10-6 .~/)1 moll-' ///,o 10-/" / // / -,XABAmo,,-'

Material and methods

Plant material. For most experiments 20- to 25-d-old sunflower seedlings (Helianthus annuus L. cv. Bismarckianus) were used. They were germinated in sand in a greenhouse for 9-10 d and then transferred to aerated black Plexiglas boxes filled with 0.51 Hoagland nutrient solution (pH 5.6) which was diluted (1:4, v/v) and renewed every fifth day. Subsequently, they were grown in a chamber at a thermoperiod of 25~ ~ C. The photoperiod was 12 h, the relative humidity 70%. The plants were irradiated with 250 lamol photons-m-Z.s -1 from 215-W Sylvania (Danvers, Mass., USA) Cool-white lamps. For special experiments, a cooling system allowed the temperature of the nutrient solution to be kept at 5 ~ C.

=o 3

r~ .o

//

/ ABA,0-8

2

"8 :3 X

uJ

1

Application of ABA. (_+)-Abscisic acid (Fluka, Basel, Switzerland) was dissolved in the nutrient solution and then applied to the root. Apical buds were treated with 5 lag (_+)-ABA dissolved in 200 lal water.

0

Hydrostatic pressure. Root exudation was measured with and without applied hydrostatic pressure. The roots were enclosed with their nutrient solution in a modified Scholander bomb which allowed continuous aeration of the nutrient solution as well as continuous application of ABA. Care was taken not to injure the roots during the transfer to the Scholander bomb (Miller 1987). Hydrostatic pressures up to 0.5 MPa were applied.

Quantitative and qualitative determination of root exudate. In order to obtain the root exudate, seedlings were decapitated below their cotyledons so that 3 cm of the hypocotyl stump, which was 2 8 cm long, was outside the Scholander bomb. For exudate collection up to 1 h, the cut surface was brought into contact with a strip of filter paper which had been impregnated with COC12 and was moved over the stump at constant speed. The exudate formed a sharply limited trace. The diameter of this trace was dependent on the amount of exudate produced and on the speed of the paper movement. The trace was calibrated with known amounts of water so that an exact determination of exudate production with time became possible. Since the exudation rate differed greatly from plant to plant, all exudate measurements with applied ABA were based on preceding control measurements, during which the root was immersed in nutrient solution and subjected to increasing hydrostatic pressures. The preceding control measurements had no influence on the subsequent exudation rates with applied ABA. When large amounts of exudate had to be collected for further analysis (osmotic potential, ion concentration), another method was used. Without applying hydrostatic pressure, the exudate appearing up to 3 h at the cut surface of decapitated plants with their roots in nutrient medium was collected with a 250-lal microsyringe and frozen until it was analyzed. Osmotic potential was measured with a freezing-point osmometer (Roebling, Berlin, West Germany). The ion concentration (K +, Ca 2+, Mg 2+) of the exudate was determined with an atomic absorption spectrophotometer (model 180 - 7 0 ; Hitachi; Tokyo, Japan).

Studies with intact seedlings. The water potential of the hypocotyls of intact seedlings treated with ABA was measured with

I

I

I

0.1

0.2

0.3

Applied Hydrostotic Pressure MPo Fig. 1. Relation between exudation rate and applied hydrostatic pressure in root systems of sunflower plants treated with ABA. Ordinate: exudation relative to the control value at 0.1 MPa. Each point is the mean of at least 15 measurements a Scholander bomb (Roth Gergtebau, Baiersdorf, FRG). The diffusion resistance of the leaves of such seedlings was measured with a Li-Cor diffusion resistance meter (Lambda-Instruments, Lincoln, Neb., USA).

Osmotic stress. To induce a water deficit in the shoots as well as in the roots, polyethyleneglycol (PEG) 6000 (Merck, Darmstadt, F R G ) was added to the nutrient solution. The concentration used was 150 g.1-1, equivalent to - 0 . 5 2 MPa. The osmotic potential of the diluted Hoagland nutrient solution was -0.073 MPa. Determination of ABA. The ABA content of the shoots and roots of seedlings under osmotic stress was measured by means of on-column electron-capture gas chromatography on fused silica quartz capillary columns. Purification of the ethanol extracts followed a procedure modified by Kettner and D6rffling (1987). Recovery was measured by means of added radioactive standards.

Results

Dose-response relationship o f the A B A effect on volume f l o w in decapitated seedlings. T h e e x u d a t i o n rate was nearly linearly proportional to the applied hydrostatic p r e s s u r e s b e t w e e n 0 a n d 0.3 M P a ( F i g . 1). E v e n w i t h o u t a p p l i e d p r e s s u r e a m e a s u r able volume flow of about 0.03 ~tl.s-1 per hypocotyl occurred. Increased amounts of exudate were o b t a i n e d w h e n A B A w a s a p p l i e d t o t h e root, especially when the root was simultaneously pressurized. There was a clear dose-response relation-

M. Ludewig et al. : A B A and water transport in sunflower

100

1oo -- 200 18o

/ 0.1 (HPo)

|

12o

~,

100 200

o,J

,,,'-.i:::1 c:z::: ,z,,,, 0

.~

and kinetin) as well as indoleacetic acid, were found to decrease the exudation rate in concentrations ranging from 10 -8 to 10 - s mol-1-1, but the effects were weak and did not exceed 10% of the control (data not shown).

f-

i II

0

I~0

/~'I"~,,,~

,~, I 0.2 (MPo)

.X~-~T

T

150 lz, O

120

-o,,-i

100

II

x

;~oo

0.3(MPo)

180 160

120 I00

327

,~

J

i

I

i

J

~

I

10-10 10-9 lO-B 10-7 10-6 10-5 lO-t, ABA

Hydraulic conductivity and reflexion coefficient. The results obtained with the optimal ABA concentration, ] 0 - 6 mol. 1- ~, at different hydrostatic pressures were used to calculate the hydraulic conductivity (Lp) according to the equation given in Table 1. Table 1 also shows the measured osmotic potentials of the exudates. It is evident that without applied pressure, A B A causes a significant increase ( = less negative values) in the osmotic potential of the exudate (~zcE). With applied pressure the osmotic potential of both control and treated plants is further increased, but differences between the applied pressures and between ABA-treated and untreated plants are small and not significant. Application of ABA to the root caused a significant rise in Lp, which was independent of the applied hydrostatic pressures. The reflexion coefficient a, calculated according to the equations in Table 1, was found to be rather small and not different in control (o-=0.138) and in ABA-treated plants (o-=0.143). A rough estimation of a using another method (the tangent test of Fiscus 1977) gave a value of 0.13 for the controls.

( moI.F 11

Fig. 2. Dose-response relationship between A B A and exudation rate in sunflower root systems at different hydrostatic pressures. Ordinate." exudation rate as % o f preceding control measurement. Each point is the m e a n o f at least 11 measurements. Vertical bars = SD

ship, with ABA 10- 6 tool. l- 1 being more effective than 10 -4 and 10 -9 tool.1-1. A detailed dose-response curve (Fig. 2) shows that concentrations as low as 10 - l ~ mol'1-1 significantly increased the exudation rate, even without pressure. Optimal concentrations were 10- s to 10- 7 mol. 1-1. Significant evidence for a biphasic nature of the doseresponse curve (with a second optimum at 10-Z~ as indicated in Fig. 2) was not found. The most pronounced increase in exudation (200% and more in relation to the control) was obtained with 10-6 mol. 1-1 at 0.1 MPa, but even in the absence of exogenous pressure, increased rates up to 140% were measured. The effects obtained were observed with racemic ABA. Whether the ( + ) - or the ( - ) - i s o m e r or both are active, is not known, nor are data available as to whether trans-ABA possesses activity. Other hormones, for example cytokinins (zeatin

Time-course studies. For short-term kinetic studies, root systems were held for ] h in A B A ] 0 - 6 mol' 1-1 at a constant hydrostatic pressure (0.1 MPa). The exudation rate increased almost immediately after application of ABA without a measurable lag-phase and continued to increase for about 20 to 30 min, reaching 150-160% after 45 to 60 min (Fig. 3). Long-term kinetic studies (up to 72 h) were performed with ABA applied to the root, as well as with A B A applied to the apical bud. Abscisic acid (10 -6 m o l . l - 1 ) was again used for root application. Within 24 h the concentration of ABA in the root medium declined linearly, as a result of uptake and metabolism, to about one third of the original concentration. Studies with radioactive ABA (data not shown) indicated that between 0.5 and 2.4% of the A B A applied to the apical bud was transported to the root. With both application techniques, a promotion of exudation for up to 12 h (with and without hydrostatic pressure) could be achieved (Fig. 4). Twentyfour h after the application, the exudation rate had decreased and was 10-20% below the control level. However, exchanging the root medium

328

M. Ludewig et al. : ABA and water transport in sunflower

Table 1. Measurement of volume flow (Jv) and osmotic potential ( ~ E ) of the exudate from decapitated sunflower plants under the influence of ABA (10 -6 mol.1 l, 1 h) and hydrostatic pressure (alP) applied to the root. Each value is the m e a n • of at least 19 measurements. A gt~ (line 3) is the difference in the osmotic potentials between nutrient solution (~P~= - 0 . 0 7 3 MPa) and exudate. From these data the hydraulic conductivity Lp was calculated according to the equation L p = J v (AP-A gj~)-i (line 4). In line 5, Lp values are presented which were calculated according to the equation Lp = Jv (AP-aA gt~)- 1. ~ is the reflexion coefficient. Calculation of c~: from eqn. 5 it follows that G = AP. A 7J~- 1-Jv. Lp 1A ~u~- 1. At zero hydrostatic pressure, ~ is therefore = - Jv. L p - 1A g'~- ~. Assuming that Lp is constant at all applied hydrostatic pressures, the exudation rates Jv at zero hydrostatic pressure and the mean Lp values can be used to calculate ~, which was found to be 0.138 for control plants and 0.143 for ABA-treated plants Hydrostatic Pressure AP [MPa] 0

0.1

1

Jv [gl 10 -3 S -1]

Control ABA 10 -6 mol.1-1

2

~E[MPa'I0

-3]

Control ABA10-6mol-1-1

--162 --147

3

Ag'~[MPa-10 3]

Control ABA 10-6 mol.1-1

-

4 5

30.9_+1.6 38.8_+1.5

--

+4.06 -+4.51

91 76

0.2

0.3

219.4_+ 9.6 334.8_+12.5

442.5_ 9.9 608.4_+16.6

669.0_+16.4 898.7+28.8

- 8 7 . 7 - + 2.86 --92.7_+ 3.57

- - 8 5 . 8 _ 2.86 - - 8 6 . 0 _ 3.58

- 9 0 . 7 - + 2.86 --86.2-+ 3.34

-16 -21

-15 --15

-19 --15

Lp = Jv(AP-A ~7c) -~ [gl. s - 1. M P a - 1]

Control ABA l0 6 mol. 1-1

0.3395 0.5105

2.6119 4.2379

2.3919 3.2886

2.3808 3.1533

Lp = Jv(AP aA 7tg)- 1 [gl- s - 1. M P a - l]

Control A B A 10 6 mol. 1- 1

2.425 3.646

2.244 3.449

2.236 3.074

2.250 3.017

i

-

i

-

i

i

i

160 0,1 M

120

=o /Control

100 I.U

,

0

10

,

, ,

20

,

,

30

,

,

/.,0

50

6

'0

Time (min)

Fig. 3. Time course of exudation of sunflower root systems held at 0.1 M P a hydrostatic pressure and treated with A B A 10 -6 mol.1-1. Time zero: application of ABA. The first measurement was made after 8 min ( = 100%). Each point is the mean_+ SD of five measurements

for a fresh nutrient solution, or continuous flow of fresh medium with and without ABA, restored or maintained the promotive effect on the exudation (Fig. 5). It seems that during the 24-h treatment, substances are formed in the medium which counteract the promotive effect of ABA on the exudation. The nature of these substances is unknown.

Ion concentrations andjTuxes. As stated before (Table i), the osmotic potential of the exudates from plants treated with ABA at zero hydrostatic pressure was found to be less negative than that of untreated control plants. The changes in the concentrations and fluxes of some ions therefore were studied in a series of short-term and long-term experiments. For short-term kinetics, decapitated seedlings were treated (without pressure) with ABA 10-6 tool. 1-1 for 3 h. The exudate was collected with a microsyringe and analyzed. Results are presented in Figs. 6 and 7. It is evident that, in accordance with the already mentioned increase in the osmotic potential ( = change to less negative values), the concentrations of the analyzed cations were lowered in response to the ABA treatment. This reduction was most pronounced between 1-2 h after application of ABA. After 3 h, the differences between ABAtreated and untreated plants became smaller or disappeared. When the fluxes of transported osmotically active substances and analyzed ions are calculated from these data, it turns out that they are significantly higher 1-3 h after the ABA treatment. This is obviously a consequence of the strongly enhanced exudation rate (Fig. 7). A long-term experiment in which ABA was applied in a continuous fresh nutrient stream for 24 h (Fig. 8) clearly showed that after a longer duration the effect of ABA on the flux of ions disappeared,

M. Ludewig et al. : ABA and water transport in sunflower

329

160

-

1/,0

(~

811

.

I'LE-T-rl"~,T,-'T'-,T

Iv

ABA

120 OMPa

*E 140

,,

120

2

o o o

"3 E

160

~Control I

g

L

i,

i

I

I

I

I

E

[

O..

120

80

w

T

/,Q

4.

100

o -o

11,0

0

UJ

120

30Q

10o

,,,T r

." - *

] . .. . ... ... . . .

60 o

,

!

, 1

3

,

,

,

, 2

O t_J

Time (h} Fig. 4. Effect of ABA (5 lag per plant) applied to the apical bud on the exudation rate of the root system9 The sunflower plants were decapitated 0, 3, 12, and 24 h after application of the hormone and the roots were subjected to hydrostatic pressures. Each point is the mean_+ SD for at least 12 plants

.I

(Control

T

"~, o . , , I . = ~ . . . = . . .

kABA I

I

I

,

....... "........

i

i

I

i

Ca 2+ //,Control

-T

20C

....... t ...... ....

I0C 0

1500

,

,AtBAt

§ I

. .

""

E

O.

80

I

Mg2+

A

X 140 ~5

~---~....... , - - - - + . . ..... ! .......

100

~J

rr"

....

I

I

I

I

I

t I

:--4---

i

" ,J-...t. [ r't"-~-, I , [Control

i

I

i

I

K"

E

R. 1000 +

v

500

200

0

8 "E

O

150

2

8 100

to

50

__.,._, 1

r-z--1

I

'

__J-~

I

IAIB C 0

___r..~l~__.,-z--

'

0 MPo

IAIB C[

[

i

0,1 M P o

I

IA BIC

.

02

MPo

!ABCI 0.3

MPo

E

80

O

60

._.

t~0

E

20

C)

[

i

t

i

I

i

I

IT

rt.:-t-,t"--}-!....t..., ('Control

~a+~+,i

I i' ' ~I9

I

{

, ....... 9 .........

~ABA

i

20

i

i

i

I

60 100 Time (min)

I

l

I/,0

I

i

180

Fig. 5. Effect of a second treatment (1 h) 24 h after a first treatment with ABA 10 -~ mol.1-1 on the exudation rate. A: control; (root medium exchanged for fresh root medium). B: root m e d i u m + A B A exchanged for root m e d i u m - A B A . C: root medium + ABA exchanged for root medium + ABA. Each point is the mean _+SD of at least six measurements

Fig. 6. Time course of the effect of A B A 10-6 tool 9 1 on volume flow Jr, osmotic value mOs and the concentrations of K +, Ca 2+, and Mg z+ in the exudate derived from sunflower root systems within 3 h after application of the hormone to the roots and simultaneous decapitation. Each point is the mean +_SD of seven plants

but the flux of water was still 40% greater than the control.

bud. The addition of PEG 6000 to the nutrient solution for 2 h caused a decrease in the leaf water potential from - 0.3 MPa to - 0.9 MPa. After this time, the seedlings were returned to the normal nutrient solution, decapitated at different time intervals and the exudation rate measured9 Osmotic stress caused a drastic reduction in the exudation rate (Fig. 9). After the stress phase, exu-

Effect o f osmotic stress on exudation rate and A B A content. This experiment was designed to test the possibility that an increased endogenous ABA level promotes volume flow in a manner similar to that of ABA applied either to the root or to the apical

M. Ludewig et al. : A B A and water transport in sunflower

330

140

--

per cent

Z,OC

Z E

-

100

20c

!

O kJ

I

I

I

I

I

I

I

60

I

J

20

I .... I

ir

,Ld.

T-I

[co2"]

[M9 2"]

[Kq

|

l&01 per

O

cent

E

"i"!

T

§

~00

I

I

i

C

B,

"nc

~nc

;-I

;-I

i---i-/ . "----Z--.] .... ~... ...... } ......

Control j

60

Mg 2 § 20

1C97ABA C ABA 1619 98 103

75 74

Ipmo, p~_ol pmoLpmok

pmo[ pmo[

i s-1

s1

i= t-,,

'IT

§

IE

O|

'

5-

'

I

i

i

I

. ......

[ ........

I

I

I

"T[[~/'-ABA

~000 o

i= o

s-

s-

s-

s-

].13

I I

O

2000

I,

E L==

C~176

J

20

I

I

f

I

I

60 100 Time (rain)

i

9 ......

I .......

I

I

140

180

Fig. 7. Time course o f the effect o f A B A 10 6 m o l . l - t on the molar fluxes o f Ca 2 +, K +, M g 2+ and the sum o f osmotically active substances (~) in exudates from sunflower root systems. Abscisic acid was added at time zero to the root medium. Simultaneously the plants were decapitated. Each point is the mean_+ SO o f seven measurements

Co 2+

C ~BA

!1,62

/Jmol I umol

,-1 !s-1 I

I Mg 2*

K§

I

Jv

Fig. 8. Changes in Jv, ~z, K +, Ca z+, a n d M g 2+ in the exudate 24 h after application o f A B A 10 -6 tool.1 i to sunflower root systems. Above: concentrations. Below: fluxes, calculated f r o m the above data. Each value is the mean o f 40 measurements plus standard deviation

dation increased again and, after about 6h, reached levels which were much higher (up to 50%) than the controls. During the stress period the osmotic potential was slightly lowered but increased again during the period of recovery. The molar fluxes of Mg2+, K+, and Ca z + were reduced during the stress period compared with the control, but increased during the recovery phase (data not shown). Moreover, osmotic stress caused a significant rise (about fivefold) in the level of ABA in the root as well as in the shoot (Fig. 10). Maximal levels were reached about 2 h after the stress period. They remained at an enhanced level for at least 6 h. Surprisingly, there were no differences in the amounts of A B A between the root and the shoot. Thus, it appears that the increase of the exudation rate after osmotic stress coincides with increased endogenous ABA levels.

M. Ludewig et al. : ABA and water transport in sunflower

331

I

16 150

I

E u u~

12

o c O

~00

"5 co

= o

\\\ ,x

5o

/

O,.,~

"s

4

O

1 2

I 0

n 2

i 4

~ 6

#

i 2/.

0,

0

J 48 h

,//

// Stress

#

4,

8,

28

,

Recovery

Fig. 9. Effect of osmotic stress on exudation rate Jv and osmotic value n of the exudate. Sunflower root systems were treated for 2 h with P E G 6000 (0.5 MPa). Each point is the mean of at least 12 plants

-0,1

:E

-5

-

-0,3

................. , .

e--

o /I\//'~176

2000

//4'8

32

Time(h)

/''~

-g

Shoot

\

.............. o - 2 }

a_ -0,5

(21

*6