Planta 151, 95 102(1981)
P l a n t a 9 Springer-Verlag 1981
Turgor Pressure and Water Transport Properties of Suspension-Cultured Cells of Chenopodium rubrum L. K.-H. Btichner, U. Zimmermann, and F.-W. Bentrup 1 Arbeitsgruppe Membranforschung am Institut fiir Medizin, Kernforschungsanlage J61ich, Postfach 1913, D-5170 Jtilich 1, and 1 Institut fiir Biologie I der UniversitS.t, Auf der Morgenstelle 1, D-7400 Ttibingen, Federal Republic of Germany
Abstract. The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgot pressure in suspension-cultured cells was in the range of between 3 and 5 bar. F r o m the turgot pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20_+ 10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, e, determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. e increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5-2.10- 6 cm s- 1 b a r - 1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a crosslinked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgot pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgot pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophylMike suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.
Key words: Cell suspension culture - Chenopodium - Elasticity - Immobilization - Pressure probe Water relations.
Introduction Cell suspension cultures from higher plant tissues seem prima faci.e well suited for studies of cell membrane transport on the cellular level. The problem arises, however, that such cells and hence their membrane properties cannot be unambiguously related to a particular type of differentiated cell in situ. Recently obtained suspension cells of Chenopodium rubrum, however, grow strictly photoautotrophically, that is, require no organic carbon source and resemble mesophyll cells by their photosynthetic capacity (Hfisemann and Barz 1977). Also, the lipid composition of photoautotrophically grown cultures closely resembles that of intact mesophyll cells, while the lipid composition of heterotrophically grown cultures is very distinct (Hiisemann et al. 1980). These Chenopodium suspension cells seem particularly well suited for studies of turgor relaxation processes and of the water transport parameters in leaf cells using the miniaturized pressure probe (Hiisken et al. 1978, Zimmermann et al. 1980, Zimmermann and Steudle 1980). Such measurements on suspended higher plant cells lend themselves to a straightforward analysis of turgor pressure relaxation curves on the basis of the phenomenological equations of the thermodynamics of irreversible processes, because these equations are presently derived only for single cells bathed in a large volume of solution. In addition, cell volume and surface area can be determined quite accurately, while this does not apply to cells within a leaf tissue. Hitherto, inaccuracy in the calculation of volume and surface area of tissue cells prevented
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K.-H. Bfichner et al.: Water Relations of Suspension-cultured cells
the precise d e t e r m i n a t i o n o f t h e h y d r a u l i c m e m b r a n e c o n d u c t i v i t y , L p , a n d its p o s s i b l e d e p e n d e n c e u p o n p h y s i o l o g i c a l c o n d i t i o n s like w a t e r stress o r a l i g h t / d a r k r e g i m e . ( Z i m m e r m a n n et al. 1980). In this c o m m u n i c a t i o n w e p r e s e n t m e a s u r e m e n t s o f t u r g o r p r e s s u r e a n d w a t e r r e l a t i o n p a r a m e t e r s in p h o t o a u t o t r o p h i c a l l y g r o w n s u s p e n s i o n cells a n d in i n d i v i d u a l cells o f i n t a c t leaves o f Ch. rubrum. In a d d i t i o n , s i m i l a r e x p e r i m e n t s are r e p o r t e d f o r s u s p e n s i o n c u l t u r e d cells o f Ch. rubrurn w h i c h h a v e b e e n i m m o b i lized w i t h i n a c r o s s - l i n k e d a l g i n a t e m a t r i x , as des c r i b e d p r e v i o u s l y f o r g u a r d cells a n d m e s o p h y l l cell p r o t o p l a s t s o f V i c i a f a b a a n d red b l o o d cells ( P i l w a t et al. 1980, S c h e u r i c h et al. 1980, S c h n a b l et al. 1980). By m e a n s o f this t e c h n i q u e , t h e cells are s u r r o u n d e d b y an a r t i f i c i a l u n s t i r r e d layer. T h i s m o d e l e x p e r i m e n t t h e r e f o r e s i m u l a t e s the s i t u a t i o n o f a cell w i t h i n the l e a f tissue a n d s h o u l d p r o v i d e i n f o r m a t i o n c o n c e r n i n g the v a l i d i t y o f the w a t e r t r a n s p o r t e q u a t i o n s to indiv i d u a l cells w i t h i n a tissue. A p r e l i m i n a r y a c c o u n t o f o u r results h a s b e e n g i v e n a l r e a d y ( B f i c h n e r et al. 1980).
Pressure Probe Technique. Cell turgor pressure, P, turgor pressure
Materials and Methods Cell Suspension Cultures. Photoautotrophically growing cell suspensions of Chenopodium rubrum were obtained from Dr. W. Hfisemann, Mfinster. These cells were derived from a hypocotyl callus culture. The suspension culture technique, according to Hfisemann and Barz (1977), employed a two-compartment culture vessel. The upper compartment contains the cell suspension culture ; the lower compartment provides the CO 2 from a 2M K2COJKHCO 3 solution to the cell suspension. The culture medium contained the mineral ions and vitamines of Murashige and Skoog (1962) (MSmedium), supplemented with 10 -v M 2.4-dichlorophenoxyacetic acid. The cultures were grown at 25~176 under 19Wm -2 of white fluorescent light on a gyratory shaker at 120 rpm. The cultures were transferred into new medium every 14 days.
Test Media Most experiments were carried out in the above quoted MS-medium. Alternatively, for some experiments a low ionic strength test medium (CH-medium) was used. The ionic strength of this medium is comparable to the ionic strength of the solution in the cell wall free space of leaves, according to a study on leaf tissue of Ricinus, Helianthus, and Brassica (Bernstein 1971). The composition of the CH-medium is given in Ohkawa et al. (1980), except for the concentration of KNO3 which is 0.01 mM in the present study. The Chenopodium suspension ceils continued to grow in this medium for at least 8 days without changing their microscopic appearance. The cells were transferred from the MS-medium to the CH-medium 16h before the experiments.
Osmotarity Assay. The osmolarity of the media and of the cell sap of Ch. rubrum ceils was determined cryoscopically (Knauer Osmometer). A cell sample was washed with distilled water freezed to 186~ and centrifuged at 12,000 rpm for 15 min using an Eppendorf centrifuge. The internal osmolarity of the cells depended on medium. It was 198 mosmol when the cells were kept in the MS-medium and 168 mosmol when kept for 16 h in the CH-medium. The osmolarity of the MS- and CH-medium was 81 and 9 mosmol, respectively.
relaxation processes, and the volumetric elastic modulus, e, were determined using the miniaturized pressure probe technique (H/isken et al. 1978; Zimmermann and Steudle 1978; Zimmermann 1978, I980; Zimmermann et al. 1980). For impalement with the microcapillary, the cell was arrested at the edge of a piece of glass glued to a cover slip, as used previously for electrophysiological measurements (Bentrup 1970; Ohkawa et al. 1980). Upon successful impalement the microcapillary is withdrawn from the edge so that the cell is completely surrounded by the medium. The cell turgot is transmitted from the cell interior to a pressure transducer by an oil-filled microcapillary with a 2-5 pm tip diameter. 1"he oil/ceil sap boundary within the capillary can be shifted manually or electronically by measuring the electrical resistance between a silver wire electrode inserted into the very tip of the microcapillary and an external reference electrode. From the shift in the boundary the change in cell volume, A V, can be calculated. If the capillary tip were clogged, this shift of the boundary would be restricted. From the volume change, A V, and the measured change in turgor pressure, A P, the volumetric elastic modulus, e, is obtained (Philip 1958): dP ~ = A ~ V.
(1)
The determination of e requires that the time for the change in volume is small compared with the half-time of water exchange. (see Results).
Immobilization Technique. A commercial alginate polymer (Manucol DH, Alginate Industries, London) with a molecular weight of 90,000 was used. The alginate was dissolved in the MS-culture medium at a concentration of 6 and 8% w/w, respectively. The cells were mixed thoroughly with a small amount of this alginate solution. Cross-linking of the alginate polymers was achieved by adding some drops of a 30 mM CaC12 solution. The reaction was completed after 30 rain at room temperature. After replacement of the CaCla/MS-medium by the culture medium the pressure probe was inserted into the immobilized cells.
Results T h e n o r m a l t u r g o r p r e s s u r e in cells o f Ch. rubrum was a b o u t 3 to 5 bar. O c c a s i o n a l l y , v a l u e s o f a b o u t 2 b a r w e r e r e c o r d e d . A s t e a d y t u r g o r p r e s s u r e was n o r m a l l y e s t a b l i s h e d w i t h i n 10 to 20 rain a f t e r insert i o n o f the p r e s s u r e p r o b e tip. A c o n s t a n t t u r g o r pressure v a l u e c o u l d b e m o n i t o r e d f o r s e v e r a l h o u r s if n o m a n i p u l a t i o n o f the t u r g o r p r e s s u r e was perf o r m e d . T h e t u r g o r p r e s s u r e r e l a x a t i o n was g e n e r a t e d by c h a n g i n g t h e t u r g o r p r e s s u r e by m e a n s o f t h e pressure p r o b e . In s o m e e x p e r i m e n t s (see T a b l e 1) the t u r g o r p r e s s u r e was c h a n g e d o s m o t i c a l l y b y a d d i t i o n o f a 50 m M s u c r o s e s o l u t i o n to the e x t e r n a l m e d i u m . Typical pressure relaxation curves for both end- and e x o s m o t i c w a t e r f l o w i n i t i a t e d by d i r e c t t u r g o r press u r e c h a n g e s are s h o w n in Fig. 1. T h e i n d u c e d t u r g o r p r e s s u r e c h a n g e at the o n s e t o f the r e l a x a t i o n p r o c e s s was a b o u t 1 - 2 b a r in b o t h d i r e c t i o n s . T h e f l u c t u a t i o n s t h a t are s u p e r i m p o s e d on t h e r e c o r d e d p r e s s u r e v a l u e s result f r o m the m a n u a l o r a u t o m a t i c r e g u l a t i o n o f the o i l / c e l l sap b o u n d a r y in t h e c a p i l l a r y tip. It is
K.-H. Bfichner et al.: Water Relations of Suspension-cultured cells
97 AV [p[l
A Q.
%.08 57.25 ?0.72 53.88 r 26.94---1] 16.84q],
B
74.08p!
C
a3~:g~Jill It60.61
. . . . . . 47.14J il "33.67 avlptj 67 35--11-~94.29
94.290I I I
I
I
I
I
I
I
I
50
100
150
200
250
300
350
t [s] Fig. 1 A-C. Typical recording of cell turgor pressure changes of a suspension cultured cell of Chenopodium rubrum (V= 0.98 nl) induced by the pressure probe. Exosmotic (A) and endosmotic (B) waterflow are recorded as pressure relaxations due to increasing and decreasing cell volume by the pressure probe. The noise of the trace is due to the electronic control of the pressure probe device which adjusts the cell-sap/oil boundary in the microcapillary. (C) shows a subsequent e-measurement from which an e-value of 35 bar was calculated
evident that the pressure-relaxation curve follows an approximately exponential time course. For evaluation of the data, a curve was drawn by hand into the turgor-relaxation curves in order to average out the fluctuations. From the semilogarithmic plot of the data shown in Fig. 2 the rate constant, k, and consequently the half-time for water transport, T1/2 = in 2/k, is calculated to be Tu2=22.0 s (exosmotic) and T1/2=25.4 s (endosmotic). The agreement of these two values (within the limits of accuracy) demonstrates that there is no polarity in water flow. This conclusion was supported by 19 independent experiments, yielding an average value for the half-time of water exchange of 20 + 10 s (see Table 1). A change in the half-time was not observed when the cells were transferred from the MS-medium into the CH-medium (Table 1). It should also be noted that the halftime for the water exchange initiated by changes in the external osmolarity (i.e., by addition of sucrose) yields similar values, as indicated in Table 1. In some cases it was possible to perform several end- and exosmotic runs on the same cell (Table 1). The halftimes for end- and exosmotic water flow, respectively, agree within 10%, indicating the accurate reproducibility of the measurements and excluding the possibility of leakages. The finding that for exosmotic runs the final turgor pressure was higher and that for endosmotic runs the final turgor pressure was lower than the initial turgor pressure (see Fig. 1) is an additional proof that in both cases no leakages occurred during the measurements. If leakages had occurred the final pressure would be lower than the original one in exosmotic experiments. In any case, as men-
0
P l a n t a 9 Springer-Verlag 1981
Turgor Pressure and Water Transport Properties of Suspension-Cultured Cells of Chenopodium rubrum L. K.-H. Btichner, U. Zimmermann, and F.-W. Bentrup 1 Arbeitsgruppe Membranforschung am Institut fiir Medizin, Kernforschungsanlage J61ich, Postfach 1913, D-5170 Jtilich 1, and 1 Institut fiir Biologie I der UniversitS.t, Auf der Morgenstelle 1, D-7400 Ttibingen, Federal Republic of Germany
Abstract. The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgot pressure in suspension-cultured cells was in the range of between 3 and 5 bar. F r o m the turgot pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20_+ 10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, e, determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. e increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5-2.10- 6 cm s- 1 b a r - 1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a crosslinked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgot pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgot pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophylMike suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.
Key words: Cell suspension culture - Chenopodium - Elasticity - Immobilization - Pressure probe Water relations.
Introduction Cell suspension cultures from higher plant tissues seem prima faci.e well suited for studies of cell membrane transport on the cellular level. The problem arises, however, that such cells and hence their membrane properties cannot be unambiguously related to a particular type of differentiated cell in situ. Recently obtained suspension cells of Chenopodium rubrum, however, grow strictly photoautotrophically, that is, require no organic carbon source and resemble mesophyll cells by their photosynthetic capacity (Hfisemann and Barz 1977). Also, the lipid composition of photoautotrophically grown cultures closely resembles that of intact mesophyll cells, while the lipid composition of heterotrophically grown cultures is very distinct (Hiisemann et al. 1980). These Chenopodium suspension cells seem particularly well suited for studies of turgor relaxation processes and of the water transport parameters in leaf cells using the miniaturized pressure probe (Hiisken et al. 1978, Zimmermann et al. 1980, Zimmermann and Steudle 1980). Such measurements on suspended higher plant cells lend themselves to a straightforward analysis of turgor pressure relaxation curves on the basis of the phenomenological equations of the thermodynamics of irreversible processes, because these equations are presently derived only for single cells bathed in a large volume of solution. In addition, cell volume and surface area can be determined quite accurately, while this does not apply to cells within a leaf tissue. Hitherto, inaccuracy in the calculation of volume and surface area of tissue cells prevented
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K.-H. Bfichner et al.: Water Relations of Suspension-cultured cells
the precise d e t e r m i n a t i o n o f t h e h y d r a u l i c m e m b r a n e c o n d u c t i v i t y , L p , a n d its p o s s i b l e d e p e n d e n c e u p o n p h y s i o l o g i c a l c o n d i t i o n s like w a t e r stress o r a l i g h t / d a r k r e g i m e . ( Z i m m e r m a n n et al. 1980). In this c o m m u n i c a t i o n w e p r e s e n t m e a s u r e m e n t s o f t u r g o r p r e s s u r e a n d w a t e r r e l a t i o n p a r a m e t e r s in p h o t o a u t o t r o p h i c a l l y g r o w n s u s p e n s i o n cells a n d in i n d i v i d u a l cells o f i n t a c t leaves o f Ch. rubrum. In a d d i t i o n , s i m i l a r e x p e r i m e n t s are r e p o r t e d f o r s u s p e n s i o n c u l t u r e d cells o f Ch. rubrurn w h i c h h a v e b e e n i m m o b i lized w i t h i n a c r o s s - l i n k e d a l g i n a t e m a t r i x , as des c r i b e d p r e v i o u s l y f o r g u a r d cells a n d m e s o p h y l l cell p r o t o p l a s t s o f V i c i a f a b a a n d red b l o o d cells ( P i l w a t et al. 1980, S c h e u r i c h et al. 1980, S c h n a b l et al. 1980). By m e a n s o f this t e c h n i q u e , t h e cells are s u r r o u n d e d b y an a r t i f i c i a l u n s t i r r e d layer. T h i s m o d e l e x p e r i m e n t t h e r e f o r e s i m u l a t e s the s i t u a t i o n o f a cell w i t h i n the l e a f tissue a n d s h o u l d p r o v i d e i n f o r m a t i o n c o n c e r n i n g the v a l i d i t y o f the w a t e r t r a n s p o r t e q u a t i o n s to indiv i d u a l cells w i t h i n a tissue. A p r e l i m i n a r y a c c o u n t o f o u r results h a s b e e n g i v e n a l r e a d y ( B f i c h n e r et al. 1980).
Pressure Probe Technique. Cell turgor pressure, P, turgor pressure
Materials and Methods Cell Suspension Cultures. Photoautotrophically growing cell suspensions of Chenopodium rubrum were obtained from Dr. W. Hfisemann, Mfinster. These cells were derived from a hypocotyl callus culture. The suspension culture technique, according to Hfisemann and Barz (1977), employed a two-compartment culture vessel. The upper compartment contains the cell suspension culture ; the lower compartment provides the CO 2 from a 2M K2COJKHCO 3 solution to the cell suspension. The culture medium contained the mineral ions and vitamines of Murashige and Skoog (1962) (MSmedium), supplemented with 10 -v M 2.4-dichlorophenoxyacetic acid. The cultures were grown at 25~176 under 19Wm -2 of white fluorescent light on a gyratory shaker at 120 rpm. The cultures were transferred into new medium every 14 days.
Test Media Most experiments were carried out in the above quoted MS-medium. Alternatively, for some experiments a low ionic strength test medium (CH-medium) was used. The ionic strength of this medium is comparable to the ionic strength of the solution in the cell wall free space of leaves, according to a study on leaf tissue of Ricinus, Helianthus, and Brassica (Bernstein 1971). The composition of the CH-medium is given in Ohkawa et al. (1980), except for the concentration of KNO3 which is 0.01 mM in the present study. The Chenopodium suspension ceils continued to grow in this medium for at least 8 days without changing their microscopic appearance. The cells were transferred from the MS-medium to the CH-medium 16h before the experiments.
Osmotarity Assay. The osmolarity of the media and of the cell sap of Ch. rubrum ceils was determined cryoscopically (Knauer Osmometer). A cell sample was washed with distilled water freezed to 186~ and centrifuged at 12,000 rpm for 15 min using an Eppendorf centrifuge. The internal osmolarity of the cells depended on medium. It was 198 mosmol when the cells were kept in the MS-medium and 168 mosmol when kept for 16 h in the CH-medium. The osmolarity of the MS- and CH-medium was 81 and 9 mosmol, respectively.
relaxation processes, and the volumetric elastic modulus, e, were determined using the miniaturized pressure probe technique (H/isken et al. 1978; Zimmermann and Steudle 1978; Zimmermann 1978, I980; Zimmermann et al. 1980). For impalement with the microcapillary, the cell was arrested at the edge of a piece of glass glued to a cover slip, as used previously for electrophysiological measurements (Bentrup 1970; Ohkawa et al. 1980). Upon successful impalement the microcapillary is withdrawn from the edge so that the cell is completely surrounded by the medium. The cell turgot is transmitted from the cell interior to a pressure transducer by an oil-filled microcapillary with a 2-5 pm tip diameter. 1"he oil/ceil sap boundary within the capillary can be shifted manually or electronically by measuring the electrical resistance between a silver wire electrode inserted into the very tip of the microcapillary and an external reference electrode. From the shift in the boundary the change in cell volume, A V, can be calculated. If the capillary tip were clogged, this shift of the boundary would be restricted. From the volume change, A V, and the measured change in turgor pressure, A P, the volumetric elastic modulus, e, is obtained (Philip 1958): dP ~ = A ~ V.
(1)
The determination of e requires that the time for the change in volume is small compared with the half-time of water exchange. (see Results).
Immobilization Technique. A commercial alginate polymer (Manucol DH, Alginate Industries, London) with a molecular weight of 90,000 was used. The alginate was dissolved in the MS-culture medium at a concentration of 6 and 8% w/w, respectively. The cells were mixed thoroughly with a small amount of this alginate solution. Cross-linking of the alginate polymers was achieved by adding some drops of a 30 mM CaC12 solution. The reaction was completed after 30 rain at room temperature. After replacement of the CaCla/MS-medium by the culture medium the pressure probe was inserted into the immobilized cells.
Results T h e n o r m a l t u r g o r p r e s s u r e in cells o f Ch. rubrum was a b o u t 3 to 5 bar. O c c a s i o n a l l y , v a l u e s o f a b o u t 2 b a r w e r e r e c o r d e d . A s t e a d y t u r g o r p r e s s u r e was n o r m a l l y e s t a b l i s h e d w i t h i n 10 to 20 rain a f t e r insert i o n o f the p r e s s u r e p r o b e tip. A c o n s t a n t t u r g o r pressure v a l u e c o u l d b e m o n i t o r e d f o r s e v e r a l h o u r s if n o m a n i p u l a t i o n o f the t u r g o r p r e s s u r e was perf o r m e d . T h e t u r g o r p r e s s u r e r e l a x a t i o n was g e n e r a t e d by c h a n g i n g t h e t u r g o r p r e s s u r e by m e a n s o f t h e pressure p r o b e . In s o m e e x p e r i m e n t s (see T a b l e 1) the t u r g o r p r e s s u r e was c h a n g e d o s m o t i c a l l y b y a d d i t i o n o f a 50 m M s u c r o s e s o l u t i o n to the e x t e r n a l m e d i u m . Typical pressure relaxation curves for both end- and e x o s m o t i c w a t e r f l o w i n i t i a t e d by d i r e c t t u r g o r press u r e c h a n g e s are s h o w n in Fig. 1. T h e i n d u c e d t u r g o r p r e s s u r e c h a n g e at the o n s e t o f the r e l a x a t i o n p r o c e s s was a b o u t 1 - 2 b a r in b o t h d i r e c t i o n s . T h e f l u c t u a t i o n s t h a t are s u p e r i m p o s e d on t h e r e c o r d e d p r e s s u r e v a l u e s result f r o m the m a n u a l o r a u t o m a t i c r e g u l a t i o n o f the o i l / c e l l sap b o u n d a r y in t h e c a p i l l a r y tip. It is
K.-H. Bfichner et al.: Water Relations of Suspension-cultured cells
97 AV [p[l
A Q.
%.08 57.25 ?0.72 53.88 r 26.94---1] 16.84q],
B
74.08p!
C
a3~:g~Jill It60.61
. . . . . . 47.14J il "33.67 avlptj 67 35--11-~94.29
94.290I I I
I
I
I
I
I
I
I
50
100
150
200
250
300
350
t [s] Fig. 1 A-C. Typical recording of cell turgor pressure changes of a suspension cultured cell of Chenopodium rubrum (V= 0.98 nl) induced by the pressure probe. Exosmotic (A) and endosmotic (B) waterflow are recorded as pressure relaxations due to increasing and decreasing cell volume by the pressure probe. The noise of the trace is due to the electronic control of the pressure probe device which adjusts the cell-sap/oil boundary in the microcapillary. (C) shows a subsequent e-measurement from which an e-value of 35 bar was calculated
evident that the pressure-relaxation curve follows an approximately exponential time course. For evaluation of the data, a curve was drawn by hand into the turgor-relaxation curves in order to average out the fluctuations. From the semilogarithmic plot of the data shown in Fig. 2 the rate constant, k, and consequently the half-time for water transport, T1/2 = in 2/k, is calculated to be Tu2=22.0 s (exosmotic) and T1/2=25.4 s (endosmotic). The agreement of these two values (within the limits of accuracy) demonstrates that there is no polarity in water flow. This conclusion was supported by 19 independent experiments, yielding an average value for the half-time of water exchange of 20 + 10 s (see Table 1). A change in the half-time was not observed when the cells were transferred from the MS-medium into the CH-medium (Table 1). It should also be noted that the halftime for the water exchange initiated by changes in the external osmolarity (i.e., by addition of sucrose) yields similar values, as indicated in Table 1. In some cases it was possible to perform several end- and exosmotic runs on the same cell (Table 1). The halftimes for end- and exosmotic water flow, respectively, agree within 10%, indicating the accurate reproducibility of the measurements and excluding the possibility of leakages. The finding that for exosmotic runs the final turgor pressure was higher and that for endosmotic runs the final turgor pressure was lower than the initial turgor pressure (see Fig. 1) is an additional proof that in both cases no leakages occurred during the measurements. If leakages had occurred the final pressure would be lower than the original one in exosmotic experiments. In any case, as men-
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