Planta

Planta 139, 227--237 (1978)

9 by Springer-Verlag 1978

Auxin Increases the Hydraulic Conductivity of Auxin-sensitive Hypocotyl Tissue J.S. Boyer and Gloria Wu Departments of Botany and Agronomy, University of Illinois, Urbana, IL 61801, USA

Abstract. The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine m a x (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( ~ ) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. ~bw was below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, ~bw was similar to the water potential of the environment when cell enlargement was not occurring. The low ~,~ in enlarging tissue indicates that there was a low conductivity for water entering the cells. The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (L'p). Despite the low L~v of growing cells, L'p decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and L'p increased when auxin was resupplied exogenously. The auxin-induced increase in L'p was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in L'p appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to L~p remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded. Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the ceil wails and by

simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.

Key words: Cell enlargement - Cell-wall extensibility - GIycine - Turgor - Water potential.

Introduction Water absorption is essential for cell enlargement in plants. Absorption increases when growth increases under the action of auxins. In cells, this absorption has been considered to occur readily with osmotic gradients so small as to be inconsequential (Ray et al., 1972). In plant tissues, however, cell enlargement is accompanied by substantial osmotic gradients (Boyer, 1968, 1974; Meyer and Boyer, 1972), which imply that water encounters a significant resistance as it moves through the tissue. In this situation, auxinmediated cell enlargement could be associated with important changes in water transmission properties of the tissue. These changes would act in concert with the well-known effect of auxin on cell-wall extensibility (Cleland, 1971). Auxin seemed to increase tissue permeability to water in some instances (Ruge, 1937; Buffel, 1952; von Guttenberg and Meinl, 1952; Thimann and Samuel, 1955; Kang and Burg, 1971) but not in others (Ketellapper, 1953; Ordin and Bonner, 1956; Dowler et al., 1974). Unfortunately, each of these studies required the tissue to be immersed in solutions. This has two consequences. First, simultaneous measurements of water absorption and tissue water potential were not possible. Second, because the experiments utilized solutes or labeled water, the penetration of

228

J.S. Boycr and G. Wu: Auxin and Hydraulic Conductivity

the tissue by these molecules u n d o u b t e d l y h a d a large diffusion c o m p o n e n t a n d w o u l d have been affected by u n s t i r r e d layers of s o l u t i o n adjacent to or within the tissue (Dainty, 1963; Ray a n d R u e s i n k , 1963). These p r o b l e m s can be c i r c u m v e n t e d by m e a s u r ing water a b s o r p t i o n a n d water potential in tissue that r e m a i n s attached to the p l a n t a n d is growing in h u m i d air. There are detectors of tissue water potential that operate in h u m i d air (Boyer a n d K n i p ling, 1965 ; Boyer, 1969). They are based o n the principle that water v a p o r in e q u i l i b r i u m with liquid water in tissue will have the same water p o t e n t i a l as the liquid. Since the detector is in t h e r m o d y n a m i c equilib r i u m with the tissue a n d does n o t c o n t a c t the tissue, the effects of u n s t i r r e d layers associated with external solutions are avoided. W a t e r a b s o r p t i o n a n d water potential m a y then be measured s i m u l t a n e o u s l y in a n e n v i r o n m e n t that assures n o r m a l p l a n t b e h a v i o r insofar as possible. The following study uses this app r o a c h to determine whether the n a t u r a l l y - o c c u r r i n g auxin, indole-3-acetic acid (IAA), alters the hydraulic c o n d u c t i v i t y of auxin-sensitive p l a n t tissue.

Theory The rate at which water enters a cell depends o n 1) the difference in water p o t e n t i a l between the cell interior a n d the external m e d i u m , a n d 2) the water t r a n s m i s s i o n properties of the cell, here defined in terms of the hydraulic conductivity. F o r tissues, the water potential m a y be considered to be a n average for all the cells, a n d water u p t a k e in the steady state is then described by the u s u a l e q u a t i o n :

dV dt

ALp(Ow-q/o)

(1)

where V is the v o l u m e of water m o v i n g into the tissue (cm3), t is the time (s), A is the area of the limiting, often complex, barrier for flow (cm2), ~t w is the average tissue water potential (bar), q/0 is the water potential of the external m e d i u m (bar), a n d Lp is the hydraulic c o n d u c t i v i t y (cm s - 1 b a r - ~ ) . F o r c o n v e n i e n c e of m e a s u r e m e n t , E q u a t i o n 1 m a y be modified to p e r m i t v o l u m e changes to be evaluated f r o m tissue dimensions. As will be shown, the cylindrical tissue used in this study was of virtually constant radius (r). A c o n s t a n t fraction (c) of the tissue v o l u m e was occupied by water. Therefore, changes in tissue length (h) could be related to changes in the v o l u m e of water c o n t a i n e d within the tissue according to cT~rZdh= dV. T h u s :

dh ~ = ctALp(q/o - q/w) = ~E P(q/0 - q/w)

(2)

where c~=l/c~zr2=dh/dV in units of cm cm -3. L'p, the a p p a r e n t hydraulic c o n d u c t i v i t y (cm 3 s 1 b a r - 1), equals ALp a n d therefore c o n t a i n s b o t h the geometric factors c o n t r i b u t i n g to A a n d the t r a n s p o r t factors c o n t a i n e d in Lp. Since the m a g n i t u d e of A was u n k n o w n , m e a s u r e m e n t s p e r m i t t e d only the c a l c u l a t i o n of L~p. However, there is evidence e n u m e r a t e d below that A r e m a i n s constant. Evidence is also presented that the g r o w i n g region was of c o n s t a n t length. Therefore, dh/dt of E q u a t i o n 2 represented the e l o n g a t i o n of a c o n s t a n t u n i t of tissue.

Material and Methods

Plant Material Soybean (Glycinemax (L.) Merr., cv. Wayne) seedlings were grown from seed in vermiculite in a dark, humid chamber (temperature29_+0.5~ C). The vermiculite was watered with 10 4 M CaC12. After 48 h, uniform seedlings were transplanted either to fresh vermiculite and watered as above, or were suspended with their roots directly in aerated 10 4M CaClz. Growth measurements were begun 20 h after transfer to the seedlings to the new conditions. All tissue manipulation was clone under a green safelight (green fluorescent bulb wrapped in green plastic sheet having maximum transmission at 525 nm and negligible transmission below 475 nm and above 575 nm).

Growth Measurements Experiments were conducted under growth conditions to assure that transpiration was negligible and the only water movement that occurred was utilized for cell enlargement. In order to determine the proportionality c~ between V and h (Eq. 2), the water volume (V) of hypocotyl tissue from both the zone of elongation and the zone of maturation was determined from the weight lost during drying at 100~ C, and micrometer measurements of the radius and length of the tissue were made. Hypocotyl elongation (dh/&) was determined in intact seedlings, seedlings with the apical 0.7 cm of the hypocotyl removed (hookless seedlings),and occasionally seedlingswith both the apical 0.7 cm and the roots removed. Rates were measured with a linear variable displacement transducer (Model 7DCDT-500; HewlettPackard, Waltham, Mass., USA) connected by a fine wire (0.076 mm diameter) to a clip attached to the hypocotyl ca. 0.8 cm below the cotyledons (Fig. 1A). The base of the hypocotyl was held in constant position by another clip attached to a rigid bar. The body of the transducer was mounted in the barrel of a microscope so that it could be raised or lowered around the transducer core. Changing the position of the transducer body permitted the system to be calibrated at any time without disturbing the seedling. With this apparatus, dh/dt could be measured when the seedlings were growing [n vermiculite or 1 0 - 4 M CaCI2 (Fig. IA). The chamber enclosing the shoot was kept dark and humid or, for some experiments, was filled with sufficient 10-4M CaCI2 to cover the entire hypocotyl. For certain experiments, hypocotyl elongation was also measured inside a thermocouple psychrometer (Fig. 1B) in which entire soybean seedlings could be placed. A mark was made with India ink on each hypocotyl base before the seedlings were inserted in the psychrometer chamber. Upon completion of the water potential measurement with the psychrometer, the seedlings were removed and the difference in length of the hypocotyls was determined.

J.S. Boyer and G. W u : Auxin and Hydraulic Conductivity

A

Air

Rigid~I reference bar

i

Pulleyl~ elongatmoves iondUringC~

In one case, water m o v e m e n t through detached hypocotyls was measured directly by excising the meristem and the roots, and forcing water through the hypocotyl under pressure. The hypocotyl had been sealed in the top of a pressure chamber (Scholander et al., 1965) and pressure was applied to the base of the hypocotyl, which was under water. The water moving through the tissue at constant pressure was collected from the exposed tip using a microliter syringe. Water Potential

~Seedting ~J/-I 0-4 M CoCl2

229

for cotibration Recorder Power

Supply

Thermocouple Waferfor ~., temperoture control Water for temperature~ control / / Petrolatum10-4M \ CaCI2 10-4 M CoCI2Petrolatum Seedling

In intact seedlings, ~w of various regions of the hypocotyl were measured by placing four seedlings in a thermocouple psychrometer chamber (Boyer, 1968) large enough to hold the entire plants (Fig. IB). Petrolatum (vaseline) was placed in a ring around the 2.5-cm region of the hypocotyl to be exposed to the thermocouple (Fig. 1 B). The petrolatum screened the thermocouple sensor from other portions of the plant. Outside the petrolatum screen, 10 . 4 M CaC12 bathed the roots. ~w was measured by the isopiestic technique (Boyer and Knipling, 1965); the thermocouple chamber had been coated with melted and resolidified petrolatum (Boyer, 1967) and measurements were corrected for the heat of respiration (Barrs, 1965). For detached hypocotyl segments, ~'w was measured with a thermocouple psychrometer for excised tissue (Boyer and Knipling, 1965). Four hypocotyl segments were placed in the bottom of the thermocouple chamber, and ~,. was measured as in the intact system. In cases where the entire seedling was surrounded by solution, segments excised from the seedlings had to be dried before the measurement of water potential. This was done by first rolling the tissue on the surface of a slightly clamp sponge, then on dry, absorbent tissue paper in a h u m i d chamber. The osmotic potential ( ~ ) was frequently determined in the hypocotyl tissue immediately after ~9,,, had been measured. The tissue was frozen in the covered psychrometer chamber on dry ice. After thawing, the sap was expressed from the tip of each hypocotyl by squeezing between the fingers. The expressed sap was stored in a capped vial and placed on a thermocouple for m e a s u r e m e n t of ~ . All manipulations for measurement of ~,,, and +s were carried out in a h u m i d chamber. Transient Water Uptake

Fig. 1A and B. A p p a r a t u s for measuring hypocotyl length and water potential in soybean seedlings. A Linear variable displacement transducer connected by a fine wire to a clip attached below the hypocotyl hook. The lower end of the hypocotyl was clipped to a rigid bar. The roots were bathed in aerated 10 4 M CaC12 or were growing in vermiculite. Moist filter paper covered the walls of the container in which the seedling was growing. The volume of solution around the roots could be increased to cover the entire seedling when desired. For calibration, the transducer body could be raised or lowered around the central core. The position of the core relative to the body determined the output of the device. Air temperature around the apparatus was 29 +_0.5 ~ C. B Thermocouple psychrometer for measuring the water potential of hypocotyls of intact seedlings. Four seedlings were used in each measurement. The circular petrolatum barrier shielded the thermocouple from peripheral parts of the chamber. Outside the barrier, the roots were bathed in 10 -4 M CaCI> By changing the position of the seedlings in the chamber, water potential could be measured at any position along the hypocotyl. Temperature was 26+0.0005 ~ C

Single seedlings were m o u n t e d in the variable displacement transducer and were immersed in 10 4 M CaC12. Elongation rates were measured in 10 4 M CaC12, then 10 -~ M CaC12 containing 0.19 M sucrose (~0 was - 5 bars), and finally in 10 4 M CaC12. This series of determinations was carried out in the following sequence for a given seedling: 1) rootless, 2) rootless and hookless, 3) rootless and hookless with 10 4 M IAA, p H 7, in each solution. All solutions were preequilibrated to the temperature of the tissue before being placed in contact with the tissue. In a different experiment, growing regions of 20 hypocotyls were excised and threaded cross-wise on a thin wire (0.076 m m diameter). The segments were partially desiccated in air, were then shortened to 1.5 cm by cutting rapidly with a razor blade under water, and were permitted to rehydrate in 10 ~ M CaC12 with or without 10 ~ M IAA at p H 7. Water uptake during rehydration was measured by weighing the hypocotyl segments after quickly blotting excess solution with sponges. Solutions I A A solutions were prepared daily by slowly titrating to pH 7 with N a O H .

230

J.S. Boyer and G. W u : Auxin and Hydraulic Conductivity i

i

Table 1. Water potentials and growth rates of the elongation zone and the mature zone of hypocotyls of intact soybean seedlings

i

SOYBEAN

Water potentials and growth rates were measured simultaneously in the same seedlings in the thermocouple psychrometer shown in Figure 1 B. Initial hypocotyl length was 8-9 cm

"E 125 E

w d

r o 0.75 o o~

Hypocotyl region (3 cm)

Growth rate ( m m h - 1)

Water potential (bars)

Elongation zone

1.2 2.1

-- 1.7 -2.1

Mature zone

0.004 0.004

- 0.3 -0.5

z_ 050 w z~

0.25

,b

o

2b

3b

4;

TIME

( rnin l

5'0

6~

7o

Fig. 2. Effects of root excision on hypocotyl growth in soybean seedlings. Hypocotyl length was measured with a linear variable displacement transducer. Excision was carried out under the 10 4 M CaC12 solution bathing the roots

1.2.0 m

i

i

i

i

SOYBEAN Excised Hypocotyl (5.3cm)

E E 10.0

i

Table 2. Water potentials and growth rates of the elongation zone and mature zone excised from hypocotyls of soybean seedlings Water potentials were measured in a thermocouple psychrometer for excised tissue and growth rates were measured just prior to excision. Initial hypocotyl length was 6-8 cm Hypocotyl region (2.5-cm segments)

Growth rate ( m m h - t)

Water potential (bars)

Elongation zone

1.2-1.6

- 3.0 -2.0 -1.7 -3.1 -2.7 -2.2 --2.7 -2.5+0.5

0.004-0.005

-0.2 -0.4 -0.5 -0.3 -0.3 -0.4 -0.6 -0.4•

,/ ~ "

v

w I-




,~ 2.0 FO I---

o

o

i

I

i

I

2

4

6

8

TIME

I0

12

Fig, 3, Exudate produced by an excised soybean hypocotyl having one end under pressurized water. The hypocotyl was 5.3 cm in length (upper cut at upper end of zone of elongation) and was exposed to pressure of 1.7 bars. The exudation rate was equivalent to an elongation rate of 18 m m h 1. In intact seedlings, rapid elongation rates were 1.3 to 2 m m h - 1

Results

1. Preliminary Measurements The radius r was 0.143 0.158 cm and the fraction c of tissue volume occupied by water was 0.47 in the elongation zone and 0.45 in the mature zone. The hypocotyl length:water-volume ratio ~ calculated from the average of these measurements was 31 cm c m - 3.

Average + S.D. a

(min)

a

Standard deviation

In order to locate the position of A within the plant, the root and hypocotyl were progressively excised. Excision of the roots or the lower, mature region of the hypocotyl under 10 4 M CaClz did not alter the rate of cell elongation (Fig. 2). Also, water forced through an excised hypocotyl at pressures similar to Ow in growing tissue ( - 1 . 5 to about - 2 . 5 bars) gave exudation rates about 10 times the rate of water uptake for rapid elongation in intact hypocotyls (Fig. 3). Therefore, the roots and xylem of the hypocotyl did not constitute significant barriers to water movement to the growing cells, and the barrier controlling the rate of water movement must have been along the radial path from the xylem to the interior of the cells of the growing tissue itself.

J.S. Bayer and G. Wu: Auxin and Hydraulic Conductivity

SO BEAN

,g .

V--2

.

.

.

.

.

.

.

.

.

.

.

231

.

.

.

\

. ., . . *

"..._

,..-.~o --

F-

/d O.17mm hr "~ /K-ExciseHypocotyl

o

/~Intact

0_

-I0

g

I

.

.

.

lie

.

Water Potential of Excised Hypocotyl%.

\\,,

~--6

.

Hypocotyl

~

~ r O 0 0 m m hr -t

0.75 ~

9

"--;

4 Length of Excised Hypocotyl

Bose and Hook

/

0.50

10.25

1.25mm hr -I

I z

J

I

I

I

I

I

I

I

i

I

l

I

I

0

I0

20

30

40

50

60

70

80

90

I00

I10

TIME

AFTER

EXCISION

I

I

120

l

I

I

150 140 150

I

I

160 170

WUJ

180 190 z

(rain)

Fig. 4. Water potential and tissue growth after excision of zone of elongation of soybean hypocotyl. Tissue growth was measured under conditions sinmlating those in a thermocouple psychrometer for excised tissue. Growth was measured with a linear variable displacement transducer, water potential with a thermocouple psychrometer for excised tissue from similar seedlings. The first 30 rain of the water potential measurement represents thermal equilibration of the psychrometer, and is represented by a dashed line

i

E

i

i

i

i

i

i

i

i

i

i

i

i

i

i

SOYBEAN

E ~p=+B7bars d/s = - 5 . 2 bars ~'w =- 1.5 bars G = O . 2 1 m m hr-i L'p=O.t6 x 10"6cm 3 sec-[. b a r I

T I-Z 1.25

i

i

i

i

i

~ p = + 4 . 1 bars ~s = - 5 2 bars ~w = - I . I bars G = 0 . 2 6 mm .hr -~ L'p= 0 . 2 9 x I O-6crn3" sec I. bar -I }~...~_.-.~ /

5 I.oo %=+30 ~) r~7~

~ w -_ - 2 . 4 bars

"3-

~= . . . . m m . . r / 1,2-p - O . 5 6 x l O-6 c m 3 .sec-i .bur -I I / I /

--

Z

0501/

/

Z

I- ~ ~ z ~ mocouple psychrometer (Fig. 1 B). Cell elongation was negligible in this region. However, in the elongating zone, Ow was - 1 . 7 to - 2 . 1 bars (Table 1) when measured with the same instrument, and cell elongation was rapid. Because the measurements used intact seedlings enclosed in the psychrometer chamber with vapor in equilibrium with the tissue water (Fig. 1 B),

transpiration was zero and there were no excision effects. The measurements had the further advantage that cell elongation and tissue water potentials were determined simultaneously. Water potentials were similar to those in the intact tissue when the measurements of ~w were repeated with an isopiestic thermocouple psychrometer that used tissue excised from hypocotyls. Table 2 shows that the mature zone of the hypocotyls had average ~Ow of - 0 . 4 bars whereas the elongation zone had average ~ , of - 2 . 5 bars. It is possible that elongation might have continued for a time after excision because of redistribution

232

J.S. Boyer and G. Wu: Auxin and Hydraulic Conductivity I

f

I

~

I

I

I

I

I

I

I

I

SOYBEAN

I

q"s = - 4.9 bars o/w = - 2.3 bars

Seedlings

26 ~ C

-~ 1.5C

I

~p= +2,~ b0~s j

G--~132m~hrv~ _ = 9 p = + :3.0 bars qSs= - 5 . 2 bars 9~o = - 2,2 bars G = 0 . 0 6 m m ' h r -I

t

-,4

LO0

S;roIAA IO -4 M pH 70

~b

0.75 Vp = + 2.3 bars g,s = - 5 . 4 bars

~

HYDRAULIC CONDUCTIVITY (cm 3 .sec -I , bor-I )

V,'~; - 3. l bars 18

Ila

Expl

Hook

lntoc!

+IAA

0.5C

0.2~

~'~

I

0 . 6 0 X I0 - 6 0 . 0 3 X

2

0 5 6 X 1 0 -s O. 1 3 X I O -e 3 . 4 4 X 1 0 -c

IO-e ' 0 . 5 9 X 1 0 -e

5

0 . 6 5 X 1 0 -s 0 . 1 1 X l O -e 3 . 4 1 X 1 0 -~

/ E xcise I / Hypoeoiyl

y

Hook

N

"-IO

I

I

I

I

I

I

0

I0

20

30

40

50

60

I

I

I

I

I

I

I

70

80

90

I00

120

130

140

150

Time After Excision (min)

Fig. 6. Hypocotyl length, water potential ( ~ ) , osmotic potential (~), turgor (@), and apparent hydraulic conductivity (L'p) before and after hook excision, and before and after supplying IAA to the hookless seedling. Measurements were made as in Figure 5. Inset shows comparison of L'p in the seedlings before excision of the hook, after excision of the hook, and after addition of IAA in 3 replicate experiments

i

1.6 x I 0 - 6

i

of water within the tissue. In this situation, Ow of excised tissue would represent Ow of tissue developmentally altered from that in an intact hypocotyl. Therefore, we studied elongation after the elongation zone had been excised in a dark, humid environment to simulate the environment encountered by the tissue during a psychrometer measurement. Figure 4 shows that cell elongation was markedly inhibited immediately after excision and became zero after about 1 h. Psychrometer readings typically became stable after about 2.5 h (Fig. 4), and the stable output was considered to represent the water potential of the excised tissue at internal equilibrium. Since equivalent portions of intact and excised tissue were similar in both ~w and length, excision effects were small. Excised tissue was therefore used for the remainder of the measurements of ~ .

SOYBEAN

.-.-. is

1.4xlO -8

m"

1,2 x l O - 6

E u v

>- 1 , 0 x l 0 -6

o

/

0 . 8 x IO "6

-

a z

0 O

o

9 ~

0 . 6 x I 0 "6

0.4x

9

e /

9 ./! . / Y 2"

I0 "6

-i-

Auxin-depleted ro tes = 0 . 0 8 - 0 . 3 24 27~

0.2 x I0" 6 ~II | I

growth rnm.hr"

-

I

I

I

I

:3

5

7

9

It

RATIO OF AU XIN -ENHAN CED : AUXIN-DEPLETED GROWTH RATES

Fig. 7. Apparent hydraulic conductivity at various levels of growth enhancement by auxin (10-* M). A ratio of 1 for auxin-enhanced: auxin-depleted growth indicates growth rate prior to enhancement by auxin. Ratios > 1 indicate enhancement by auxin during steady growth 15 to 130 rain after IAA was supplied. Data are from 16 experiments

2. Apparent Hydraulic Conductivity during Steady Growth Hypocotyl elongation was rapid when intact seedlings grew in vermiculite in a dark, humid chamber (transpiration was less than 1/50 the rate of water uptake during rapid growth). About 30 rain after the hypocotyl hook was excised, elongation decreased (Fig. 5).

J.S. Bayer and G. Wu: Auxin and Hydraulic Conductivity t01

233

effect of excising the hook (with cotyledons and epicotyl) must have been the removal of the auxin supply for the hypocotyl, and spraying auxin on the stump must have replaced the endogenous auxin. L~p decreased when the hook was excised but increased when auxin was sprayed onto the auxin-depleted hypocotyl (Fig. 6, inset). As before, ~Ps increased slightly, and turgor varied inversely with the rate of growth (Fig. 6). The increase in L ~ was large when the enhancement of growth by auxin was large (Fig. 7). Furthermore, L'p increased soon after the auxin-induced increase in elongation (Fig. 8).

Io

SOYBEAN

91

24.0 - 27.0 ~ C

~h

Auxin-Depleted Growth Rates=

8

0 1 4 - 0 3 1 m r n h r -~ Auxin-Depleted Hydroulic Conductivities = 0 I I - 0 . 3 3 cm 3 9sec TM b(]r -I o Hydraulic Conductivity 9 Growth

~

7~ o

~

9

Spray IAA

~6

,

5~

o

10-4M, pH 7 0

o

I D--! -I0

-5

1~5

IO TIME

SINCE I A A

ADDITION

2LO

(min)

Fig. 8. Growth rates and L'p after spraying hookless hypocotyls with IAA. Ratios of 1 indicate growth rates and L'p before IAA effects. Ratios greater than 1 were observed after IAA enhancement of growth

3. Transient Water Uptake Because the dependence of the hydraulic conductivity on psychrometer measurements could lead to error if the psychrometer were in error, we also tested whether the decreased conductivity affected water movement in hypocotyl tissue recovering from a water deficit. This approach had the advantage that water potential did not need to be determined. A rootless seedling was immersed in a - 5 bar sucrose solution, which was a sufficiently low ~)0 to just prevent elongation. When the sucrose solution was replaced with 10 -4 M CaC12, a lag was observed before an increase in tissue length occurred (Fig. 9, bottom trace) because water had to reenter the tissue to establish the water potential gradient and the turgor necessary for a length increase. It should be noted that the elastic, reversible recovery in tissue water status had to precede the resumption of growth in this experiment.

The IPw of the elongation zone was - 2 . 4 bars in intact seedlings but it became less negative after excision, eventually approaching - 1 bar (Fig. 5, inset). The ~s also became slightly less negative, but turgor (@, the difference between ~ and ~s) was inversely related to growth rate (Fig. 5, inset). Lip calculated from Equation 2 was 0.56x 10 -6 cm 3 s -1 bar 1 in the intact hypocotyls, but decreased to half or less after the hypocotyl hook was excised. Rapid hypocotyl elongation resumed if auxin (10 4 M, pH 7.0) was sprayed on the hypocotyl stump after hook excision (Fig. 6). Concentrations of 10 -5 and 10 .6 M IAA were equally effective but were more variable than 10-4 M. Spraying with water at similar pH had no effect. Therefore, the immediate

I

I

I

i

I

I

t

I

I

I

SOYBEAN 28 + 025 ~ C

/~H~oo' M e s s

LAG TIMES (min)

+IAA

Sucrose (-5 bors) T A A (10 "4

/

Plant

Hookless / -L -/

/

Merislem Meristem Meristem I Present Absent Absent § IAA

I 2 3

I0

5

7

5 8 5

8

16

I0 9

6

0.5

Sucrose S bor~) e e

1

1- 5 bars )

J

-20

d T-lag , h =17n rain g

-

(I

_/

5

17 17 18

14 9 7

15

II

5.5 t4 I0 27

55 II

3 17.5

;,mi0

Sucrose

I

I

I

I

I

-tO

0

I0

20

50

1

40

[

I

I

50

60

70

Time Since RemovGI of Sucrose (min)

Fig. 9. Resumption of growth after soybean seedlings were exposed to a solution having a growth-inhibiting concentration of sucrose. The sucrose solution had an osmotic potential of - 5 bars. Hypocotyl elongation was measured with a linear variable displacement transducer attached to rootless seedling (lower trace), the same seedling 60 rain after excising the hook (middle trace), and the same seedling exposed to exogenous auxin in all solutions (upper trace). Inset shows 8 replicate experiments

234

J.S, Boyer and G. Wu: Auxin and Hydraulic Conductivity

.-~ 15

SOYBEAN

~- 12

I'-T

9

ua 6

/

/P

"

-IAA

,

Z

m 5

q~ < Ld nO

0

_z o

,;

2'o

5'o

4'0

5'0

6'o

7'o

TIME (rain)

Fig. 10. Water uptake by excised soybean hypocotyls that had been desiccated prior to rehydration with and without auxin. The hypocotyls had been desiccated in air to 85% of their original weight. Rehydration was initiated by recutting segments (1.5 cm) from the hypocotyl zone of elongation under water with or without 10 -4 M IAA, pH %0

Possible secondary effects of the sucrose on wall extensibility would be of little consequence, therefore, since only the time until the initial, elastic change was recorded. After the seedling was depleted of auxin by excising the hypocotyl hook, the lag became longer in the same seedling (Fig. 9, middle trace). When exogenous IAA was supplied to the same seedling, the lag shortened (Fig. 9, top trace). The control, in which the intact seedling was exposed to - 5 bar sucrose repeatedly, exhibited a constant lag time (data not shown). The ratio of lag times for the intact seedling: hookless seedling: hookless seedling+IAA were 1.00: 1.76: 1.09, on average. Therefore, water entry after an osmotic transient was substantially faster if IAA was present than if IAA was absent. If excised hypocotyls were severely desiccated by permitting the loss of 15% of their fresh weight in air (after which they appeared flaccid), the subsequent recovery was relatively unaltered by auxin (Fig. 10).

Discussion

The results show that l) enlarging plant tissue is not in osmotic equilibrium with the aqueous environment, and 2) auxin increases the ability of water to move into enlarging cells in response to the osmotic disequilibrium. These findings have several consequences for cell enlargement. First, the osmotic disequilibrium must drive water uptake for cell enlargement, since water uptake took place when ~O-~w was significant but approached zero when 0o ~ approached zero (Tables 1, 2). This

behavior has also been observed in leaves (Boyer, 1968, 1974). Water uptake for growth and osmotic disequilibrium persisted for many hours in leaves (Boyer, 1968). Since transpiration was negligible in all these experiments, water lost by evaporation could not account for the continued water uptake and osmotic disequilibrium. It should be pointed out that osmotic disequilibrium for tissue is much larger than for single cells (Green et al., 1971) during growth. Thus, substantial osmotic disequilibrium may be a tissue rather than a cell phenomenon. Second, the relationship between osmotic disequilibrium and growth rate was observed in the elongation zone of hypocotyls when the supply of auxin was changed by excising the hook and associated tissues. ~o-~w decreased about 1 bar as cell elongation decreased after hook excision (Figs. 5, 6), as would be expected if Oo-Ow drives water uptake for growth. However, since Oo-0w was not exactly proportional to growth rate but in fact changed less than the growth rate, L'p must also have changed. In our experiments, L ~valways decreased when the auxin supply to the tissue decreased. It might be argued that the psychrometer measurements were in error so that the relation between growth rate and 0o-Ow was inaccurate. Several types of evidence indicate that this is unlikely. First, the isopiestic psychrometer provided measurements at thermodynamic equilibrium (Boyer and Knipling, 1965). Second, isopiestic measurements are the only ones shown to give accurate values of water potential with tissue of known water potential (Boyer, 1966). Third, the psychrometer was able to detect Oo-0w approaching zero in leaves (Boyer, 1974) and hypocotyls (Tables 1, 2), so that water potentials close to zero should have been measureable. Finally, the measurements of osmotic disequilibrium have been confirmed with a pressure chamber, which Utilizes an entirely different principle for measuring equilibrium water potentials (Boyer, 1968, 1974). It is also important that the two types of psychrometer measurements agreed with each other. Water potentials for excised and intact tissue should have represented an average for the tissue, as required in Equation 1. With excised tissue, there was no net water movement into the sections during the measurement of Ow, and gradients in water potential would have equilibrated to some average value. With intact tissue, growth continued and water movement into the tissue would have also continued during the measurement of Ow. As a result, gradients in water potential undoubtedly persisted, but once again the vapor pressure (and hence the ~ ) of the tissue would have reflected an average because the intercellular spaces would have communicated with virtually all the cells.

J.S. Boyer and G. Wu: Auxin and Hydraulic Conductivity

235

The persistence of osmotic disequilibrium in growing tissue casts in doubt (see also Ray and Ruesink, 1963) growth experiments that involve bathing tissue in solutions of various osmotic strengths. In this situation, unit change in ~o does not imply unit change in Ow, since ~ o : / = ~ and the magnitude of disequilibrium is a function of growth rate (Tables 1, 2; Figs. 5, 6). We assume that the reason for osmotic disequilibrium is that the walls of enlarging cells yield under the action of turgor, thus placing an upper limit on turgor. As a consequence, ~ remains below ~o, and water continues to enter the cells. During steady growth, rates of wall yielding and rates of water uptake are equal. This concept, earlier presented by Lockhart (1965) and Ray et al. (1972), provides a convenient means of assessing the consequences of auxin action on L'p. Following Lockhart (1965), Ray et al. (1972), and Green et al. (1971), cell extension is considered to occur only when turgor exceeds some minimum (I1), a concept supported by extensive evidence (Cleland, 1971). Although the precise relationship of turgor to cell wall extension appears variable (Green et al., 1971), we will assume for convenience the simple form:

dh

= m ( % - Y)

(3)

where m is the extensibility of the cell walls (cm sbar 1) and 0p is the turgor (bar). Because ~ may be considered to be determined by ~ , + @ in the growing cells of soybean hypocotyls, @= ~-~,. Substituting the latter relation in Equation 3, and combining Equations 2 and 3 to eliminate w gives ;

dh

mc~Ep

dt -~Ep+m (0o- ~ - Y).

(4)

This is a general rate equation for cell enlargement. The coefficient (mc~Up/c~Up+m) determines the rate of cell enlargement in a particular water potential environment. When m is large, the coefficient approximates c~Up, which then controls growth. When czL'p is large, m controls growth. The factor ( t P 0 - O s - I1) is the net osmotic force available for growth, and represents the maximum osmotic force (~o - ~ ) diminished by Y. In oat coleoptiles, auxin appears to have little effect on Y (Green and Cummins, 1974) and the present work shows only a small effect on ~s over the short times of the experiments (Figs. 5, 6). Since (Jo is determined by the environment and c~is a constant, auxin must have affected m and/or L'p primarily, in these experiments.

Cortical Cell Length ( ~ m ) ~,

0

0

50 I00 150200 250

• [[ [ ~, II ~

0o,,on#>1, c~L'pcontrols cell enlargement. When m~ c~L'p ~ 1, m controls cell enlargement.

Eq. 5 may be evaluated if Y is assumed to be zero as a first approximation. This approximation is a conservative one, since it minimizes the contribution of c~L'p in Equation 5. Y appears to be small in soybean hypocotyls (Meyer and Boyer, 1972) and may be a constant (Green and Cummins, 1974). Since 0o was essentially zero in the steady state experiments, m/c~L'p=-~w/Op. F r o m Figure 5, m/c~L'p was 0.80 in the intact seedling, and 0.41 and 0.27 in the hookless seedling. F r o m Figure 6, m/eL'p was 1.35 in the intact seedling, 0.73 in the hookless seedling, and 0.88 in the hookless seedling supplied with exogenous auxin. Therefore, wall extensibility and hydraulic conductivity appeared to control the rate of cell enlargement about equally. The experiments that have failed to show effects of auxin on water transport have involved tissue influx or efflux of labeled water by diffusion (Ketellapper, 1953; Ordin and Bonnet, 1956; Dowler et al., 1974). A possible reason for these failures is the presence of unstirred layers of water adjacent to and within the tissue in this type of experiment. Unstirred layers represent regions where diffusion predominates and the solute (labeled water) concentration is not the same as in the bulk phase. The problem arises as an unavoidable consequence of the tortuous, microscopic pathways between cells (Dainty, 1963; Ray and Ruesink, 1963). When unstirred layers are present, the major barriers to diffusion from the bulk phase often are the unstirred layers themselves rather than the regions of the tissue where conductivity is changing, an idea recognized by several past investigators (Ordin and Bonnet, 1956; Dowler et al., 1974). In the steady-state measurements of the present work, water movement was primarily by bulk flow and external solutes were not used, so that unstirred layers were avoided. Another reason for the lack of auxin response in diffusion experiments may be the relative insensitivity of diffusion to differences in the bulk flow of water through porous structures and membranes. Careful work with osmotically-driven flow across model membranes of hydraulic conductivities similar to those of higher plants (about 10 -6 cm s -1 bar 1) indicates that bulk flow is the predominant component of membrane transport of water, and diffusion is only a minor component (Robbins and Mauro, 1960). It therefore seems that auxin effects on water movement are most likely to be observed if osmotic driving forces and' flows are measured directly, as in the present work. In view of the foregoing, it appears that water transport in growing hypocotyls consists of water movement through the roots and up the xylem along rather small gradients in water potential, so that xy-

J.S. Boyer and G. Wu: Auxin and Hydraulic Conductivity

lem ~ is close to 00. If there is little growth, the surrounding tissues also have t),~ close to ~)o, as occurs in the mature zone at the base of the hypocotyl (Fig. 11). If cell enlargement is occurring, however, water moves radially from the xylem into the surrounding tissues (Fig. 11), where it encounters a significant resistance. The resistance probably exists because of many cytoplasmic and wall barriers that must be traversed before all the cells are supplied with water. When auxin increases the hydraulic conductivity of the elongating tissue, water moves more easily through these barriers. The net result is increased water uptake with only small changes in ~,v, an uptake that approximates the increased capability for cell enlargement brought about by auxin-enhanced wall extensibility. This work was supported by National Science Foundation Grant GB41314 and Water Resources Center Grant S-032-ILL. The authors thank Kathy Zangerl for competent technical help and Dr. L.N. Vanderhoef for advice during the course of the work. We are also indebted to Dr. Fred Molz, Dr. Fred Meins, Jr., and Dr. Colin Wraight for reading the manuscript.

References Barrs, H.D. : Comparison of water potentials in leaves as measured by two types of thermocouple psychrometer. Aust. J. Biol. Sci. 18, 36-52 (1965) Boyer, J.S.: Isopiestie technique: Measurement of accurate leaf water potentials. Science 154, 1459-1460 (1966) Boyer, J.S.: Leaf water potentials measured with a pressure chamber. Plant Physiol. 42, i33-137 (1967) Boyer, J.S.: Relationship of water potential to growth of leaves. Plant Physiol. 43, 1056-1062 (1968) Boyer, J.: Measurement of the water status of plants. Ann. Rev. Plant Physiol. 20, 351-364 (1969) Boyer, J.S.: Water transport in plants: mechanism of apparent changes in resistance during absorption. Planta 117, 187-207 (1974) Boyer, J.S., Knipling, E.B.: Isopiestic technique formeasuring leaf water potentials with a thermocouple psychrometer. Proc. Natl. Acad. Sci. USA 54, 1044-1051 (1965) Buffel, K.: New techniques for comparative permeability studies on the oat coleoptile with reference to the mechanism of auxin action, Meded. Kon. Vlaamse Acad. Wettensch. Len. Schone Kunst. Belg. 14, No. 7 (1952) Cleland, R.: Cell walI extension. Ann. Rev. Plant Physiol. 22, 197-222 (1971) Cleland, R.: Auxin-induced hydrogen ion excretion from Arena coleoptiles. Proc. Natl. Acad. Sci. USA 70, 3092-3093 (1973) Dainty, J. : Water relations of plant cells. Adv. Bot. Res. 1,279 326 (1963) Dowler, M.J., Rayle, D.L., Cande, W.Z., Ray, P.M., Durand, H., Zenk, M.H. : Auxin does not alter the permeability of pea segments to tritium-labeled water. Plant Physiol. 53, 229-232 (1974)

237 Green, P.B., Erickson, R.O., Buggy, J.: Metabolic and physical control of cell elongation rate. Plant Physiol. 47, 423 430 (1971) Green, P.B., Cnmmins, W.R.: Growth rate and turgor pressure. Auxin effect studied with an automated apparatus for singIe coleoptiles. Plant Physiol. 54, 863 869 (1974) Guttenburg, H. von, Meinl, G. : ()bet den Einflul3 des pH-Wertes und der Temperatur auf die durch Heteroauxin bedingte Verfinderungen der Wasserpermeabilittit. Planta 40, 431 442 (1952) Hardin, J.W., Cherry, J.H., Mort6, D.J., Lembi, C.A.: Enhancement of RNA polymerase activity by a factor released by auxin from plasma membrane. Proc. Natl. Acad. Sci. USA 69, 3146 3150 (1972) Hertel, R., Thomson, K., Russo, V.E.A.: In vitro auxin binding to particulate cell fractions from corn coleoptiles. Planta 107, 325 340 (1972) Holm, R.E., Key, J.L.: Hormonal regulation of cell elongation in the hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis. Plant Physiol. 44, 1295-1302 (1969) Kang, B.G., Burg, S.P.: Rapid change in water flux induced by auxins. Proc. Natl. Acad. Sci. USA 68, 1730-1733 (197I) Ketellapper, H.J. : The mechanism of the action of indole~3-acetic acid on the water absorption by Arena coleoptile sections. Acta Bot. Neerl. 2, 38%444 (1953) Lockhart, J.A.: An analysis of irreversible plant cell elongation. J. Theor. Biol. 8, 264-275 (1965) Lfittge, U., Higinbotham, N., Pallaghy, C.H. : Electrochemical evidence of specific action of indole acetic acid on membranes in Mnium leaves. Z. Naturforsch. 27b, I239 1242 (1972) Meyer, R,F., Boyer, J.S. : Sensitivity of cell division and cell elongation to low water potentials in soybean hypocotyls. Planta 108, 77-87 (1972) Ordin, L., Bonnet, J.: Permeability of Arena coleoptile sections to water measured by diffusion of deuterium hydroxide. Plant Physiol. 31, 53-57 (1956) Ray, P.M., Green, P.B., Cleland, R.: Role of turgot in plant cell growth. Nature 239, 163 164 (1972) Ray, P.M., Ruesink, A.W.: Osmotic behavior of oat coleoptile tissue in relation to growth. J. Gen. Physiol. 47, 83-101 (1963) Rayle, D.L.: Auxin-induced hydrogen-ion secretion in Arena coleoptiles and its implications. Planta 114, 63-73 (1973) Rayle, D.L., Evans, M.L., Hertel, R.: Action of auxin on cell elongation. Proc. Natl. Acad. Sci. USA 65, i84 191 (1970) Robbins, E., Mauro, A. : Experimental study of the independence of diffusion and hydrodynamic permeability coefficients in collodion membranes. J. Gen. Physiol. 43, 523 532 (1960) Ruge, U.: Untersuchungen fiber den EinfluB des Hetero-Auxins auf das Streckungswachstum des Hypokotyls von Helianthus annuus. Z. Bot. 31, 1-56 (1937) Scholander, P.F., Hammel, H.T., Bradstreet, E.D., Hemmingsen, E.A.: Sap pressure in vascular plants. Science 148, 339-346 (1965) Thimann, K.V., Samuel, E.W.: The permeability of potato tissue to water. Proc. Natl. Acad. Sci. USA 41, 1029 1033 (1955) Van der Woude, W.J., Lembi, C.A., Morr6, D.J.: Auxin (2,4-D) stimulation (in vivo and in vitro) of polysaccharide synthesis in plasma membrane fragments isolated from onion stems. Biochem. Biophys. Res. Commun. 46, 245 253 (1972) Zimmermann, U., Steudle,. E., Lelkes, P.I. : Turgor pressure regulation in Valonia utrieularis. Plant Physiol. 58, 608-613 (1976)

Received 12 September; accepted 16 December 1977