Planta
Planta (1988)174:t23-t26
9 Springer-Verlag 1988
Osmotic regulation of starch synthesis in potato tubers? Karl J. Oparka and Kathryn M. Wright Department of Physiology and Crop Production, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U K
Abstract. Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 m M but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300M00 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 raM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.
Key words: Osmoticum (starch synthesis) - SoIanum (starch synthesis) - Starch synthesis - Sucrose uptake - Tuber.
Introduction
In order to maintain the pressure gradient necessary to drive the long-distance transport of sucrose through the phloem it is essential that the sucrose imported into sink tissues is physically or chemically isolated from the site of unloading. This may be achieved by a variety of mechanisms depending on the nature of the sink in question. For example, unloading may be maintained by physical compartmentation of sucrose between apoplastic and symplastic pools, by chemical alteration of sucrose
by hydrolysis to hexoses or conversion to starch, or by direct utilisation of assimilates for growth (see review by Thorne 1985 and references therein). In the potato tuber, a vegetative starch-accumulating sink, both sucrose storage and starch synthesis occur following unloading (Mares and Marschner 1980; Oparka and Prior 1987) although the factors influencing the compartmental balance between these two processes are poorly understood. Recent work by a number of groups has shown that cell turgor may play an important role in influencing solute transport within sinks (Wolswinkel 1985; Patrick et al. 1986), and the mechanism of sucrose uptake in sinks which accumulate only sucrose has received considerable attention. In the sugar-beet taproot, sucrose is accumulated against a concentration gradient and stored in the vacuole. Recently, Wyse et al. (1986) have shown that sucrose uptake into sugar-beet taproot tissue is turgor-dependent, high cell turgors inhibiting sucrose uptake, possibly by inhibition of the plasmalemma ATPase. In a recent investigation of the potato tuber (Oparka and Prior 1987) we showed that sucrose is not hydrolysed following unloading and appears to enter storage cells directly from the phloem by a symplastic pathway. In an attempt to isolate the unloading component from the sucrose compartmentation occurring within the storage cells we have begun experiments on sucrose uptake and conversion by isolated potato tuber discs. Here we demonstrate that both the uptake and the conversion of sucrose to starch are greatly influenced by the osmotic environment of the storage cells. Material and methods Plant material. Potato tubers (Solanum tuberosum L.) cv. Record were harvested from plants growing in compost in an unheated glasshouse. The growing tubers ranged in size from 50-100 g fresh weight when used for experiments. Tubers were harvested on the day of an experiment and cut transversely
124
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis? Fig,
using a hand-held potato slicer into 3-mm-thick slices. Discs (5 m m diameter, 3 m m thick) were then cut from the central, starch-accumulating perimedulla using a cork borer.
Determination of tissue osmolarity. The osmolarity of the fresh tuber tissue was determined on approximately 30 randomly selected discs. The tissue was frozen in liquid nitrogen and centrifuged at 3000.g. The osmolarity of 0.25-ml aliquots of supernatant was determined by freezing-point depression on an Advanced DigiMatic osmometer (model 3D II; Advanced Instruments Inc., Needham Heights Mass., USA). In the experiments reported here the osmolarity ranged from 285-305 mOsmolkg -1. To prevent the fresh tissue from drying out, and to remove surface debris, the discs were washed for 30 min in 25 m M 2(N-morpholino)ethanesulfonic acid (Mes) buffer containing 300 m M mannitol (pH 6.5) before being transferred to solutions of differing osmotic concentrations. This pretreatment did not affect sucrose uptake; control discs transferred directly to the treatment solutions without washing showed identical uptake kinetics to the washed discs. After washing, the discs were blotted and their fresh weights determined. These values were subsequently used to determine total sucrose uptake.
Uptake of[14C]sucrose. The osmotic-treatment solutions were similar to those employed by Wyse et al. (1986). The basic mediu m contained 25 m M Mes buffer, pH 6.5 plus the appropriate concentration o f m a n n i t o l and sucrose, depending on the experimental treatment. In all experiments, discs were equilibrated in buffered mannitol solutions of identical molarities to the treatment solutions for 90 rain before being transferred to the appropriate treatment containing mannitol and sucrose. In order to maintain the same osmotic concentrations as the equilibration media, increased sucrose concentrations were compensated for by decreased mannitol concentrations (see also Wyse et al. 1986). In some experiments, sorbitol replaced mannitol as the external osmoticum. Prior to incubation, 9.25 k B q - m l [U-I~C] sucrose (specific activity 20.7 G B q . m m o l - ~ ; Amersham International, Amersham, Bucks., U K ) was added to all treatments. Incubations were made for 3 h in 1 ml of treatment solution contained in 20-ml plastic scintillation vials. There were 10 discs per vial and three to five replicate vials per treatment. Vials were placed on a shaker throughout the incubation period to aerate the solutions. After incubation the discs were washed for 3 x 3 rain in the sucrose-free treatment solutions to remove free-space sucrose. The discs were then extracted twice in 80% ethanol at 70~ for 6 h. Radioactivity in this ethanol-soluble fraction was determined by scintillation counting and the data converted to sucrose equivalents. This represented the amount of sucrose taken up by the tissue, although it is appreciated that after 3 h not all the [t4C]sucrose may have remained in this form. However, thin-layer chromatography of the ethanol extracts showed that over 80% of the tissue ~4C remained as sucrose. Starch was extracted from the tissue as described previously (Oparka 1985). Between 90 95% of the insoluble a4C was found to be present as starch over a range of mannitol concentrations. Consequently, in all experiments reported here the discs were combusted directly on a sample oxidiser (model 306; Packard, Caversham, Berks., U K ) immediately following extraction in ethanol, and the ~4C-insoluble fraction designated as starch. All experiments were repeated at least twice.
Results Total sucrose uptake by potato tuber discs showed biphasic kinetics, exhibiting a saturable compo-
a)
6.0
v=
2.C
b)
.4.0 t.~ y
"2 1.( ~2.0
o
~ 0.~ 215 510 75 100 Sucrose concentration [mM)
25 50 75 tO0 Sucrose concentration [mM)
Fig. 1. a Effect of mannitol concentration on total sucrose uptake by potato tuber discs. The discs were equilibrated in the appropriate unlabelled mannitol solutions for 90 min prior to uptake of [a4C]sucrose. The uptake period was 3 h; 9 9 1 0 0 m M mannitol; O O 2 0 0 m M mannitol; [] [] 300 m M mannitol. Values are means + SE. b Conversion of sucrose to starch at different mannitol concentrations. Starch is expressed as sucrose equivalents. Data are from the same experiment shown in a. Symbols as in a
nent, saturating at about 25-30 m M and a linear component predominating at higher external concentrations (Fig. 1 a). Total uptake above 30 mM remained linear up to external sucrose concentrations of 300 m M (data not shown). Raising the external mannitol concentration caused a significant increase in total sucrose uptake and also changed the slope of the linear component. The amount of sucrose converted to starch was also increased by raising the osmotic concentration (Fig. 1 b), the relationship becoming increasingly curvilinear at higher mannitol concentrations. In subsequent experiments we examined the uptake of 50 m M sucrose in buffered osmotica, extending the range of external mannitol concentrations to 500 mM, 200 m M above the measured tissue osmolarity. Conversion of sucrose to starch showed a distinct osmotic optimum at, or close to, 300 m M and was reduced on either side of this (Fig. 2 a). The data show that at mannitol concentrations greater than 300 mM the inhibition of starch synthesis was greater than at low mannitol concentrations, for the same change in concentration. The same effect was demonstrated using sorbitol as the external osmoticum (data not shown). The fraction of sucrose converted to starch, i.e. the partitioning between sucrose and starch (Fig. 2b) was influenced by mannitol concentra-
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis? 25
Fig 2
b)
centrations (0-200 mM) but were relatively constant between 300 400 mM, despite a marked decline in starch synthesis (Fig. 3). Above 400 m M a marked increase in sucrose uptake was observed which was not paralleled by an increase in starch synthesis.
09
,g ~20
08
, 07 06 05
\
b 04 ~0.3
g
02
g
Discussion
o
g_
0.1 10 1;0 2;0 3;0 4;0 540
100 200 3 0 400 500 Mannitol concentration [mM)
Mannitol concentration [mM)
Fig. 2. a Conversion of 50 m M sucrose to starch by potato tuber discs over a range of external mannitol concentrations. b Percentage incorporation of sucrose to starch over a range of external mannitol concentrations. Data derived from a
Fig, 0.5
0.4 e~
~?
.
0.3
o
o
0.2
o
0.1 ~
I
200
300 400 500 Mannitol concentration (mM)
125
600
Fig. 3. Levels of soluble material (sucrose equivalents), taken up by potato tuber discs immersed in osmoticum containing 50 mM sucrose, in relation to the amount of starch at different external mannitol concentrations. 9 9 soluble; O O starch
tion as well as the total amount of sucrose taken up. At 275 m M mannitol, 24% of the sucrose was converted to starch compared with 20% at 200 m M and 9% at 400 mM. The amount of soluble material taken up, but not converted to starch, was also influenced by the external osmoticum but did not follow the same pattern observed for starch synthesis. Levels of soluble material (expressed as sucrose equivalents) were low at low mannitol con-
Altering the osmotic environment of potato tuber discs influences not only the total uptake of sucrose into the storage cells but also the partitioning between sucrose and starch. The total uptake characteristics reported here are not the same as those reported by Wyse et al. (1986) for sugar-beet taproot tissue. These authors found that as the osmotic concentration increased the biphasic pattern of uptake became more pronounced, while the slope of the linear component did not change. In our experiments raising the mannitol concentration altered the slope of the linear component, indicating a different uptake mechanism from that observed in sugar beet. This is perhaps not surprising in view of the different nature of the storage tissues concerned. In the rapidly growing tubers used in our experiments, starch synthesis was optimised at 300 m M mannitol, a value remarkably close to the measured tissue osmolarity. Starch synthesis declined markedly at mannitol concentrations above and below this optimum. The inhibition of starch synthesis on either side of the optimal mannitol concentration was not proportional to the change in osmotic concentration and it is unlikely that the same mechanism reduced starch synthesis under both high and low osmotica. Wyse et al. (1986) have shown that in sugar-beet taproot tissue, high cell turgors may reduce sucrose uptake by inhibition of the plasmalemma ATPase and it is possible that a similar effect was operating in our experiments. The potential role of cell turgor in influencing carbohydrate partitioning in the potato tuber now requires to be examined further by direct measurement of tissue turgor under different osmotic environments. Wyse et al. (1986) did not examine the uptake characteristics of sucrose at osmotic concentrations greater than those measured in their tissue. We have shown that under supra-optimal mannitol concentrations starch synthesis is greatly reduced, although this was not accompanied by a corresponding change in the soluble fraction, at least within the range 300.400 mM. These observations indicate that sucrose uptake was not dependent on starch synthesis. The marked increase in sucrose uptake above 400 m M may have
126
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis?
been related to general membrane leakiness as plasmolysis of the tissue increased. Recently Ehret and Ho (1986) reported that altering the salinity of nutrient-film solutions influenced the partitioning between sugars and starch in growing tomato fruits, high salinity favouring partitioning to starch and low salinity favouring partitioning to hexoses. They suggested that compartmentation was influenced by the osmotic status of the fruit. It seems likely that by altering the salinity of the nutrient solution these authors produced similar osmotic effects to those reported here with excised potato discs. Ehret and Ho (1986) also suggested that the raised potassium levels characteristic of their high-salinity solutions may have stimulated starch synthesis. However, in our experiments potassium was absent from the osmotic solutions and so a direct effect of this ion on starch synthesis was eliminated. The mechanisms by which the water relations of the storage cells influenced carbohydrate partitioning are not the subject of the present paper. The aim of this communication was to report a significant effect of osmotic environment on sucrose uptake and starch synthesis by potato tubers. The ways in which sucrose compartmentation and starch synthesis are altered by both sub-optimal and supra-optimal osmotica are the subjects of further investigation in our laboratory. It now requires to be demonstrated to what extent tissue
water relations influence sucrose partitioning in different plant organs which store starch either transitorily or for long periods of time. We are indebted to D. Prior for technical assistance.
References Ehret, D.L., Ho, L.C. (1986) The effects of salinity on dry matter partitioning and fruit growth in tomatoes grown in nutrient film culture. J. Hort. Sci. 61, 361-367 Mares, D.J., Marschner, H. (1980) Assimilate conversion in potato tubers in relation to starch deposition and cell growth. Ber. Dtsch. Bot. Ges. 93, 299-313 Oparka, K.J. (1985) Changes in partitioning of current assimilate during tuber bulking in potato. Ann. Bot. 55, 705-713 Oparka, K.J., Prior, D.A.M. (]987) 1~C sucrose efflux from the perimedulla of growing potato tubers. Plant Cell Environ. 10, 667-675 Patrick, J.W., Jacobs, E., Offler, C.E., Cram, W.J. (1986) Photosynthate unloading from seed coats of Phaseolus vulgaris L. - Nature and cellular location of turgor-sensitive unloading. J. Exp. Bot. 37, 1006-1019 Thorne, J.H. (1985) Phloem unloading of C and N assimilates in developing seeds. Annu. Rev. Plant Physiol. 36, 317-343 Wolswinkel, P. (1985) Phloem unloading and turgor-sensitive transport: Factors involved in sink control of assimilate partitioning. Physiol. Plant. 65, 331-339 Wyse, R.E., Zamski, E., Tomos, A.D. (1986) Turgor regulation of sucrose transport in sugar-beet taproot tissue. Plant Physiol. 81, 478481 Received 2 September; accepted 7 October 1987
Planta (1988)174:t23-t26
9 Springer-Verlag 1988
Osmotic regulation of starch synthesis in potato tubers? Karl J. Oparka and Kathryn M. Wright Department of Physiology and Crop Production, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U K
Abstract. Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 m M but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300M00 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 raM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.
Key words: Osmoticum (starch synthesis) - SoIanum (starch synthesis) - Starch synthesis - Sucrose uptake - Tuber.
Introduction
In order to maintain the pressure gradient necessary to drive the long-distance transport of sucrose through the phloem it is essential that the sucrose imported into sink tissues is physically or chemically isolated from the site of unloading. This may be achieved by a variety of mechanisms depending on the nature of the sink in question. For example, unloading may be maintained by physical compartmentation of sucrose between apoplastic and symplastic pools, by chemical alteration of sucrose
by hydrolysis to hexoses or conversion to starch, or by direct utilisation of assimilates for growth (see review by Thorne 1985 and references therein). In the potato tuber, a vegetative starch-accumulating sink, both sucrose storage and starch synthesis occur following unloading (Mares and Marschner 1980; Oparka and Prior 1987) although the factors influencing the compartmental balance between these two processes are poorly understood. Recent work by a number of groups has shown that cell turgor may play an important role in influencing solute transport within sinks (Wolswinkel 1985; Patrick et al. 1986), and the mechanism of sucrose uptake in sinks which accumulate only sucrose has received considerable attention. In the sugar-beet taproot, sucrose is accumulated against a concentration gradient and stored in the vacuole. Recently, Wyse et al. (1986) have shown that sucrose uptake into sugar-beet taproot tissue is turgor-dependent, high cell turgors inhibiting sucrose uptake, possibly by inhibition of the plasmalemma ATPase. In a recent investigation of the potato tuber (Oparka and Prior 1987) we showed that sucrose is not hydrolysed following unloading and appears to enter storage cells directly from the phloem by a symplastic pathway. In an attempt to isolate the unloading component from the sucrose compartmentation occurring within the storage cells we have begun experiments on sucrose uptake and conversion by isolated potato tuber discs. Here we demonstrate that both the uptake and the conversion of sucrose to starch are greatly influenced by the osmotic environment of the storage cells. Material and methods Plant material. Potato tubers (Solanum tuberosum L.) cv. Record were harvested from plants growing in compost in an unheated glasshouse. The growing tubers ranged in size from 50-100 g fresh weight when used for experiments. Tubers were harvested on the day of an experiment and cut transversely
124
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis? Fig,
using a hand-held potato slicer into 3-mm-thick slices. Discs (5 m m diameter, 3 m m thick) were then cut from the central, starch-accumulating perimedulla using a cork borer.
Determination of tissue osmolarity. The osmolarity of the fresh tuber tissue was determined on approximately 30 randomly selected discs. The tissue was frozen in liquid nitrogen and centrifuged at 3000.g. The osmolarity of 0.25-ml aliquots of supernatant was determined by freezing-point depression on an Advanced DigiMatic osmometer (model 3D II; Advanced Instruments Inc., Needham Heights Mass., USA). In the experiments reported here the osmolarity ranged from 285-305 mOsmolkg -1. To prevent the fresh tissue from drying out, and to remove surface debris, the discs were washed for 30 min in 25 m M 2(N-morpholino)ethanesulfonic acid (Mes) buffer containing 300 m M mannitol (pH 6.5) before being transferred to solutions of differing osmotic concentrations. This pretreatment did not affect sucrose uptake; control discs transferred directly to the treatment solutions without washing showed identical uptake kinetics to the washed discs. After washing, the discs were blotted and their fresh weights determined. These values were subsequently used to determine total sucrose uptake.
Uptake of[14C]sucrose. The osmotic-treatment solutions were similar to those employed by Wyse et al. (1986). The basic mediu m contained 25 m M Mes buffer, pH 6.5 plus the appropriate concentration o f m a n n i t o l and sucrose, depending on the experimental treatment. In all experiments, discs were equilibrated in buffered mannitol solutions of identical molarities to the treatment solutions for 90 rain before being transferred to the appropriate treatment containing mannitol and sucrose. In order to maintain the same osmotic concentrations as the equilibration media, increased sucrose concentrations were compensated for by decreased mannitol concentrations (see also Wyse et al. 1986). In some experiments, sorbitol replaced mannitol as the external osmoticum. Prior to incubation, 9.25 k B q - m l [U-I~C] sucrose (specific activity 20.7 G B q . m m o l - ~ ; Amersham International, Amersham, Bucks., U K ) was added to all treatments. Incubations were made for 3 h in 1 ml of treatment solution contained in 20-ml plastic scintillation vials. There were 10 discs per vial and three to five replicate vials per treatment. Vials were placed on a shaker throughout the incubation period to aerate the solutions. After incubation the discs were washed for 3 x 3 rain in the sucrose-free treatment solutions to remove free-space sucrose. The discs were then extracted twice in 80% ethanol at 70~ for 6 h. Radioactivity in this ethanol-soluble fraction was determined by scintillation counting and the data converted to sucrose equivalents. This represented the amount of sucrose taken up by the tissue, although it is appreciated that after 3 h not all the [t4C]sucrose may have remained in this form. However, thin-layer chromatography of the ethanol extracts showed that over 80% of the tissue ~4C remained as sucrose. Starch was extracted from the tissue as described previously (Oparka 1985). Between 90 95% of the insoluble a4C was found to be present as starch over a range of mannitol concentrations. Consequently, in all experiments reported here the discs were combusted directly on a sample oxidiser (model 306; Packard, Caversham, Berks., U K ) immediately following extraction in ethanol, and the ~4C-insoluble fraction designated as starch. All experiments were repeated at least twice.
Results Total sucrose uptake by potato tuber discs showed biphasic kinetics, exhibiting a saturable compo-
a)
6.0
v=
2.C
b)
.4.0 t.~ y
"2 1.( ~2.0
o
~ 0.~ 215 510 75 100 Sucrose concentration [mM)
25 50 75 tO0 Sucrose concentration [mM)
Fig. 1. a Effect of mannitol concentration on total sucrose uptake by potato tuber discs. The discs were equilibrated in the appropriate unlabelled mannitol solutions for 90 min prior to uptake of [a4C]sucrose. The uptake period was 3 h; 9 9 1 0 0 m M mannitol; O O 2 0 0 m M mannitol; [] [] 300 m M mannitol. Values are means + SE. b Conversion of sucrose to starch at different mannitol concentrations. Starch is expressed as sucrose equivalents. Data are from the same experiment shown in a. Symbols as in a
nent, saturating at about 25-30 m M and a linear component predominating at higher external concentrations (Fig. 1 a). Total uptake above 30 mM remained linear up to external sucrose concentrations of 300 m M (data not shown). Raising the external mannitol concentration caused a significant increase in total sucrose uptake and also changed the slope of the linear component. The amount of sucrose converted to starch was also increased by raising the osmotic concentration (Fig. 1 b), the relationship becoming increasingly curvilinear at higher mannitol concentrations. In subsequent experiments we examined the uptake of 50 m M sucrose in buffered osmotica, extending the range of external mannitol concentrations to 500 mM, 200 m M above the measured tissue osmolarity. Conversion of sucrose to starch showed a distinct osmotic optimum at, or close to, 300 m M and was reduced on either side of this (Fig. 2 a). The data show that at mannitol concentrations greater than 300 mM the inhibition of starch synthesis was greater than at low mannitol concentrations, for the same change in concentration. The same effect was demonstrated using sorbitol as the external osmoticum (data not shown). The fraction of sucrose converted to starch, i.e. the partitioning between sucrose and starch (Fig. 2b) was influenced by mannitol concentra-
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis? 25
Fig 2
b)
centrations (0-200 mM) but were relatively constant between 300 400 mM, despite a marked decline in starch synthesis (Fig. 3). Above 400 m M a marked increase in sucrose uptake was observed which was not paralleled by an increase in starch synthesis.
09
,g ~20
08
, 07 06 05
\
b 04 ~0.3
g
02
g
Discussion
o
g_
0.1 10 1;0 2;0 3;0 4;0 540
100 200 3 0 400 500 Mannitol concentration [mM)
Mannitol concentration [mM)
Fig. 2. a Conversion of 50 m M sucrose to starch by potato tuber discs over a range of external mannitol concentrations. b Percentage incorporation of sucrose to starch over a range of external mannitol concentrations. Data derived from a
Fig, 0.5
0.4 e~
~?
.
0.3
o
o
0.2
o
0.1 ~
I
200
300 400 500 Mannitol concentration (mM)
125
600
Fig. 3. Levels of soluble material (sucrose equivalents), taken up by potato tuber discs immersed in osmoticum containing 50 mM sucrose, in relation to the amount of starch at different external mannitol concentrations. 9 9 soluble; O O starch
tion as well as the total amount of sucrose taken up. At 275 m M mannitol, 24% of the sucrose was converted to starch compared with 20% at 200 m M and 9% at 400 mM. The amount of soluble material taken up, but not converted to starch, was also influenced by the external osmoticum but did not follow the same pattern observed for starch synthesis. Levels of soluble material (expressed as sucrose equivalents) were low at low mannitol con-
Altering the osmotic environment of potato tuber discs influences not only the total uptake of sucrose into the storage cells but also the partitioning between sucrose and starch. The total uptake characteristics reported here are not the same as those reported by Wyse et al. (1986) for sugar-beet taproot tissue. These authors found that as the osmotic concentration increased the biphasic pattern of uptake became more pronounced, while the slope of the linear component did not change. In our experiments raising the mannitol concentration altered the slope of the linear component, indicating a different uptake mechanism from that observed in sugar beet. This is perhaps not surprising in view of the different nature of the storage tissues concerned. In the rapidly growing tubers used in our experiments, starch synthesis was optimised at 300 m M mannitol, a value remarkably close to the measured tissue osmolarity. Starch synthesis declined markedly at mannitol concentrations above and below this optimum. The inhibition of starch synthesis on either side of the optimal mannitol concentration was not proportional to the change in osmotic concentration and it is unlikely that the same mechanism reduced starch synthesis under both high and low osmotica. Wyse et al. (1986) have shown that in sugar-beet taproot tissue, high cell turgors may reduce sucrose uptake by inhibition of the plasmalemma ATPase and it is possible that a similar effect was operating in our experiments. The potential role of cell turgor in influencing carbohydrate partitioning in the potato tuber now requires to be examined further by direct measurement of tissue turgor under different osmotic environments. Wyse et al. (1986) did not examine the uptake characteristics of sucrose at osmotic concentrations greater than those measured in their tissue. We have shown that under supra-optimal mannitol concentrations starch synthesis is greatly reduced, although this was not accompanied by a corresponding change in the soluble fraction, at least within the range 300.400 mM. These observations indicate that sucrose uptake was not dependent on starch synthesis. The marked increase in sucrose uptake above 400 m M may have
126
K.J. Oparka and K.M. Wright: Osmotic regulation of starch synthesis?
been related to general membrane leakiness as plasmolysis of the tissue increased. Recently Ehret and Ho (1986) reported that altering the salinity of nutrient-film solutions influenced the partitioning between sugars and starch in growing tomato fruits, high salinity favouring partitioning to starch and low salinity favouring partitioning to hexoses. They suggested that compartmentation was influenced by the osmotic status of the fruit. It seems likely that by altering the salinity of the nutrient solution these authors produced similar osmotic effects to those reported here with excised potato discs. Ehret and Ho (1986) also suggested that the raised potassium levels characteristic of their high-salinity solutions may have stimulated starch synthesis. However, in our experiments potassium was absent from the osmotic solutions and so a direct effect of this ion on starch synthesis was eliminated. The mechanisms by which the water relations of the storage cells influenced carbohydrate partitioning are not the subject of the present paper. The aim of this communication was to report a significant effect of osmotic environment on sucrose uptake and starch synthesis by potato tubers. The ways in which sucrose compartmentation and starch synthesis are altered by both sub-optimal and supra-optimal osmotica are the subjects of further investigation in our laboratory. It now requires to be demonstrated to what extent tissue
water relations influence sucrose partitioning in different plant organs which store starch either transitorily or for long periods of time. We are indebted to D. Prior for technical assistance.
References Ehret, D.L., Ho, L.C. (1986) The effects of salinity on dry matter partitioning and fruit growth in tomatoes grown in nutrient film culture. J. Hort. Sci. 61, 361-367 Mares, D.J., Marschner, H. (1980) Assimilate conversion in potato tubers in relation to starch deposition and cell growth. Ber. Dtsch. Bot. Ges. 93, 299-313 Oparka, K.J. (1985) Changes in partitioning of current assimilate during tuber bulking in potato. Ann. Bot. 55, 705-713 Oparka, K.J., Prior, D.A.M. (]987) 1~C sucrose efflux from the perimedulla of growing potato tubers. Plant Cell Environ. 10, 667-675 Patrick, J.W., Jacobs, E., Offler, C.E., Cram, W.J. (1986) Photosynthate unloading from seed coats of Phaseolus vulgaris L. - Nature and cellular location of turgor-sensitive unloading. J. Exp. Bot. 37, 1006-1019 Thorne, J.H. (1985) Phloem unloading of C and N assimilates in developing seeds. Annu. Rev. Plant Physiol. 36, 317-343 Wolswinkel, P. (1985) Phloem unloading and turgor-sensitive transport: Factors involved in sink control of assimilate partitioning. Physiol. Plant. 65, 331-339 Wyse, R.E., Zamski, E., Tomos, A.D. (1986) Turgor regulation of sucrose transport in sugar-beet taproot tissue. Plant Physiol. 81, 478481 Received 2 September; accepted 7 October 1987
