Planta
Planta 145,113 117 (1979)
9 by Springer-Verlag 1979
Effects of Osmotic Stress on Polar Auxin Transport
in Arena Mesocotyl Sections
A. R. Sheldrake
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad-500 016, A.P., India
Abstract. Segments of mesocotyls of Arena sativa L.
transported [1-t4C]indol-3yl-acetic acid (IAA) with
strictly basipetal polarity. Treatment of the segments
with solutions of sorbitol caused a striking increase
in basipetal auxin transport, which was greatest at
concentrations around 0.5 M. Similar effects were
observed with mannitol or quebrachitol as osmotica,
but with glucose or sucrose the increases were smaller. Polar transport was still detectable in segments
treated with 1.2 M sorbitol. The effects of osmotic
stress on the polar transport of auxin were reversible,
but treatment with sorbital solutions more concentrated than 0.5 M reduced the subsequent ability of
mesocotyl segments to grow in response to IAA. The
increased transport of auxin in the osmotically
stressed segments could not be explained in terms
of an increased uptake from donor blocks. The velocity of transport declined with higher concentrations
of osmoticum. The reasons for the enhancement of
auxin transport by osmotic stress are not known.
Key words: Auxin transport - Arena
segments - Osmotic stress - Sorbitol.
Material and Methods
Plant Material and Osmotic Treatment
Seeds of Arena sativa L. cv. W W 16253 (Swedish Seed Assoc.
Weibullsholm, Sweden) were soaked in water in the dark for 3 h
and then germinated and grown in vermiculite in plastic boxes,
covered with aluminium foil, in total darkness at 20 ~ C. The seedlings were used for experiments when they were 4 6 d old. For
experiments involving the pretreatment of segments with osmoticure, segments about 10 m m long were excised with a razor blade
from the apical part of the mesocotyls and the basal ends were
cut obliquely, to permit their subsequent recognition. These manipulations were performed in dim green light. The segments were
then incubated in solutions of osmoticum (5 ml) in vials kept in
a rack which was rotated for 2 h at 1 rpm around an axis inclined
at 45 ~ ; control segments were similarly incubated in distilled water.
The incubation took place in darkness. Microscopic examination
(with Nomarski differential interference optics) of sections cut from
segments which had been incubated in plasmolysing solutions
(mounted in the same solutions) showed that the period of incubation was sufficient for cells throughout the segments to be plasmolysed, although plasmolysis was rather less pronounced in cells
in the middle of the segments than at the ends.
Mesocotyl
Introduction
This work was begun in an attempt to obtain evidence, through an investigation of the effects of plasmolysis, for or against the symplastic transport of
auxin, A similar approach was adopted by Cande
and Ray (1976), who found that the polar transport
of auxin was demonstrable in segments of oat coleoptiles (Arena sativa L.) plasmolysed with 0.5 and
0.7 M mannitol. In experiments with segments of oat
mesocotyls, I found that polar transport not only took
place in plasmolysed tissues, but that it was strikingly
enhanced, This unexpected phenomenon is described
below.
Auxin Transport Experiments
For transport experiments, 7-mm segments were cut from the pretreated mesocotyl segments by pressing a glass slide, on which
segments had been aligned, onto razor blades m o u n t e d 7 m m apart.
The segments (12 per test) were then arranged horizontally on
a glass slide; they rested on a thin strip of filter paper coated
with petroleum jelly. Receiver and donor agar (1%, w/v) blocks
were applied and the transport assemblies were placed in Petri
dishes lined with moist filter paper as described by Sheldrake
(1973). Transport took place in the dark at 22 ~ C. D o n o r blocks
contained [1-14C]indol-3yl-acetic acid (IAA) (Radiochemical
Centre, A m e r s h a m , U . K . ; 52 m C i / m M ) at a concentration of
3.0 g M unless otherwise stated. Donor and receiver blocks applied
to pretreated segments contained the same concentration of the
same osmoticum used in the pretreatment. During the transport
period water uptake into the segments may have occurred to a
small extent, owing to their incubation in a saturated atmosphere,
but no deplasmolysis of previously plasmolysed tissue could be
detected by microscopical examination of sections cut from segments at the end of the transport period.
0032-0935/79/0145/0113/$01.00
114
At the end of the transport period the receiver blocks and
the segments were placed in 4 ml of scintillation fluid of the following composition: 2,5-diphenyloxazole (PPO) 5 g; naphthalene
100 g; dioxane to 1 l. The quenching caused by the agar blocks
was not affected by the varying concentrations of sorbitol which
they contained; nor was the quenching by the mesocotyI segments
affected by their pretreatment in this osmoticum. The quenching
effects were in all cases small ( < 1%). All samples were counted
on a Nuclear Chicago Unilux scintillation counter for at least
10 rain and corrected for background counts (ca. 30 cpm).
Since the growth of mesocotyls is affected by exposure to red
light, the effect on auxin transport of exposing the segments to
daylight during the cutting of the 7-ram segments and the setting
up of the transport assemblies was examined. Little or no difference was found after performing these manipulations in dim green
light or daylight; they were carried out in daylight in all the experiments reported in this paper.
Ether extracts of receiver biocks were chromatographed in isopropanol-ammonia-water (10: 1: 1, v/v), pyridine-ammonia (4: 1,
v/v) and ethanol-water (7:3, v/v) on cellulose thin-layer pIates;
the procedures for the detection of IAA marker spots and the
analysis of the distribution of radioactivity in the developed chrom a t o g r a m s were as described by Sheldrake (1971). Nearly all the
radioactivity transported into receiver blocks applied to the basal
end of Arena mesocotyl segments supplied with radioactive I A A
was found at the RF of I A A in all three solvent systems. The
transport of radioactivity can therefore be equated with the transport of IAA.
Measurements of Segment Growth
The influence of exposure to osmotic solutions on the ability of
the mesocotyl segments to grow was examined by incubating 10ram segments in osmotic solutions for 2 h as described above:
the segments were then incubated in distilled water for 1.5 h before
a 4-ram segment was cut from the apical part of each 10-ram
segment and incubated (10 segments per test) in a solution of
I A A (50 gg/l) under the conditions used for the Arena mesocotyl
extension bioassay as described by Sheldrake (1971).
A . R . Sheldrake: Osmotic Stress and Polar Auxin Transport
3600
3200
U
o
"~ 2800
-~ 2 4 0 0
U
ID
="
_! 1600
C
~ 1200
C
o
800
U
400
acropetol
0 ~
0
0.4
0.8
1.2
Sorbitol ( M )
1.6
2.0
Fig. 1. Transport of I A A by mesocotyi segments from 4-d-old
Arena seedlings after pretreatment with sorbitol. Transport by pretreated segments with the same concentration of sorbitol in donor
and receiver blocks that was used for the pretreatments: 9 basipetal, zx acropetal. Transport by segments pretreated with sorbitol
but placed in water for 1.5 h before the transport period, with
distilled water in donor and receiver blocks : 9 basipetal, [] acropetal. Concentration of I A A in donor blocks: 1.5 gM. Transport
time: 3 h
Results
4.5
Effects of Sorbitol Treatments on Auxin 7?ansport
4.0
The amount of radioactivity transported by Arena
mesocotyl segments treated with different concentrations of sorbitol is shown in Figure 1. Over twice
as much auxin was transported basipetally by segments treated with 0.4 M sorbitol as by the watertreated controls. With higher sorbitol concentrations
auxin transport declined, but even With 1.2 M sorbitol
there was considerable basipetal movement. In all
cases very little auxin moved acropetally (Fig. 1).
A second set of mesocotyl segments was incubated
in sorbitol solutions at the same time and in the same
way as those used in the experiments above, but before being tested for their ability to transport auxin
they were rinsed and placed in water for 1.5 h, during
which time cells that were previously plasmolysed underwent deplasmolysis. The results (Fig. 1) showed
basipetal
2000
C
E
3.5
3.o I
2.5
3=
o 2.o
0
1.5
1.0
0.5
0
. :
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
Sorbitol (M)
Fig. 2. Growth of mesocotyl segments (4 mm) from 5-d-old Arena
seedlings. The segments were pretreated with sorbitol solution for
2 h, then placed in water for 1.5 h before incubation in the dark
for 20 h at 20 ~ C in a medium containing 50 gg/Iitre I A A
A, R. Sheldrake: Osmotic Stress and Polar Auxin Transport
that the effects of osmotic stress on basipetal auxin
transport were more or less reversible. Again there
was very little acropetal movement of auxin.
115
E
"g
1 '0 Fx'T
0.6
0.4
O.
o
0
Effects of Pretreatment with Sorbitol
on the Ability to Grow
The effects of pretreatment with sorbitol followed
by a 1.5 h period in water on the ability of mesocotyl
segments to grow in response to I A A are shown in
Figure 2. Pretreatment with sorbitol solutions more
concentrated than 0.4 M resulted in reductions in
growth, and no growth occurred after pretreatment
with 1.0 M sorbitol.
"7,.. 1
A Growth
6
B.UDtake
0
0
0
~
12000
g 8000
O
u
4 000
0
l
I
I
I
I
!
~
I
I
I
I
I
I
I
4500
4000
Effects of Other Osmotica
The experiments described above were repeated with
mannitol as the osmoticum, with similar results.
The effects of a number of other osmotica on
basipetal auxin transport were compared with sorbitol
at concentrations of 0.5 M. Quebrachitol resulted in
increases similar to those with sorbitol (100-110%
more transport than in controls). Only small increases
(20-30%) were observed with glucose and sucrose,
perhaps because these sugars were taken up and metabolized, thus reducing their osmotic effect. If auxin
transport was increased because the osmotica acted
as a carbon source, larger increases would have been
expected with glucose and sucrose than with the sugar
alcohols sorbitol, mannitol and quebrachitol. Moreover all three sugar alcohols brought about similar
increases and it would seem improbable that all three
entered the cells and were metabolized to similar extents, if they were metabolized at all. These results
therefore indicate that the increased auxin transport
brought about by sorbitol was primarily owing to
its osmotic effect.
Further Observations on the Effects
of Different Concentrations of Sorbitol
More detailed observations on basipetal auxin transport by mesocotyl segments treated with sorbitol
showed that increased auxin transport became apparent at concentrations above 0.15 M (Fig. 3 C). Slightly
less transport than in the control occurred at concentrations of 0.05 and 0.10 M sorbitol; this effect was
observed in each of four separate experiments. The
amount of radioactivity in the segments at the end
of the transport period decreased with increasing
concentrations of sorbitol, although the total counts
in segments plus receiver blocks remained more or
3500
q.~ 3 0 0 0
E 2 500
2000
0
to
1500
I000
50O
~
0
0.I
L
i
I
I
0.2 0.3 0.4 0,5 0 6 0.7 0.8 0.9 1.0
Sorbltol ( M )
Fig. 3A-C. Basipetal transport of IAA by mesocotyl segments,
from 4-d-old Arena seedlings, pretreated with sorbitol. Transport
time: 3.5 h. A Growth of segments during the transport period.
B Total radioactivity in receiver blocks and segments at the end
of the transport period: o; radioactivity in segments at the end
of the transport period: o. C Radioactivity in receivers
less constant up to sorbitol concentrations of 0.5 M
(Fig. 3B). The growth of the segments which took
place during the transport period is shown in Figure 3 A.
Microscopical examination of sections of mesocotyl segments after incubation in sorbitol solutions
showed that cortical and epidermal cells were plasmolysed in concentrations of 0.4 M sorbitol and
above, in such a way that the protoplasts had pulled
away from one or both of the end walls of the cells.
It was difficult to observe plasmolysis in cells within
the stele; in some of the cells protoplasts appeared
to have remained in contact with both end walls and
retracted from the side walls. No plasmolysis was
observed in 0.3 M sorbitol. Increased auxin transport
occurred with concentrations of sorbitol lower than
those necessary to plasmolyse the cells or to suppress
growth completely (Fig. 3).
116
A . R . Sheldrake: Osmotic Stress and Polar Auxin Transport
2400,
2800
2000
2400
2000
"7
,.-
. . ,2oo
~, 800
L.
_r
,__o
1200
400
o
0
C
o
.6M
E ~ 1600
'7 1600
.E
L3
.3M
Sorbitol in donors
800
0
I
I
I
I
0.3
0.6
0.9
1.2
Sorbitol
2
time {h)
3
4
Fig. 5. Rates of basipetal IAA transport by mesocotyl segments
400
0
l
(7 ram) from 4.5-d-old Arena seedlings. Controls (pretreated with
water) o ; segments pretreated with 0.3 M sorbitol zx; 0.6 M sorbitol e ; 0,9 M sorbitol Ez; 1.2 M sorbitol A. The slopes of the
lines and positions of intersects were calculated by the "least
squares" method. The velocities (in ram/h) calculated from these
intercepts were: control, 5.3; 0.3 M sorbitol, 4.1; 0.6 M sorbitoI,
3.5; 0.9 M sorbitol, 3.4; 1.2 M sorbitol, 3.1
(M)
Fig. 4. Basipetal transport of IAA by non-pretreated mesocotyl
segments from 5-d-old Arena seedlings. Distilled water in receiver
blocks, sorbitol in donor blocks o ; distilled water in donor blocks,
sorbitol in receiver blocks []. Transport time: 3.5 h
Sorbitol in Donor or Receiver Blocks Applied
to Non-pretreated Segments
The data in Figure 3 show that the increased transport
of auxin cannot be explained in terms of an increased
uptake of auxin from donor blocks into segments
treated with osmoticum. The relative effects of the
osmoticum on the uptake of auxin from donor blocks
and on the transport of auxin through the segments
were further investigated by applying donor or receiver blocks containing different concentrations of
sorbitol to freshly cut, non-pretreated segments. If
osmotic stress primarily affected the uptake of auxin,
sorbitol in the donor blocks should have much more
effect than in the receiver blocks. On the other hand,
if osmotic stress modified auxin transport through
the segments, the transport process should be affected
by sorbitol in both donor and receiver blocks. In
fact, a greater enhancement of basipetal auxin transport was observed with increased sorbitol concentrations in the receiver blocks than in the donor blocks
(Fig. 4).
In this experiment, there was an efflux of water
from the ends of the segments in contact with the
agar blocks containing sorbitol, because these blocks
were not in osmotic equilibrium with the segments.
The smaller effect of sorbitol in donor than in receiver
blocks may be explained at least in part by the flow
of water from the segments into the blocks, diluting
the I A A and also carrying some I A A from the segments back towards the donors.
In the other experiments described in this paper, the
segments and agar blocks should have been in osmotic
equilibrium, and no such flow of fluid was observed.
It could perhaps be argued that nevertheless there
may have been slight osmotic differences between segments and agar blocks resulting in a small flow of
water into receiver blocks and thus to an increased
movement of I A A into receivers. However even if
this took place to some extent, it could not adequately
explain the increases in auxin transport in osmotically
stressed segments, for two reasons. Firstly these increases were at least as large (Figs. 1, 3) as those
which occurred when sorbitol was present only in the
receiver blocks (Fig. 4); the latter increases should
have been much larger if water movement into receiver blocks were of primary importance. Secondly,
auxin transport was enhanced when sorbitol was present only in donor blocks (Fig. 4).
Effects of Osmotic Stress on the Velocity
of Auxin Transport
The velocity of basipetal auxin transport, measured
by the classical method of van der Weij (1932), declined as the concentrations of osmoticum was raised
(Fig. 5). The increased transport of auxin into the
receiver blocks by osmotically stressed segments indicates that the overall capacity of the transport system
was increased, either because more auxin which had
already been taken up into the tissue entered the
transport stream, and/or because the efflux of auxin
from the segments into the receivers was enhanced.
Discussion
An enhancement of auxin transport by osmotic stress
has been observed with segments of Phaseolus petioles
A. R. Sheldrake: Osmotic Stress and Polar Auxin Transport
s u p p l i e d with d o n o r a n d receiver blocks c o n t a i n i n g
0.2 M m a n n i t o l ( M c C r e a d y and Jacobs, 1967) a n d
with segments of Nicotiana stem tissue with d o n o r
b l o c k s c o n t a i n i n g 0.6 M s o r b i t o l (Sheldrake, 1973).
By c o n t r a s t , t r e a t m e n t o f Arena coleoptile segments
with 0.5 M m a n n i t o l resulted in a slight r e d u c t i o n
in the a m o u n t of auxin t r a n s p o r t e d ( C a n d e a n d R a y ,
1976). These o b s e r v a t i o n s indicate t h a t o s m o t i c
stress m a y n o t result in increased a u x i n t r a n s p o r t
in all tissues or at least that the c o n c e n t r a t i o n o f
o s m o t i c u m resulting in e n h a n c e d auxin t r a n s p o r t m a y
vary f r o m tissue to tissue.
The r e d u c e d ability o f Arena m e s o c o t y l segments
to grow in r e s p o n s e to auxin after they h a d been
e x p o s e d to s o r b i t o l c o n c e n t r a t i o n s o f 0.5 M or m o r e
(Fig. 2) m a y have been a result of d a m a g e to the
tissue, b u t such d a m a g e c o u l d n o t have been severe
since the ability to t r a n s p o r t a u x i n was little affected
(Fig. 1). In a given tissue the c o n c e n t r a t i o n of o s m o t i c u m r e q u i r e d to increase p o l a r auxin t r a n s p o r t w o u l d
p r e s u m a b l y d e p e n d on the sensitivity o f the tissue
to d a m a g e by o s m o t i c stress a n d i n d e e d in very sensitive tissues it m a y n o t be possible to observe any
increase at all.
T w o negative c o n c l u s i o n s are i n d i c a t e d by the results d e s c r i b e d above. Firstly, the increased auxin
t r a n s p o r t which o c c u r r e d with s o r b i t o l c o n c e n t r a t i o n s
a b o v e 0.3 M shows t h a t the effect c a n n o t simply be
e x p l a i n e d in terms o f a s u p p r e s s i o n of g r o w t h by
the segments d u r i n g the t r a n s p o r t p e r i o d (Fig. 3). Secondly, t h e effect does n o t seem to d e p e n d on the
s e p a r a t i o n o f the p l a s m a l e m m a of the p r o t o p l a s t s
f r o m the cell walls, since it was o b s e r v e d b o t h in
segments which were n o t p l a s m o l y s e d (in 0.20-0.25 M
s o r b i t o l : Fig. 3) a n d also with higher c o n c e n t r a t i o n s
o f s o r b i t o l (0.8 M : Fig. 1) which were sufficient to
p l a s m o l y s e m o s t if n o t all of the cells.
One possible r e a s o n for the e n h a n c e m e n t of auxin
t r a n s p o r t by o s m o t i c a l l y stressed segments m a y be
t h a t o s m o t i c stress s o m e h o w reduces the i m m o b i l i z a tion or m e t a b o l i s m o f auxin within the cells, leaving
m o r e auxin a v a i l a b l e for t r a n s p o r t .
Two further possible types o f e x p l a n a t i o n are
suggested by a recent h y p o t h e s i s of auxin t r a n s p o r t
( R u b e r y a n d S h e l d r a k e , 1974; G o l d s m i t h , 1977), acc o r d i n g to which there is a p H - d e p e n d e n t u p t a k e of
a u x i n into the cells resulting in an a c c u m u l a t i o n of
auxin a n i o n s within the c y t o p l a s m , a n d a carrierm e d i a t e d efflux o f these a n i o n s d o w n an e l e c t r o c h e m ical gradient. It is s u p p o s e d t h a t the carriers m e d i a t i n g
this efflux are p r e f e r e n t i a l l y localized at the b a s a l ends
o f the cells a n d t h a t auxin moves f r o m cell to cell
by diffusion t h r o u g h the e x t r a c e l l u l a r fluid. The rates
117
o f u p t a k e a n d efflux o f a u x i n are affected b y the
e x t r a c e l l u l a r p H a n d the i n t r a c e l l u l a r a n d extracellular c o n c e n t r a t i o n s o f a u x i n ( R u b e r y a n d Sheldrake,
1973, 1974). O s m o t i c stress results in an increase in
the v o l u m e of the e x t r a c e l l u l a r space a n d a decrease
in the v o l u m e o f the cells ( W a l t e r a n d K r e e b , 1970).
These changes in relative v o l u m e c o u l d influence the
rates o f auxin u p t a k e , m o v e m e n t a n d efflux f r o m
the cells d u r i n g the t r a n s p o r t process a n d thus influence the a m o u n t o f auxin t r a n s p o r t e d . H o w e v e r ,
t o o little is k n o w n a b o u t the kinetics o f these
processes to p r e d i c t w h a t these effects w o u l d be.
A l t e r n a t i v e l y , or a d d i t i o n a l l y , o s m o t i c stress c o u l d
influence auxin t r a n s p o r t t h r o u g h an effect on ext r a c e l l u l a r pH. In Avena coleptiles, o s m o t i c stress is
k n o w n to affect the e x c r e t i o n o f H + ions f r o m the
cells into the e x t r a c e l l u l a r fluid (Cleland, 1975).
O n l y f u r t h e r research will be able to d i s t i n g u i s h
b e t w e e n these, a n d other, possibilities: the p h e n o m ena d e s c r i b e d a b o v e m a y then be able to shed s o m e
light on the m e c h a n i s m of auxin t r a n s p o r t . A t present,
however, the e n h a n c e m e n t o f auxin t r a n s p o r t by osm o t i c stress can only be r e g a r d e d as u n e x p l a i n e d .
This work was carried Out in the Department of Biochemistry
of the University of Cambridge during the tenure of the Royal
Society Rosenheim Research Fellowship 9 I thank Professor Sir
Frank Young, F.R.S., for making research facilities available to
me.
References
Cande, W. Z., Ray, P. M. : Nature of cell-to-cell transfer of auxin
in polar transport. Planta 129, 43-52 (1976)
Cleland, R. E. : Auxin-induced hydrogen ion excretion: correlation
with growth, and control by external pH and water stress 9
Planta 127, 233-242 (1975)
Goldsmith, M. H. M.: The polar transport of auxin 9Ann. Rev.
Plant Physiol. 28, 439-478 (1977)
McCready, C. C., Jacobs, W. P. : Movement of growth regulators
in plants. V. A further note on the relationship between polar
transport and growth. New Phytol. 66, 485488 (1967)
Rubery, P., Sheldrake, A. R.: Effect of pH and surface charge
on cell uptake of auxin. Nature New Biol. 244, 285-288 (1973)
Rubery, P., Sheldrake, A. R.: Carrier-mediated auxin transport 9
Planta118, 101 121 (1974)
Sheldrake, A. R.: The occurrence and significance of auxin in
the substrata of bryophytes. New Phytol. 70, 519 526 (1972)
Sheldrake, A.R.: Auxin transport in secondary tissues. J. Exp.
Bot. 24, 87 96 (1973)
van der Weij, H. G. : Der Mechanismus des Wuchsstofftransportes.
Rec. Tray. Bot. N6erl. 29, 379496 (1932)
Walter, H., Kreeb, K. : Die Hydratation and Hydratur des Protoplasmas der Pflanzen und ihre 6ko-physiologische Bedeutung.
Protoplasmatologia 11 C 6, 59-67 (1970)
Received 16 January; accepted 20 June 1978