Planta
Planta (1982)156:16-20
9 Springer-Verlag 1982
The Effect of glucose on chloride uptake by Chlorella II. Effect of intracellular acidification on chloride uptake
R. Doblinger and H.W. Tromballa* Institut ffir Physikalische Chemie der Universitgt, Wghringerstrasse 42, A-1090 Wien, Austria
Abstract. Glucose at 5 mM inhibited chloride uptake by C h l o r e l l a f u s c a in the light and in the dark by 30-t-10% and acidified the cell interior by 0.2 units (measured with 5.5-dimethyl-oxazolidine-2.4-dione; DMO). Neither effect was shown by the glucose analogues 3-O-methylglucose and 6-desoxyglucose which are transported into the cell but not metabolized. As it was surmised that inhibition of C1- transport was the result of intracellular acidification, the action of other agents on C1transport and intracellular pH was examined. The Tris salts of the permeant acids DMO and propionic acid inhibited C1- transport. The concentration and pH dependence of this effect were consistent with the free acids being the effective agents. At pH 6.7 the 15 mM Tris salts had about the same effect on C1- transport as 5 mM glucose. Under these conditions the intracellular pH (measured by the DMO method) was lowered by 0.2 units with glucose as well as, in presence of the Tris salts. When the algae were gassed with 1.5% CO2 in air, C1- uptake in the light was depressed to 40-50% and the intracellular pH was lowered by 0.4 units. On the basis of these observations and the results from the preceding study, inhibition of C1 transport by glucose is interpreted as inhibition of an ATP-dependent C1-/OH- exchange by intracellular acidification, due to glucose metabolism. Key words: Acid, permeant - Carbon dioxide C h l o r e l l a - Chloride transport - Glucose (and C L transport) - pH, intracellular
Introduction
There is much evidence that in plants cations (mainly K +) are exchanged against protons and anions against hydroxyl ions (or cotransported with protons) (see reviews by Poole 1978 and Bentrup 1978). K+/H + exchange is believed to play a central role in the pH regulation of the cell as a "biophysical pH stat" (see Smith and Raven 1979). In C h l o r e l l a K+/H + exchange is well established, as it has been shown that intracellular acidification by various independent methods caused net K + uptake, examples including permeant acids (Tromballa 1978), the uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) (Tromballa 1981a), glucose (Tromballa 1981b), and CO2 in the light (Tromballa, in preparation). It can be expected that anion uptake responds differently to intracellular acidification, i.e., that it is inhibited by the same agents. In our laboratory it has been found that active uptake of the C1 analogue Br- is inhibited by glucose (Paschinger and Broda 1967). For this effect no satisfactory mechanism could be given. The development of knowledge on ion transport in plants and the recent studies with C h l o r e l l a briefly summarized above - now offer a possible explanation: glucose acidifies the cell interior and this acidification inhibits C1-/OH- exchange. The present study tests this hypothesis by examining the effects of glucose, permeant acids, and COz on C1- transport and intracellular pH. Materials and methods
* To whom correspondence should be addressed
Abbreviations: CCCP = carbonyl cyanide-m-chlorophenylhydrazone; D C M U = 3-(3.4-dichlorophenyl)-1.1-dimethylurea; D M O - 5.5-dimethyl-oxazolidine-2.4-dione; Tris = tris-hydroxymethylaminomethane
0032-0935/82/0156/0016/$01.00
Culture of Chlorellafusca (211-8b, G6ttingen) and experimental procedures were as described earlier (Doblinger and Tromballa 1982). Intracellular pH was determined with D M O using a filter technique (Tromballa 1981 a, Doblinger and Tromballa 1982). In the control experiments it was established that the amount
R. Doblinger and H_W. Tromballa: Chloride uptake by Chlorella II
17
30
30 uo
~X-'---X--------~
u
_~
20
~20 c v
9
z~ i
20
40
60
80
30
100 rain
Fig. 1. Inhibition of C1- uptake by glucose. 2-10-5 M chloride, 2.5 ml cells 1-1, pH 7.0. Open symbols, light; closed symbols, dark. o 9 control, zx 9 5 m M glucose added at t = 0
60
90
120 rnin
Fig. 3. Inhibition of C1- uptake by 5 mM glucose. Dependence on time of glucose additon. 2.10-5 M chloride, 2.5 ml cells 1-1, pH 7.0, light, e control, xglucose added at $, + a t t = 0 , 9 at t = - 10 min, 9 at t = - 60 min
z E 5 24
_•30
E X ~3
~20 2
lO
o
D O 30
60
90
120
150 mm
F i g . 2. Inhibition of C1- uptake by glucose. Dependence on
glucose concentration. 2 . 1 0 - S M chloride, 2.5ml cells 1-, pH7.0, light, o control, ix 1.5raM, n 5 m M ,
Planta (1982)156:16-20
9 Springer-Verlag 1982
The Effect of glucose on chloride uptake by Chlorella II. Effect of intracellular acidification on chloride uptake
R. Doblinger and H.W. Tromballa* Institut ffir Physikalische Chemie der Universitgt, Wghringerstrasse 42, A-1090 Wien, Austria
Abstract. Glucose at 5 mM inhibited chloride uptake by C h l o r e l l a f u s c a in the light and in the dark by 30-t-10% and acidified the cell interior by 0.2 units (measured with 5.5-dimethyl-oxazolidine-2.4-dione; DMO). Neither effect was shown by the glucose analogues 3-O-methylglucose and 6-desoxyglucose which are transported into the cell but not metabolized. As it was surmised that inhibition of C1- transport was the result of intracellular acidification, the action of other agents on C1transport and intracellular pH was examined. The Tris salts of the permeant acids DMO and propionic acid inhibited C1- transport. The concentration and pH dependence of this effect were consistent with the free acids being the effective agents. At pH 6.7 the 15 mM Tris salts had about the same effect on C1- transport as 5 mM glucose. Under these conditions the intracellular pH (measured by the DMO method) was lowered by 0.2 units with glucose as well as, in presence of the Tris salts. When the algae were gassed with 1.5% CO2 in air, C1- uptake in the light was depressed to 40-50% and the intracellular pH was lowered by 0.4 units. On the basis of these observations and the results from the preceding study, inhibition of C1 transport by glucose is interpreted as inhibition of an ATP-dependent C1-/OH- exchange by intracellular acidification, due to glucose metabolism. Key words: Acid, permeant - Carbon dioxide C h l o r e l l a - Chloride transport - Glucose (and C L transport) - pH, intracellular
Introduction
There is much evidence that in plants cations (mainly K +) are exchanged against protons and anions against hydroxyl ions (or cotransported with protons) (see reviews by Poole 1978 and Bentrup 1978). K+/H + exchange is believed to play a central role in the pH regulation of the cell as a "biophysical pH stat" (see Smith and Raven 1979). In C h l o r e l l a K+/H + exchange is well established, as it has been shown that intracellular acidification by various independent methods caused net K + uptake, examples including permeant acids (Tromballa 1978), the uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) (Tromballa 1981a), glucose (Tromballa 1981b), and CO2 in the light (Tromballa, in preparation). It can be expected that anion uptake responds differently to intracellular acidification, i.e., that it is inhibited by the same agents. In our laboratory it has been found that active uptake of the C1 analogue Br- is inhibited by glucose (Paschinger and Broda 1967). For this effect no satisfactory mechanism could be given. The development of knowledge on ion transport in plants and the recent studies with C h l o r e l l a briefly summarized above - now offer a possible explanation: glucose acidifies the cell interior and this acidification inhibits C1-/OH- exchange. The present study tests this hypothesis by examining the effects of glucose, permeant acids, and COz on C1- transport and intracellular pH. Materials and methods
* To whom correspondence should be addressed
Abbreviations: CCCP = carbonyl cyanide-m-chlorophenylhydrazone; D C M U = 3-(3.4-dichlorophenyl)-1.1-dimethylurea; D M O - 5.5-dimethyl-oxazolidine-2.4-dione; Tris = tris-hydroxymethylaminomethane
0032-0935/82/0156/0016/$01.00
Culture of Chlorellafusca (211-8b, G6ttingen) and experimental procedures were as described earlier (Doblinger and Tromballa 1982). Intracellular pH was determined with D M O using a filter technique (Tromballa 1981 a, Doblinger and Tromballa 1982). In the control experiments it was established that the amount
R. Doblinger and H_W. Tromballa: Chloride uptake by Chlorella II
17
30
30 uo
~X-'---X--------~
u
_~
20
~20 c v
9
z~ i
20
40
60
80
30
100 rain
Fig. 1. Inhibition of C1- uptake by glucose. 2-10-5 M chloride, 2.5 ml cells 1-1, pH 7.0. Open symbols, light; closed symbols, dark. o 9 control, zx 9 5 m M glucose added at t = 0
60
90
120 rnin
Fig. 3. Inhibition of C1- uptake by 5 mM glucose. Dependence on time of glucose additon. 2.10-5 M chloride, 2.5 ml cells 1-1, pH 7.0, light, e control, xglucose added at $, + a t t = 0 , 9 at t = - 10 min, 9 at t = - 60 min
z E 5 24
_•30
E X ~3
~20 2
lO
o
D O 30
60
90
120
150 mm
F i g . 2. Inhibition of C1- uptake by glucose. Dependence on
glucose concentration. 2 . 1 0 - S M chloride, 2.5ml cells 1-, pH7.0, light, o control, ix 1.5raM, n 5 m M ,
