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Effect of absorbable and non-absorbable solutes on the elongation of barley coleoptiles
Department of Soil Science and Agricultural Engineering, University of California, Riverside, California 92502, USA
Effect of Absorbable and Non-Absorbable Solutes on the Elongation of Barley Coleoptiles
J. J. OERTLI With 8 figures Received October 15, 1974
Summary The effect of combinations of mannitol and KCI on the elongation of barley coleoptile sections was investigated. During initial phases KCl reduced the lengths of coleoptile sections but after a few hours, a stimulatory effect became progressively more evident and an optimum KCI concentration at which elongation was at a maximum was clearly indicated. In time, the position of the optimum was shifted toward higher KCl concentrations. Mannitol depressed the elongation rate at all times and it caused a shift of the optimum KCl concentration toward higher values. This response of elongating colcoptiles to added osmotica is caused by a dual effect of absorbable solutes: (1) the external water potential is reduced and through it the turgor pressure and (2) after absorption of the solutes the vacuolar osmotic component of the water potential is reduced and consequently the turgor pressure is increased. During initial phases, only the depressing effect of an osmoticum is possible but as the consequences of solute uptake become effective, the stimulation becomes noticeable. For elongating tissues, continuous solute uptake is necessary to maintain favorable cellular water relations. The observed optimum is due to the hyperbolic nature of the KCI absorption isotherm. Since little mannitol enters the cells, only a depressing effect by this osmoticum is observed. The elongation of coleoptiles could reasonably be predicted from a model of cell elongation in which solute uptake was partly rate limiting. Solute uptake is essential for maintaining a small water potential gradient favorable for water uptake which itself is necessary to maintain turgor pressure and cause elongation.
Introduction In a previous communication (OERTLI, 1975) it was shown that external solutes exert a dual effect on cell elongation in barley coleoptiles. One effect was that solutes reduced the water potential, and turgor pressure was maintained only if the internal solute concentration was balanced by an adequate solute uptake. In contrast to mature cells, growing tissue had a continuous demand for solutes. This solute requirement, necessary to maintain turgor pressure, increased linearly with external moisture stress. The second effect was on the rate of solute uptake, a measure for the rate of
z.
Pflanzenphysiol. Bd. 75. S. 287-295. 1975.
288
J. J.
OERTLI
fulfillment of the solute requirement, it also increased with the external concentration. However, this latter relation was hyperbolic and the uptake tended toward saturation at high substrate levels. It was then shown theoretically and experimentally that growth rates resulting from a linear solute requirement and a hyperbolic pattern of fulfillment must have a maximum at an optimum concentration. We now report elongation studies with barley coleoptiles in the presence of external solutes part of which are taken up and part of which are essentially non-absorbable. Non-absorbable solute produce an adequate simulation of nonosmotic moisture stress, because in either case, the solute requirement for turgor maintenance is increased but the fulfillment can be performed only by absorbable solutes. These studies are, thus, a more adequate simulation of natural conditions of moisture stress. Methods and Materials Preparation of the plant material and the experimental procedure have been described previously (OERTLI, 1975). Coleoptile sections, 6.2 mm in length, were taken from 3-cm-long barley seedlings and exposed for various periods to continuously shaken solutions containing 0.1 Ofo sucrose, 1 ppm IAA (indoleacetic acid), 0.1 mM potassium phosphate buffer (pH 7), and various levels of Kcl and mannitol. Elongation was obtained as the difference between the initial and final lengths. The osmotic water potential component of expressed sap was determined with a Hewlett-Packard vapor pressure osmometer, and the pressure potential component was calculated as the difference to the total water potential.
Results and Discussion
The average final lengths of barley coleoptiles after 19-hour exposures to treatment solutions is progressively reduced by increasing levels of the non-absorbable osmoticum mannitol (Fig. 1). The addition of KCI has a stimulatory effect and for each level of mannitol an optimum concentration of KCl is clearly indicated. The position of the optimum is shifted to higher KCl concentrations by increasing mannitol levels. The growth depression by mannitol is, of course, not unexpected (MARRE et aI., 1973) and even a stimulatory effect of KCI and NaCI on the elongation of Avena coleoptiles in presence of mannitol has been reported (ORDIN et aI., 1956). In terms of our model (OERTLI, 1975), the solute requirement for maintenance of turgor pressure is increased by mannitol without changing the rate of fulfillment; hence, growth must be reduced. KCI, on the other hand, increased both the solute requirement and the rate of fulfillment by solute uptake. Because of the hyperbolic nature of the solute absorption isotherm, an optimum KCI concentration at which growth is maximum is observed. Figs. 2 to 6 present the time curves of coleoptile lengths for various mannitol and KCI treatments. Optimum KCl concentrations and their shift to higher levels with increasing stress caused by mannitol are again apparent. In addition, these data clearly indicate a shift of the optimum with time toward higher KCl concentrations.
z.
Pjlanzenphysiol. Ed. 75. S. 287-295. 1975.
Effect of Solutes on the Elongation of Barley Coleoptiles
289
120 , - - - - - - - - - - - - - - - - - - - ,
110
MANNITOL mM 100
o
E E I f-
co
90
z
w -'
80
70
o
o 60~L---~---~--~---~~
o
20
40
60
80
mMKCL
Fig. 1: Effect of mannitol and KCl levels on the final length of barley coleoptiles. Initial length 6.2 mm, 19-hour experiment.
During the initial phases - within the first two hours (Fig. 6) - coleoptile lengths are greatest in the absence of KCl and are depressed by increasing levels of KCl. Subsequently, a stimulatory effect of KCl becomes progressively more apparent. In the absence of KCl, little growth was observed at mannitol concentrations of 200 mM and higher and addition of the absorbable solute KCl usually caused a considerable stimulation of coleoptile elongation. This shift in optimum with time is again explicable through the dual role of external KCl. One effect which is fully noticeable immediately is the reduction of water potential and consequently of turgor pressure of cells; hence, the initial shrinking which was more pronounced with increasing KCl. As time proceeds, the cellular osmotic pressure increases because of solute uptake and the turgor pressure is restored. Thereafter, continuous solute uptake assures maintenance of turgor pressure during elongation.
z.
P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
290
]. ]. OERTLl
9
30 50 10 20
100 mM MANNITOL KCL mM
8
0
0
0
70
0
•
E E
•
I l-
0
•
t!)
z W
7
...J
6
o
15
5
20
HOURS
Fig. 2: Effect of various KCI levels on the elongation of barley coleoptiles in presence of 0.1 M mannitol.
9
200 mM MANNITOL 8
10
0
•
E E
30 20 70 50
0
0
20
10
I
I-
~7 w
0
...J
6
o
5
10
15
20
25
HOURS
Fig. 3: Effect of various KCI levels on the elongation of barley coleoptiles in presence of 0.2 M mannitol. Z. Pjlanzenphysiol. Bd. 75. S. 287-295. 1975.
292
]. ].
OERTLI
AVERAGES
620
610
E E
600 I
I-
'" Z W ....J
590
580
o
20
40
60 80 MINUTES
100
120
Fig. 6: Details of the first two hours of Fig. 4.
Which effects of mannitol on cell elongation can now be predicted from our earlier model of cell elongation in which solute uptake played - at least partly - a rate-limiting role? A mathematical analysis of this model has to include solute requirements for cell elongation due to absorbable and non-absorbable external solutes, whereas the rate of fullfillment is given by the uptake of absorbable solutes. Thus, if the effect of non-absorbable solutes on the external water potential is expressed by 6. P (this term can also include non-osmotic water stress) one obtains by an analogous analysis as before (OERTLI, 1975). Relative elongation rate: G
=
dV Vdt
Optimum water potential due
to
KCl:
.
.
P"o
Optimum external concentration: Copt =
=
-
{
C
e
tI 1/2
1J- Kmoce(Ll P - P p) It 1/2
Maximum elongation at optimum concentration: G ma " Z. P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
Kmo (.1P - Pr,)
Effect of Solutes on the Elongation of Barley Coleoptiles where V t
293
Volume,
=
time,
=
Vmax Kmo
=
lJl~u = Co =
maximum solute uptake rate in Michaelis-Menten kinetics,
=
"Michaelis-Menten" constant of absorb abies solutes, osmotic component of absorbable solutes of water potential, and
proportionally constant between osmolality and osmotic potential component.
With increasing mannitol concentrations, the term"'" P becomes progressively more negative. Consequently, the growth rate G is reduced, the optimum concentration of KCI is increased and the maximum elongation rate at the optimum concentration is decreased. All these qualitative statements are in agreement with the observations (Figs. 1 to 5). Results of a quantitative analysis are shown in Fig. 8 for which final coleoptile lengths have been calculated with help of the above equation for the elongation rate G. V max was taken as 5560 mosmols/kg/19 hours and Kmo as 29.8 mosmols/l (OERTLI, 1975). The final turgor pressure was assumed 4.5 bars in the absence of mannitol (OER TLI, 1975) and 4 bars in the presence of mannitol (Fig. 7). The initial length of coleoptiles (6.2 mm) was also corrected for a change in turgor pressure from the original 6 bars to 4.5 and 4 bars. respectively. The similarity between Figs. 1 and 8 is evident. The agreement between these two figures could have been improved in several ways. For example, the variability of 18
OP
16
14 12
fJ)
10
a:
lD
8 6
TP
4 2 0
01
03
MANNITOL
04
05
M
Fig. 7: Effect of mannitol in presence and absence of 10 mM KCI on osmotic pressure (OP) and turgor pressure (TP) of barley coleoptiles. Z. P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
294
J. J.
OERTLI
14
x
13 MANNITOL mM
12
o
/I
x______________
10 I
J--
100
9
(})
z
w 8
...J
200
E 7 E
300
6
/".- - - - - - -x- - - - _
400
5
4 0
40
80
120
mMKCI
Fig. 8: Elongation of barley coleoptile sections as a function of KCl supply for different levels of mannitol. Calculated from the solute absorption isotherm and from turgor pressure changes for an assumed experimental period of 19 hours. turgor pressure with treatment could have been measured and taken into account or, secondly, a correction should have been made for solutes in the free space of coleoptiles when the osmolality of the expressed sap was measured (Fig. 7) or, thirdly, an effect of high mannitol levels on the absorption isotherm of KCl should have been taken into consideration (GREENWAY et aI., 1968; SMITH et aI., 1973). Other processes that may have contributed to the difference between Figs. 1 and 8 are a possible slight permeability of membranes to mannitol and changes in vacuolar osmolality due to e. g. carbohydrate metabolism. Even without these refinements the qualitative and quantitative agreement between observed and calculated elongation rates is sufficient to support the hypothesis that solute transport necessary for maintenance of turgor pressure can have a rate-limiting effect on cell elongation. One might expect that the addition of the osmoticum KCI to mannitol solutions further depresses cell elongation. Instead, as a net effect, elongation is stimulated because through solute uptake, favorable
z.
Pflanzenphysiol. Bd. 75. S. 287-295. 1975.
Effect of Solutes on the Elongation of Barley Coleoptiles
295
internal osmotic water relations are created and in particular a decrease in turgor pressure due to growth is prevented. References GREENWAY, H., B. KLEPPER, and P. G . HUGHES: Planta 80, 129 (1968). MARRE, E., P. LADO, F. R. CALDAGNO, and R. COLOMBO: Plant Science Letters 1, 179 (1973). OERTLl, ]. ].: Z. Pflanzenphysiol. 74, 440 (1975). ORDIN, L., T. H. ApPLEWHITE, and J. BONNER: Plant Physiol. 31, 44 (1956). SMITH, R. c., B. H. ST. JOHN, and R. PARRONDO: Amer. J. Bot. 60, 839 (1973). Dr. J. J. OERTLI, Department of Soil Science and Agricultural Engineering, University of California, Riverside, California 92502, USA.
Z. PJlanzenphysiol. Bd. 75 . S. 287-295. 1975.
Effect of Absorbable and Non-Absorbable Solutes on the Elongation of Barley Coleoptiles
J. J. OERTLI With 8 figures Received October 15, 1974
Summary The effect of combinations of mannitol and KCI on the elongation of barley coleoptile sections was investigated. During initial phases KCl reduced the lengths of coleoptile sections but after a few hours, a stimulatory effect became progressively more evident and an optimum KCI concentration at which elongation was at a maximum was clearly indicated. In time, the position of the optimum was shifted toward higher KCl concentrations. Mannitol depressed the elongation rate at all times and it caused a shift of the optimum KCl concentration toward higher values. This response of elongating colcoptiles to added osmotica is caused by a dual effect of absorbable solutes: (1) the external water potential is reduced and through it the turgor pressure and (2) after absorption of the solutes the vacuolar osmotic component of the water potential is reduced and consequently the turgor pressure is increased. During initial phases, only the depressing effect of an osmoticum is possible but as the consequences of solute uptake become effective, the stimulation becomes noticeable. For elongating tissues, continuous solute uptake is necessary to maintain favorable cellular water relations. The observed optimum is due to the hyperbolic nature of the KCI absorption isotherm. Since little mannitol enters the cells, only a depressing effect by this osmoticum is observed. The elongation of coleoptiles could reasonably be predicted from a model of cell elongation in which solute uptake was partly rate limiting. Solute uptake is essential for maintaining a small water potential gradient favorable for water uptake which itself is necessary to maintain turgor pressure and cause elongation.
Introduction In a previous communication (OERTLI, 1975) it was shown that external solutes exert a dual effect on cell elongation in barley coleoptiles. One effect was that solutes reduced the water potential, and turgor pressure was maintained only if the internal solute concentration was balanced by an adequate solute uptake. In contrast to mature cells, growing tissue had a continuous demand for solutes. This solute requirement, necessary to maintain turgor pressure, increased linearly with external moisture stress. The second effect was on the rate of solute uptake, a measure for the rate of
z.
Pflanzenphysiol. Bd. 75. S. 287-295. 1975.
288
J. J.
OERTLI
fulfillment of the solute requirement, it also increased with the external concentration. However, this latter relation was hyperbolic and the uptake tended toward saturation at high substrate levels. It was then shown theoretically and experimentally that growth rates resulting from a linear solute requirement and a hyperbolic pattern of fulfillment must have a maximum at an optimum concentration. We now report elongation studies with barley coleoptiles in the presence of external solutes part of which are taken up and part of which are essentially non-absorbable. Non-absorbable solute produce an adequate simulation of nonosmotic moisture stress, because in either case, the solute requirement for turgor maintenance is increased but the fulfillment can be performed only by absorbable solutes. These studies are, thus, a more adequate simulation of natural conditions of moisture stress. Methods and Materials Preparation of the plant material and the experimental procedure have been described previously (OERTLI, 1975). Coleoptile sections, 6.2 mm in length, were taken from 3-cm-long barley seedlings and exposed for various periods to continuously shaken solutions containing 0.1 Ofo sucrose, 1 ppm IAA (indoleacetic acid), 0.1 mM potassium phosphate buffer (pH 7), and various levels of Kcl and mannitol. Elongation was obtained as the difference between the initial and final lengths. The osmotic water potential component of expressed sap was determined with a Hewlett-Packard vapor pressure osmometer, and the pressure potential component was calculated as the difference to the total water potential.
Results and Discussion
The average final lengths of barley coleoptiles after 19-hour exposures to treatment solutions is progressively reduced by increasing levels of the non-absorbable osmoticum mannitol (Fig. 1). The addition of KCI has a stimulatory effect and for each level of mannitol an optimum concentration of KCl is clearly indicated. The position of the optimum is shifted to higher KCl concentrations by increasing mannitol levels. The growth depression by mannitol is, of course, not unexpected (MARRE et aI., 1973) and even a stimulatory effect of KCI and NaCI on the elongation of Avena coleoptiles in presence of mannitol has been reported (ORDIN et aI., 1956). In terms of our model (OERTLI, 1975), the solute requirement for maintenance of turgor pressure is increased by mannitol without changing the rate of fulfillment; hence, growth must be reduced. KCI, on the other hand, increased both the solute requirement and the rate of fulfillment by solute uptake. Because of the hyperbolic nature of the solute absorption isotherm, an optimum KCI concentration at which growth is maximum is observed. Figs. 2 to 6 present the time curves of coleoptile lengths for various mannitol and KCI treatments. Optimum KCl concentrations and their shift to higher levels with increasing stress caused by mannitol are again apparent. In addition, these data clearly indicate a shift of the optimum with time toward higher KCl concentrations.
z.
Pjlanzenphysiol. Ed. 75. S. 287-295. 1975.
Effect of Solutes on the Elongation of Barley Coleoptiles
289
120 , - - - - - - - - - - - - - - - - - - - ,
110
MANNITOL mM 100
o
E E I f-
co
90
z
w -'
80
70
o
o 60~L---~---~--~---~~
o
20
40
60
80
mMKCL
Fig. 1: Effect of mannitol and KCl levels on the final length of barley coleoptiles. Initial length 6.2 mm, 19-hour experiment.
During the initial phases - within the first two hours (Fig. 6) - coleoptile lengths are greatest in the absence of KCl and are depressed by increasing levels of KCl. Subsequently, a stimulatory effect of KCl becomes progressively more apparent. In the absence of KCl, little growth was observed at mannitol concentrations of 200 mM and higher and addition of the absorbable solute KCl usually caused a considerable stimulation of coleoptile elongation. This shift in optimum with time is again explicable through the dual role of external KCl. One effect which is fully noticeable immediately is the reduction of water potential and consequently of turgor pressure of cells; hence, the initial shrinking which was more pronounced with increasing KCl. As time proceeds, the cellular osmotic pressure increases because of solute uptake and the turgor pressure is restored. Thereafter, continuous solute uptake assures maintenance of turgor pressure during elongation.
z.
P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
290
]. ]. OERTLl
9
30 50 10 20
100 mM MANNITOL KCL mM
8
0
0
0
70
0
•
E E
•
I l-
0
•
t!)
z W
7
...J
6
o
15
5
20
HOURS
Fig. 2: Effect of various KCI levels on the elongation of barley coleoptiles in presence of 0.1 M mannitol.
9
200 mM MANNITOL 8
10
0
•
E E
30 20 70 50
0
0
20
10
I
I-
~7 w
0
...J
6
o
5
10
15
20
25
HOURS
Fig. 3: Effect of various KCI levels on the elongation of barley coleoptiles in presence of 0.2 M mannitol. Z. Pjlanzenphysiol. Bd. 75. S. 287-295. 1975.
292
]. ].
OERTLI
AVERAGES
620
610
E E
600 I
I-
'" Z W ....J
590
580
o
20
40
60 80 MINUTES
100
120
Fig. 6: Details of the first two hours of Fig. 4.
Which effects of mannitol on cell elongation can now be predicted from our earlier model of cell elongation in which solute uptake played - at least partly - a rate-limiting role? A mathematical analysis of this model has to include solute requirements for cell elongation due to absorbable and non-absorbable external solutes, whereas the rate of fullfillment is given by the uptake of absorbable solutes. Thus, if the effect of non-absorbable solutes on the external water potential is expressed by 6. P (this term can also include non-osmotic water stress) one obtains by an analogous analysis as before (OERTLI, 1975). Relative elongation rate: G
=
dV Vdt
Optimum water potential due
to
KCl:
.
.
P"o
Optimum external concentration: Copt =
=
-
{
C
e
tI 1/2
1J- Kmoce(Ll P - P p) It 1/2
Maximum elongation at optimum concentration: G ma " Z. P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
Kmo (.1P - Pr,)
Effect of Solutes on the Elongation of Barley Coleoptiles where V t
293
Volume,
=
time,
=
Vmax Kmo
=
lJl~u = Co =
maximum solute uptake rate in Michaelis-Menten kinetics,
=
"Michaelis-Menten" constant of absorb abies solutes, osmotic component of absorbable solutes of water potential, and
proportionally constant between osmolality and osmotic potential component.
With increasing mannitol concentrations, the term"'" P becomes progressively more negative. Consequently, the growth rate G is reduced, the optimum concentration of KCI is increased and the maximum elongation rate at the optimum concentration is decreased. All these qualitative statements are in agreement with the observations (Figs. 1 to 5). Results of a quantitative analysis are shown in Fig. 8 for which final coleoptile lengths have been calculated with help of the above equation for the elongation rate G. V max was taken as 5560 mosmols/kg/19 hours and Kmo as 29.8 mosmols/l (OERTLI, 1975). The final turgor pressure was assumed 4.5 bars in the absence of mannitol (OER TLI, 1975) and 4 bars in the presence of mannitol (Fig. 7). The initial length of coleoptiles (6.2 mm) was also corrected for a change in turgor pressure from the original 6 bars to 4.5 and 4 bars. respectively. The similarity between Figs. 1 and 8 is evident. The agreement between these two figures could have been improved in several ways. For example, the variability of 18
OP
16
14 12
fJ)
10
a:
lD
8 6
TP
4 2 0
01
03
MANNITOL
04
05
M
Fig. 7: Effect of mannitol in presence and absence of 10 mM KCI on osmotic pressure (OP) and turgor pressure (TP) of barley coleoptiles. Z. P/lanzenphysiol. Bd. 75. S. 287-295. 1975.
294
J. J.
OERTLI
14
x
13 MANNITOL mM
12
o
/I
x______________
10 I
J--
100
9
(})
z
w 8
...J
200
E 7 E
300
6
/".- - - - - - -x- - - - _
400
5
4 0
40
80
120
mMKCI
Fig. 8: Elongation of barley coleoptile sections as a function of KCl supply for different levels of mannitol. Calculated from the solute absorption isotherm and from turgor pressure changes for an assumed experimental period of 19 hours. turgor pressure with treatment could have been measured and taken into account or, secondly, a correction should have been made for solutes in the free space of coleoptiles when the osmolality of the expressed sap was measured (Fig. 7) or, thirdly, an effect of high mannitol levels on the absorption isotherm of KCl should have been taken into consideration (GREENWAY et aI., 1968; SMITH et aI., 1973). Other processes that may have contributed to the difference between Figs. 1 and 8 are a possible slight permeability of membranes to mannitol and changes in vacuolar osmolality due to e. g. carbohydrate metabolism. Even without these refinements the qualitative and quantitative agreement between observed and calculated elongation rates is sufficient to support the hypothesis that solute transport necessary for maintenance of turgor pressure can have a rate-limiting effect on cell elongation. One might expect that the addition of the osmoticum KCI to mannitol solutions further depresses cell elongation. Instead, as a net effect, elongation is stimulated because through solute uptake, favorable
z.
Pflanzenphysiol. Bd. 75. S. 287-295. 1975.
Effect of Solutes on the Elongation of Barley Coleoptiles
295
internal osmotic water relations are created and in particular a decrease in turgor pressure due to growth is prevented. References GREENWAY, H., B. KLEPPER, and P. G . HUGHES: Planta 80, 129 (1968). MARRE, E., P. LADO, F. R. CALDAGNO, and R. COLOMBO: Plant Science Letters 1, 179 (1973). OERTLl, ]. ].: Z. Pflanzenphysiol. 74, 440 (1975). ORDIN, L., T. H. ApPLEWHITE, and J. BONNER: Plant Physiol. 31, 44 (1956). SMITH, R. c., B. H. ST. JOHN, and R. PARRONDO: Amer. J. Bot. 60, 839 (1973). Dr. J. J. OERTLI, Department of Soil Science and Agricultural Engineering, University of California, Riverside, California 92502, USA.
Z. PJlanzenphysiol. Bd. 75 . S. 287-295. 1975.