Planta
Planta 138, 229 235 (1978)
9 by Springer-Verlag 1978
Sucrose and Proton Cotransport in Ricinus Cotyledons I. H + Influx Associated with Sucrose Uptake
Vanessa M. Hutchings Department of Botany, University of Cambridge, Cambridge CB2 1QW, U.K.
Abstracts. Ricinus cotyledons were used to investigate whether sucrose uptake by the phloem could be due to H+-sucrose cotransport. The addition of sucrose to the medium bathing Ricinus cotyledons created a pH shift to more alkaline values. The effect was not observed on the addition of glucose or fructose. Net H + influx and sucrose influx were both shown to be temperature sensitive, dependent on pH and external sucrose concentration. However, net H + influx was far more sensitive to pH than sucrose influx and was abolished at high pH; it was also observed for a shorter time period than sucrose uptake. The stoichiometry of H + :sucrose was less than one and declined with increasing sucrose concentration and increasing pH. Two possible models are proposed to account for this low and changing stoichiometry. Given an H + pump, recirculation would seem an inevitable consequence of sucrose and proton cotransport. Key words: Phloem Proton cotransport - Ricinus - Sucrose cotransport.
Introduction
There is good evidence for the widespread occurrence of active proton transport in non-animal cells. It has been suggested that the function of the H + pump at the plant cell plasmalemma is to regulate cytoplasmic pH and provide energy for secondary active transport of solutes (reviews by MacRobbie, 1975; Raven and Smith, 1976a; Smith and Raven, 1976). An electrogenic H § pump will provide a proton electrochemical potential gradient as an energy source Present address: V.M. Hutchings, Department of Biochemistry,
University of Cambridge, Tennis Court Road, Cambridge, CB2 IQW, U.K.
for secondary active solute transport. A carrier cotransporting H + and a neutral solute, such as a sugar or amino acid, may move in response to the electrical and pH gradient. There is evidence for protons as the major driving force for cotransport systems in non-animal cells (Slayman, 1974), most clearly defined in bacteria (reviews by Boos, 1974; Simoni and Postma, 1975; Hamilton, 1975). Proton dependent cotransport systems for sugars and amino acids have also been described for yeasts Saccharomyces cerevisiae, S. carlsbergenis and S.fragilis by Eddy and Nowacki (1971) and Seaston et al. (1973, 1976). An H+-cotransport system for glucose and its analogues has been well characterized in Neurospora (Slayman and Slayman, 1974) and in Chlorella ( K o m o r and Tanner, 1974a, b). In higher plant cells, evidence for a depolarising effect of sugars and amino acids on the membrane potential suggests that there may be an H + cotransport system present (Etherton and Nuovo, 1974; Jones et al., 1975; Anderson, 1976; Racusen and Galston, 1977). In higher plants, solute accumulation into the phloem is an important active process (Geiger, 1975), although the mechanism is not understood. It has been suggested that an H + pump provides the driving force for solute transport (Ziegler, 1975; Giaquinta, 1977) but as yet there is no experimental evidence. Developing Ricinus cotyledons are adapted to take up sugar, mostly sucrose, from the endosperm and transfer it to the translocation stream for the growing embryonic axis. The endosperm may be removed from the cotyledons, which retain the capacity to absorb sucrose at a high uptake rate. Sucrose is accumulated against high concentration gradients, and is sensitive to pH, and metabolic inhibitors (Kriedemann and Beevers, 1967a). There is therefore good evidence for active transport of sucrose in Ricinus cotyledons. The existence of H+-sucrose cotransport in Ricinus cotyledons is the subject of the present investigation.
0032-0935/78/0138/0229/$01.40
230
V.M. Hutchings: Sucrose and Proton Cotransport.I
I I ~ r e f e r e n c e electrode hL - H ~ pH / I J/Itaerator
electrode
to water ~ cot ledons
~ H H~-~
:I-JL-LfTII
i, blackperspex
L i ~ - - - ~11
/i
L~---= --~ II -
~
chamber from
L~_aLerbath
~
stirrer
Fig. 1. Plan of the chamber used in pH and flux measurements. The reference and pH electrodes shown in the diagram are part of the pH stat used in flux measurements
Radioactivity was determined by liquid scintillation spectrometry in toluene/triton X-100, as described by Patterson and Greene (1965). Scintillation fluid was composed of 2 volumes toluene, containing 0.4% PPO, 0.01% POPOP + l volume Triton X-100. Scintillation fluid (15ml) was added to samples, in glass vials, and counted on a Tracerlab Corumatic 11 Liquid Scintillation Spectrometer. Efficiency was estimated by the channels ratio method and was 45-55%.
Results H + Influx on Addition o f Sucrose
Materials and Methods Ricinus communis var. Gibsonii seeds were soaked overnight at room temperature and grown in sterile moist micafil for 24 h at 30 ~ C and 4 days at 25 ~ C, in the dark. p H and Flux Measurements A black perspex chamber surrounded by a water jacket was used throughout experiments for measurement of pH, H + and sucrose fluxes (Fig. 1). The embryonic axis was removed from the germinated Ricinus seedlings, so that 10 m m of hypocotyl remained, and the endosperm was removed, leaving a pair of Ricinus cotyledons. Cotyledons were chosen at the same stage of development and at the same size. The average weight of each cotyledon pair was 77 rag. Five cotyledon pairs were immersed in 14.5ml CaSO 4 (max. final volume after additions 15 ml). The solution was continuously aerated and maintained at a temperature of 24 ~ C. The chamber was open to the air and experiments were carried out in the dark. A Cecar micro-combination electrode (Beckman R I I C Ltd.) was connected to a Beckman phasar 1 pH meter. Changes in pH were shown on a chart recorder.
Automatic Titration o f H + Protons absorbed (or O H - released) by cotyledons were automatically titrated with 1 x 10 .3 mol 1-1 HC1 so that the pH remained constant at its initial value. Equipment consisted of a Radiometer type T T T II Titrator with an SBU I Syringe Burette and an SBR 2 Titrigraph. A calomel reference electrode (Radiometer type K401) and a glass pH electrode (Radiometer type G200C) were connected to a pH meter P H M 2 7 through to the titrator. Rates of H + flux were estimated from the trace of the Titrigraph chart.
Labelling with [ U- 14C]Sucrose Cotyledon pairs were allowed to equilibrate in unbuffered 0.1 mmol 1 1 CaSO~, initially at pH 6.5, for 20 min. During this time the pH of the solution became progressively acid, until it finally reached a steady value. When necessary 0.1 tool 1 i N a O H or 0.1 tool I- ~ HCI were added to adjust the pH. Constant pH was then maintained via the pH stat. The labelling solution consisted of [U-14C] sucrose (0.1 ~tCi/ ml), non-radioactive sucrose solution and 0.1 m m o l l 1 CaSO4. After 11 min, the cotyledon pairs were removed from the chamber, blotted lightly, and washed in unlabelled sucrose solution for a total of 5 rain. Cotyledons, blotted dry, were immersed in 1 ml Koch-Light solubiliser TS-I, in glass vials, and incubated for 48 h at 50 ~ C.
On addition of sucrose to the medium bathing the Ricinus cotyledons, there was an initial pH transient and then, after 2-3 rain, there was a steady increasing alkalinity in the external solution. This increase continued for between 20-30 min before a steady pH was reached. Typical pH traces are shown in Figure 2a, b. When the cotyledons were added at the start of the experiment, the pH decreased rapidly in the CaSO4 solution, but after 15-20 rain, the pH of the solution came to a steady value. The initial release of H + was interpreted as resulting from the displacement of H + in the cell walls by Ca 2+. In all the experiments described, sucrose was added only when a steady pH was maintained in the medium. Addition of sucrose appeared to be associated with an increase in alkalinity in the medium. For accurate measurement of H + influx on addition of sucrose, H § flux was estimated by automatic titration, to a constant pH, of a solution bathing the cotyledons. At any increase in pH above a set value, there was addition of 1 • 10-3 tool 1-1 HC1. Figures 3 a, b are typical pH stat traces, showing acid consumed on addition of sucrose to the medium bathing five cotyledon pairs. There was an initial lag of 2-3 min, followed by increasing acid consumption for 20-30 min. This was similar to the observed pattern of pH change. The pH stat traces also show that acid consumption depends on the pH of the medium and the final sucrose concentration. Thus, addition of sucrose appeared to be associated with H + influx.
Characteristics of Sucrose-linked H +Influx
Sucrose-linked H + influx and sucrose uptake in Ricinus cotyledons took place for different time periods. Net H + influx was observed for 20-30 rain. Sucrose uptake occurred for several hours. Thus, in further experiments carried out, the rates were measured over the same time period of 11 min. When a temperature of 3~ was maintained in the experimental chamber containing the cotyledons,
V.M. Hutchings: Sucrose and Proton Cotransport.I
Table 1. The effect of low temperature, Rates of sucrose uptake were measured at 3 ~ and 24 ~ C, from 10 m m o l l 1 sucrose at the stated pHs. The inhibition of sucrose uptake at 3 ~ C is expressed as a percentage of sucrose uptake at 24 ~ C. The activation energy for the process is shown at each pH. ( R e s u l t s + s t a n d a r d error of the mean)
a
sucrose final ,conc. 125mM
1
__
_
..--
/ /
_ _
02pH
9
231
unit
Sucrose uptake (nmol m i n - t (5 cotyledons 1))
pH
2.5min b ~sucrose final conc. lOmM
6.0 6.3 6.7
J :~..j~
I0033pH unit
24 ~ C
3~ C
% inhibition of uptake at 3 ~ C
246_+11 (3) 240+ I0 (1) 206+6(2)
43.8_+2.8 (1) 36.0_+2.3 (1) 34.3_+3.7(1)
82 85 83
Activation energy (kJ mol 1)
56 61 58
2.Smm Fig. 2a and b. The change in pH of the external medium due to the addition of sucrose to Ricinus cotyledons. In these experiments, a similar method was used to that described in Methods except that one cotyledon pair was bathed in 4.5 ml of 0.1 mmol 1 ~ CaSO~, in a smaller model of the chamber shown in Figure 1. Sucrose was added to a m a x i m u m volume of 5 ml. a Sucrose added to a final concentration of 125 mmol 1 1 at initial pH 6.09 b Sucrose added to a final concentration of 10mmol 1 ~ at initial pH 6.3.
~60 o
9 lOmMsucrose
~40
95 mM sucrose 2mM sucrose
T
._= E
-
9
Ez0
v a
800
h:
/ //
0
/
5OmM sucrose
g ~400
constant PH60
/
E .,,..-
o b
~
6.6
6.8
%0
3b
10time(rain) 2b
E ~400
6-4 PH
,00[
2mM sucrose
b 600 t'
6.2
are the results of a m i n i m u m of 17 experiments at each concentration of sucrose used. Each point is the mean value of 2 5 experiments.
t
/
60
Fig. 4. Sucrose-dependent H + influx plotted against pH. These
pH6.2
-~200
10mMsucrose
r tf3
~susucrosefinal conc.lOmM -6 E =
200
/
r
~/'"'~P 0
,,/ 0
t
100 5raM sucrose
H6.5 ID
lOtime(min) 20
3()
sucrose added
2mMsucrose 0
6'.0
612
6~4 pH
6'-6
4.8--
Fig. 3. a pH stat trace on addition of sucrose at constant pH 6.0. b pH star trace on addition of sucrose at constant pH 6.2 and pH 6.5
Fig. 5. Sucrose influx plotted against pH, These are the results of a m i n i m u m of 11 experiments at each concentration of sucrose used. Each point is the mean value of 1-6 experiments
no increase in pH was observed on addition of sucrose to the medium. For example, in an experiment in
there was no detectable
which sucrose was added to a final concentration o f 10 m m o l 1 t a t p H 6 . 0 , t h i s i n d u c e d a n H + i n f l u x of 37.5 nmol min-
t (5 c o t y l e d o n s
t) a t 2 4 ~ C w h e r e a s
change
ment was repeated three times. Table 1 shows that over the
at 3 ~ C. This experipH
range
6 . 0 6.7,
sucrose uptake was inhibited, at 3 ~ C, by an average of 83% of uptake at 24 ~ C. Activation energies for
232
the process are shown and are typical of an enzyme reaction, rather than passive diffusion. The results show that low temperature inhibited net H + influx and sucrose uptake, which indicates that both were dependent on metabolism. D a t a in Figure 4 shows that sucrose-linked H § influx was strongly dependent upon the p H of the medium in the presence of a final concentration of 10 mmol 1 t, 5 m m o l 1 1 and 2 m m o l 1- ~ sucrose. At all sucrose concentrations there was m a x i m u m H § influx at p H 6.2, with H + influx falling to zero rate at p H 6.6. In contrast, sucrose influx appeared to be insensitive to p H in the presence of final concentrations of sucrose of 1 0 m m o l l 1, 5 m m o l l ~ and 2 m m o l 1 1 sucrose (Fig. 5). Sucrose influx was larger than net H + influx, for example, at 2 m m o l 1- ~ sucrose concentration, the highest sucrose flux was 9.3 lamol h - 1 (5 cotyledons-1) compared to net H + influx of 3.4 gmol h 1 ( 5 cotyledons- 1). Figure 6 shows net H + influx plotted against sucrose concentration at p H 6.0. Clearly, net H + influx was dependent on the concentration of sucrose in the medium. Figure 7 shows that the rate of sucrose uptake was also dependent on the final concentration of sucrose, but whereas H § influx was saturated below 50 mmol 1- ~ sucrose, sucrose uptake was not fully saturated below 100 m m o l 1 ~ sucrose. Sucrose influx from 100 m m o l l -~ sucrose, at p H 6.0, was 179 lamol h -1 (5 cotyledons-i), compared to a net H + influx of 8.6 pmol h - 1 (5 cotyledons 1). The insets in Figures 6 and 7 show data transposed to Lineweaver-Burke plots, and the calculated K~ and Vma~values from the graphs are shown in Table 2. Results at p H 6.4 and p H 6.5 are also given. The Km and V~,~ values for sucrose influx at p H 6.0 and p H 6.5 were similar. The K~ and Vm~x values for net H + influx at p H 6.0 and p H 6.4 were similar. However, the Km and Vm,x values for sucrose influx and the 1"
Planta 138, 229 235 (1978)
9 by Springer-Verlag 1978
Sucrose and Proton Cotransport in Ricinus Cotyledons I. H + Influx Associated with Sucrose Uptake
Vanessa M. Hutchings Department of Botany, University of Cambridge, Cambridge CB2 1QW, U.K.
Abstracts. Ricinus cotyledons were used to investigate whether sucrose uptake by the phloem could be due to H+-sucrose cotransport. The addition of sucrose to the medium bathing Ricinus cotyledons created a pH shift to more alkaline values. The effect was not observed on the addition of glucose or fructose. Net H + influx and sucrose influx were both shown to be temperature sensitive, dependent on pH and external sucrose concentration. However, net H + influx was far more sensitive to pH than sucrose influx and was abolished at high pH; it was also observed for a shorter time period than sucrose uptake. The stoichiometry of H + :sucrose was less than one and declined with increasing sucrose concentration and increasing pH. Two possible models are proposed to account for this low and changing stoichiometry. Given an H + pump, recirculation would seem an inevitable consequence of sucrose and proton cotransport. Key words: Phloem Proton cotransport - Ricinus - Sucrose cotransport.
Introduction
There is good evidence for the widespread occurrence of active proton transport in non-animal cells. It has been suggested that the function of the H + pump at the plant cell plasmalemma is to regulate cytoplasmic pH and provide energy for secondary active transport of solutes (reviews by MacRobbie, 1975; Raven and Smith, 1976a; Smith and Raven, 1976). An electrogenic H § pump will provide a proton electrochemical potential gradient as an energy source Present address: V.M. Hutchings, Department of Biochemistry,
University of Cambridge, Tennis Court Road, Cambridge, CB2 IQW, U.K.
for secondary active solute transport. A carrier cotransporting H + and a neutral solute, such as a sugar or amino acid, may move in response to the electrical and pH gradient. There is evidence for protons as the major driving force for cotransport systems in non-animal cells (Slayman, 1974), most clearly defined in bacteria (reviews by Boos, 1974; Simoni and Postma, 1975; Hamilton, 1975). Proton dependent cotransport systems for sugars and amino acids have also been described for yeasts Saccharomyces cerevisiae, S. carlsbergenis and S.fragilis by Eddy and Nowacki (1971) and Seaston et al. (1973, 1976). An H+-cotransport system for glucose and its analogues has been well characterized in Neurospora (Slayman and Slayman, 1974) and in Chlorella ( K o m o r and Tanner, 1974a, b). In higher plant cells, evidence for a depolarising effect of sugars and amino acids on the membrane potential suggests that there may be an H + cotransport system present (Etherton and Nuovo, 1974; Jones et al., 1975; Anderson, 1976; Racusen and Galston, 1977). In higher plants, solute accumulation into the phloem is an important active process (Geiger, 1975), although the mechanism is not understood. It has been suggested that an H + pump provides the driving force for solute transport (Ziegler, 1975; Giaquinta, 1977) but as yet there is no experimental evidence. Developing Ricinus cotyledons are adapted to take up sugar, mostly sucrose, from the endosperm and transfer it to the translocation stream for the growing embryonic axis. The endosperm may be removed from the cotyledons, which retain the capacity to absorb sucrose at a high uptake rate. Sucrose is accumulated against high concentration gradients, and is sensitive to pH, and metabolic inhibitors (Kriedemann and Beevers, 1967a). There is therefore good evidence for active transport of sucrose in Ricinus cotyledons. The existence of H+-sucrose cotransport in Ricinus cotyledons is the subject of the present investigation.
0032-0935/78/0138/0229/$01.40
230
V.M. Hutchings: Sucrose and Proton Cotransport.I
I I ~ r e f e r e n c e electrode hL - H ~ pH / I J/Itaerator
electrode
to water ~ cot ledons
~ H H~-~
:I-JL-LfTII
i, blackperspex
L i ~ - - - ~11
/i
L~---= --~ II -
~
chamber from
L~_aLerbath
~
stirrer
Fig. 1. Plan of the chamber used in pH and flux measurements. The reference and pH electrodes shown in the diagram are part of the pH stat used in flux measurements
Radioactivity was determined by liquid scintillation spectrometry in toluene/triton X-100, as described by Patterson and Greene (1965). Scintillation fluid was composed of 2 volumes toluene, containing 0.4% PPO, 0.01% POPOP + l volume Triton X-100. Scintillation fluid (15ml) was added to samples, in glass vials, and counted on a Tracerlab Corumatic 11 Liquid Scintillation Spectrometer. Efficiency was estimated by the channels ratio method and was 45-55%.
Results H + Influx on Addition o f Sucrose
Materials and Methods Ricinus communis var. Gibsonii seeds were soaked overnight at room temperature and grown in sterile moist micafil for 24 h at 30 ~ C and 4 days at 25 ~ C, in the dark. p H and Flux Measurements A black perspex chamber surrounded by a water jacket was used throughout experiments for measurement of pH, H + and sucrose fluxes (Fig. 1). The embryonic axis was removed from the germinated Ricinus seedlings, so that 10 m m of hypocotyl remained, and the endosperm was removed, leaving a pair of Ricinus cotyledons. Cotyledons were chosen at the same stage of development and at the same size. The average weight of each cotyledon pair was 77 rag. Five cotyledon pairs were immersed in 14.5ml CaSO 4 (max. final volume after additions 15 ml). The solution was continuously aerated and maintained at a temperature of 24 ~ C. The chamber was open to the air and experiments were carried out in the dark. A Cecar micro-combination electrode (Beckman R I I C Ltd.) was connected to a Beckman phasar 1 pH meter. Changes in pH were shown on a chart recorder.
Automatic Titration o f H + Protons absorbed (or O H - released) by cotyledons were automatically titrated with 1 x 10 .3 mol 1-1 HC1 so that the pH remained constant at its initial value. Equipment consisted of a Radiometer type T T T II Titrator with an SBU I Syringe Burette and an SBR 2 Titrigraph. A calomel reference electrode (Radiometer type K401) and a glass pH electrode (Radiometer type G200C) were connected to a pH meter P H M 2 7 through to the titrator. Rates of H + flux were estimated from the trace of the Titrigraph chart.
Labelling with [ U- 14C]Sucrose Cotyledon pairs were allowed to equilibrate in unbuffered 0.1 mmol 1 1 CaSO~, initially at pH 6.5, for 20 min. During this time the pH of the solution became progressively acid, until it finally reached a steady value. When necessary 0.1 tool 1 i N a O H or 0.1 tool I- ~ HCI were added to adjust the pH. Constant pH was then maintained via the pH stat. The labelling solution consisted of [U-14C] sucrose (0.1 ~tCi/ ml), non-radioactive sucrose solution and 0.1 m m o l l 1 CaSO4. After 11 min, the cotyledon pairs were removed from the chamber, blotted lightly, and washed in unlabelled sucrose solution for a total of 5 rain. Cotyledons, blotted dry, were immersed in 1 ml Koch-Light solubiliser TS-I, in glass vials, and incubated for 48 h at 50 ~ C.
On addition of sucrose to the medium bathing the Ricinus cotyledons, there was an initial pH transient and then, after 2-3 rain, there was a steady increasing alkalinity in the external solution. This increase continued for between 20-30 min before a steady pH was reached. Typical pH traces are shown in Figure 2a, b. When the cotyledons were added at the start of the experiment, the pH decreased rapidly in the CaSO4 solution, but after 15-20 rain, the pH of the solution came to a steady value. The initial release of H + was interpreted as resulting from the displacement of H + in the cell walls by Ca 2+. In all the experiments described, sucrose was added only when a steady pH was maintained in the medium. Addition of sucrose appeared to be associated with an increase in alkalinity in the medium. For accurate measurement of H + influx on addition of sucrose, H § flux was estimated by automatic titration, to a constant pH, of a solution bathing the cotyledons. At any increase in pH above a set value, there was addition of 1 • 10-3 tool 1-1 HC1. Figures 3 a, b are typical pH stat traces, showing acid consumed on addition of sucrose to the medium bathing five cotyledon pairs. There was an initial lag of 2-3 min, followed by increasing acid consumption for 20-30 min. This was similar to the observed pattern of pH change. The pH stat traces also show that acid consumption depends on the pH of the medium and the final sucrose concentration. Thus, addition of sucrose appeared to be associated with H + influx.
Characteristics of Sucrose-linked H +Influx
Sucrose-linked H + influx and sucrose uptake in Ricinus cotyledons took place for different time periods. Net H + influx was observed for 20-30 rain. Sucrose uptake occurred for several hours. Thus, in further experiments carried out, the rates were measured over the same time period of 11 min. When a temperature of 3~ was maintained in the experimental chamber containing the cotyledons,
V.M. Hutchings: Sucrose and Proton Cotransport.I
Table 1. The effect of low temperature, Rates of sucrose uptake were measured at 3 ~ and 24 ~ C, from 10 m m o l l 1 sucrose at the stated pHs. The inhibition of sucrose uptake at 3 ~ C is expressed as a percentage of sucrose uptake at 24 ~ C. The activation energy for the process is shown at each pH. ( R e s u l t s + s t a n d a r d error of the mean)
a
sucrose final ,conc. 125mM
1
__
_
..--
/ /
_ _
02pH
9
231
unit
Sucrose uptake (nmol m i n - t (5 cotyledons 1))
pH
2.5min b ~sucrose final conc. lOmM
6.0 6.3 6.7
J :~..j~
I0033pH unit
24 ~ C
3~ C
% inhibition of uptake at 3 ~ C
246_+11 (3) 240+ I0 (1) 206+6(2)
43.8_+2.8 (1) 36.0_+2.3 (1) 34.3_+3.7(1)
82 85 83
Activation energy (kJ mol 1)
56 61 58
2.Smm Fig. 2a and b. The change in pH of the external medium due to the addition of sucrose to Ricinus cotyledons. In these experiments, a similar method was used to that described in Methods except that one cotyledon pair was bathed in 4.5 ml of 0.1 mmol 1 ~ CaSO~, in a smaller model of the chamber shown in Figure 1. Sucrose was added to a m a x i m u m volume of 5 ml. a Sucrose added to a final concentration of 125 mmol 1 1 at initial pH 6.09 b Sucrose added to a final concentration of 10mmol 1 ~ at initial pH 6.3.
~60 o
9 lOmMsucrose
~40
95 mM sucrose 2mM sucrose
T
._= E
-
9
Ez0
v a
800
h:
/ //
0
/
5OmM sucrose
g ~400
constant PH60
/
E .,,..-
o b
~
6.6
6.8
%0
3b
10time(rain) 2b
E ~400
6-4 PH
,00[
2mM sucrose
b 600 t'
6.2
are the results of a m i n i m u m of 17 experiments at each concentration of sucrose used. Each point is the mean value of 2 5 experiments.
t
/
60
Fig. 4. Sucrose-dependent H + influx plotted against pH. These
pH6.2
-~200
10mMsucrose
r tf3
~susucrosefinal conc.lOmM -6 E =
200
/
r
~/'"'~P 0
,,/ 0
t
100 5raM sucrose
H6.5 ID
lOtime(min) 20
3()
sucrose added
2mMsucrose 0
6'.0
612
6~4 pH
6'-6
4.8--
Fig. 3. a pH stat trace on addition of sucrose at constant pH 6.0. b pH star trace on addition of sucrose at constant pH 6.2 and pH 6.5
Fig. 5. Sucrose influx plotted against pH, These are the results of a m i n i m u m of 11 experiments at each concentration of sucrose used. Each point is the mean value of 1-6 experiments
no increase in pH was observed on addition of sucrose to the medium. For example, in an experiment in
there was no detectable
which sucrose was added to a final concentration o f 10 m m o l 1 t a t p H 6 . 0 , t h i s i n d u c e d a n H + i n f l u x of 37.5 nmol min-
t (5 c o t y l e d o n s
t) a t 2 4 ~ C w h e r e a s
change
ment was repeated three times. Table 1 shows that over the
at 3 ~ C. This experipH
range
6 . 0 6.7,
sucrose uptake was inhibited, at 3 ~ C, by an average of 83% of uptake at 24 ~ C. Activation energies for
232
the process are shown and are typical of an enzyme reaction, rather than passive diffusion. The results show that low temperature inhibited net H + influx and sucrose uptake, which indicates that both were dependent on metabolism. D a t a in Figure 4 shows that sucrose-linked H § influx was strongly dependent upon the p H of the medium in the presence of a final concentration of 10 mmol 1 t, 5 m m o l 1 1 and 2 m m o l 1- ~ sucrose. At all sucrose concentrations there was m a x i m u m H § influx at p H 6.2, with H + influx falling to zero rate at p H 6.6. In contrast, sucrose influx appeared to be insensitive to p H in the presence of final concentrations of sucrose of 1 0 m m o l l 1, 5 m m o l l ~ and 2 m m o l 1 1 sucrose (Fig. 5). Sucrose influx was larger than net H + influx, for example, at 2 m m o l 1- ~ sucrose concentration, the highest sucrose flux was 9.3 lamol h - 1 (5 cotyledons-1) compared to net H + influx of 3.4 gmol h 1 ( 5 cotyledons- 1). Figure 6 shows net H + influx plotted against sucrose concentration at p H 6.0. Clearly, net H + influx was dependent on the concentration of sucrose in the medium. Figure 7 shows that the rate of sucrose uptake was also dependent on the final concentration of sucrose, but whereas H § influx was saturated below 50 mmol 1- ~ sucrose, sucrose uptake was not fully saturated below 100 m m o l 1 ~ sucrose. Sucrose influx from 100 m m o l l -~ sucrose, at p H 6.0, was 179 lamol h -1 (5 cotyledons-i), compared to a net H + influx of 8.6 pmol h - 1 (5 cotyledons 1). The insets in Figures 6 and 7 show data transposed to Lineweaver-Burke plots, and the calculated K~ and Vma~values from the graphs are shown in Table 2. Results at p H 6.4 and p H 6.5 are also given. The Km and V~,~ values for sucrose influx at p H 6.0 and p H 6.5 were similar. The K~ and Vm~x values for net H + influx at p H 6.0 and p H 6.4 were similar. However, the Km and Vm,x values for sucrose influx and the 1"
