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Proton Symport
• JOUR.AL OF •
J Plant Physiol. Vol. 154. pp. 447-453 (1999)
Plan• Physioloay
http:/ /www.urbanfischer.de/journals/jpp
© 1999 URBAN & FISCHER
Effects of ABA on the Distribution of Sucrose and Protons Across the Plasmalemma of Pea Mesophyll Protoplasts. Suggesting a Sucrose/Proton Symport CHANA 0PASKORNKUL\ SYLVIA LINDBERG 2 ,
and }AN-ERIC TILLBERG 1
1
Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden
2
Department of Plant Physiology, Swedish University of Agricultural Sciences, Box 7047, S-750 07 Uppsala, Sweden
Received December 16, 1997 · Accepted July 15, 1998
Summary The effects of abscisic acid (10- 9 -10- 6 mol/L) were studied on light induced COrdependent 0 2 evolution, sucrose synthesis, efflux of sucrose and changes in cytosolic proton concentration in mesophyll protoplasts of Pisum sativum L. cv. Fenomen. Photosynthesis did not change with increased apoplastic ABA or sucrose concentrations although sucrose synthesis decreased in the presence of ABA. In the absence of apoplastic sucrose the sucrose efflux increased and was most pronounced at 10-7 mol/L ABA. The sucrose efflux also increased in the presence of ABA and sucrose together and was most pronounced at 10- 9 mol/L ABA and 20 mmol/L apoplastic sucrose. The cytosolic proton concentration decreased upon addition of ABA with or without sucrose, but increased upon addition of p-chloromercuribenzenesulfonic acid, a sucrose transport inhibitor. The cytosolic proton concentration decreased in the presence of 10- 9 mol/L ABA in combination with various apoplastic sucrose concentrations. In the presence of 10-7-10- 6 mol/L ABA in combination with apoplastic sucrose levels higher than 1 mmol/L, the cytosolic concentration of proton increases. The results indicate that there is a relationship between the external environment and the sucrose efflux and cytosolic proton concentration in pea mesophyll protoplasts and that there is a sucrose/ proton symport at the plasmalemma of pea mesophyll cells. They also show that sucrose increases the sensitivity to ABA in its action on sucrose efflux and cytosolic proton concentration.
Key words: ABA concentration, apoplastic space, cytosolic [W ], pea, photosynthesis, Pisum sativum, protoplast, sucrose concentration, sucrose efflux, sucrose/proton symport. Abbreviations: BCECF-AM = tetra[acetoxymethyl] ester of the fluorescent dye bis-carboxyethyl-carboxyfluorescein; PCMBS = p-chloromercuribenzenesulfonic acid. Introduction
Phloem loading is the transport of photosynthate from the mesophyll into the sieve tube-companion cell complex in the minor veins of the mature leaf. It provides the driving force for long-distance phloem translocation of photoassimilates, and thus it is an important determinant of the growth and development of the plant. Two theoretical concepts for phloem loading in source leaves are usually proposed: (1) symplastic loading via the continuity of the plasmodesmata
from mesophyll into the sieve tubes (Turgeon and Beebe, 1991), and (2) apoplastic loading of the sieve tubes, which requires the transport of photosynthates from the symplastic system of the mesophyll cells into the free apoplastic space, and subsequently energy-dependent, carrier-mediated transport into the sieve tube-companion cell complex (Bush, 1992). Although the apoplastic loading concept is still the commonly accepted hypothesis, in recent years there is a growing tendency to consider phloem loading as a process that may be exerted by different mechanisms in the various 0176-1617/99/154/447$ 12.00/0
448
CHANA 0PASKORNKUL, SYLVIA LINDBERG, and ]AN-ERic TILLBERG
plant species. Sugar-beet and pea are among the species being apoplastically loaded whereas, e.g. Coleus blumei is supposedly symplastically loaded. The different strategies probably have ecophysiological significance, as they are associated with specific environmental conditions (Van Bel, 1993). Efflux of sucrose from mesophyll cells into the free apoplastic space, in the vicinity of the conducting complex, is an inevitable step in the apoplastic loading concept. Changes of physical and chemical factors in the apoplastic space, such as sucrose and abscisic acid concentrations, may have important influence on both efflux of sucrose from the mesophyll cells and on the transport of sucrose into the sieve tube-companion cell complex. The estimated concentration of the apoplastic sucrose ranges between 0.1 and 5 mmol/L in several species and has been found to vary on a diurnal basis within a plant leaf. It has been suggested that apoplastic sucrose may play an important role in the induction of a putative phloemlocated sucrose carrier, leading to vein loading and export from the leaf (Giaquinta, 1983). In a previous report (Opaskornkul et al., 1994), we demonstrated that sucrose efflux from the mesophyll cells is influenced by the apoplastic sucrose concentration. ABA may be involved in phloem unloading of photoassimilates by regulating the sink strength in several sink tissues (Brenner and Cheikh, 1995). The endogenous concentration of ABA in the apoplast can increase a hundred fold very rapidly during stress (Hartung and Radin, 1989). Inhibition of photosynthesis by ABA in an intact leaf occurs via the impact of ABA on stomatal closure. However, the primary effect of ABA on phloem loading of sucrose is not clearly demonstrated (Morris, 1996). Despite the importance of sucrose efflux in the apoplastic loading hypothesis, no direct experimental evidence for such a mechanism has been shown, the preferential site of sucrose release from the mesophyll cells into the apoplastic space is also still unknown. Moreover, a study of the environmental conditions in the free space regarding the regulation of sucrose efflux has not been reported. To contribute to a better knowledge of an apoplastic loading mechanism and of the sucrose-efflux step from mesophyll cells adjacent to the minor vein, we investigated the efflux of newly-formed sucrose from the mesophyll protoplasts of source leaves under different conditions of an artificial apoplastic sucrose and ABA pool. We also investigated photosynthesis and sucrose synthesis as well as the cytosolic pH changes in the protoplasts upon addition of ABA and sucrose.
Materials and Methods Growth conditions Pea plants (Pisum sativum L. cv. Fenomen) were grown in an aerated Hoagland solution in a climate chamber (Eliasson, 1978). They were irradiated for 16 h with a PPFD of 300 Jlmol m- 2s-1 at 25 "C and RH 85 %, and kept in the dark for 8 h at 18 "C and RH 65%.
Protoplast isolation Protoplasts were isolated according to the method of Opaskornkul et al. (1994). The purified protoplast stock was kept on ice until
use (within 4 hours after isolation). The viability and activities of the protoplasts from the same preparation were studied before and after the experiments mentioned below by vital staining with Evans blue and by measurements of COrdependent 0 2 evolution in a photosynthetic-assay medium (see below).
Measurement ofphotosynthesis and respiration The C02-dependent 02 evolution and the 0 2 consumption of the protoplasts were followed in the presence of sucrose in combinations with ABA at different concentrations. Mesophyll protoplasts corresponding to 50-60 Jlg Chl mL -I were preincubated for 5 min in the dark at 25 "C in a photosynthetic-assay medium (0.4 mol!L sorbitol, 1 mmol!L CaC[z, 10 mmol!L NaHC0 3 , 1 mmol/L EDTA, 25 mmol/L Hepes-KOH, pH 7.8) containing ABA and/or sucrose. The final concentrations of sucrose in the medium were 1, 5 and 20 mmol!L and of ABA in the range of 10- 9 to 10- 6 mol!L. The photosynthetic experiments were carried out using a waterjacketed Clark-type oxygen electrode (Hansatech LTD, King's Lynn, UK) at 2TC and a PPFD of300J.imolm- 2 s-1, provided by a 250W-fiber optic lamp at the surface of the electrode cell. To prevent protoplast destruction, the magnetic stirring speed was set at a minimum while still maintaining a stable response. The vitality of the protoplasts was controlled afterwards. The respiration experiments were carried out in the same way but in darkness. Chlorophyll content was measured according to Sestak et al. (1971).
Measurement of 14C-sucrose synthesis The measurements of synthesis of 14C-sugars in protoplasts were carried out in the same photosynthetic-assay medium as mentioned above; however with the addition of 14C-NaHC0 3 (370 MBq mL - 1), the final concentration of NaHC0 3 was 5 mmol!L, final concentrations of sucrose ranged from 0 to 20 mmol!L, and ABA from 10- 9 to w- 6 mol!L. Each sample of 1 mL, corresponding to 100 Jlg Chl mL -I, was incubated for 20 min at 27"C in a shaking water bath at 30 strokes per min and was illuminated with incandescent spotlights from under the water bath, which provided a PPFD of 275 Jlmol m - 2s-1 at the sample level. At the end of the incubation period, the vials with the samples were immediately frozen in liquid nitrogen and kept at -18 "C until analysis. Methods for extraction and purification of sugars as well as determination of the content of 14C-sugars by HPLC and a scintillation counter technique were carried out according to Opaskornkul et al. (1994).
Determination of 14 C-sucrose efflux Protoplasts were suspended in a 100-mL Erlenmeyer-flask with photosynthetic assay medium (0.4 mol!L sorbitol, 2 mmol!L NaHC0 3, 1 mmol!L EDTA, 25 mmol/L Hepes-KOH, pH 7.8) and 14C-NaHC0 3 (740 MBq mL -I) to obtain a suspension of 10 mL corresponding to 100 Jlg Chl mL -I. After incubation for 5 min under the same conditions as above, the protoplast suspension was washed 2 X 3 min at 100 gn with ice-cold buffer. After the last centrifugation, the suspension was divided equally into 20 centrifuge tubes. The protoplasts in each tube were suspended in 1 mL photosyntheticassay medium containing ABA and/or sucrose. The final concentrations of sucrose and ABA in the medium ranged from 0-20 mmol! L and 10- 9 to w- 6 mol!L, respectively. Determination of 14C-sucrose efflux was according to Opaskornkul et al. (1994).
Determination ofintracellular pH With some modifications, determination of the cytosolic concentration of H+ was carried out according to the method of Lindberg
ABA Effect on Sucrose and Protons Distribution and Strid (1997). Mesophyll protoplasts were loaded with 8 Jlmoi!L tetra[acetoxymethyl] ester of the fluorescent dye bis-carboxyethylcarboxyfluorescein, BCECF-AM (Molecular Probes, Eugene, Ore., USA) at 4 'C in a dye loading buffer (0.5 moi!L sorbitol, 1 mmoi!L CaC1 2, 0.5 mmoi!L MgC1 2, 0.05 % PVP, 0.2% BSA, 5 mmoi!L MES-KOH, pH 5.5). The protoplast suspension was washed once with the same medium as above before dye loading. During loading, non-specific esterase activity of the cytoplasm is assumed to split the ester into an ion-binding carboxylate form, thereby changing its fluorescence excitation spectra (Gillespie, 1989). After 1 h of incubation in the loading medium, the protoplast suspension was washed once with the same medium; thereafter, the protoplast suspension was centrifuged at 100 gn for 3 min, and the supernatant was removed and replaced with a measuring buffer (0.5 mol/L sorbitol, 1 mmol/L CaClz, 5 mmol/L KCl, 0.05 % PVP, 0.5% BSA, 5 mmol!L Tris-HCl, pH 7). The dye-loaded protoplasts were kept at 4 'C until measurement. Fluorescence measurements were carried out on a single protoplast in the same buffer before and after application of ABA, sucrose or the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS; Sigma). Control tests were performed using glucose, fructose or a non-reducing disaccharide cellobiose. Micro slides were covered with poly-L-lysine (MW 150,000-300,000; Sigma) in order to attach protoplasts to their surface. Application of different solutions was done with a microsyringe and fluorescence measurements were performed at room temperature (20 ± 2 'C). An epi-fluorescence microscope (Axiovert 10; Zeiss, Oberkochen, Germany), supplied with an electromagnetic filter-exchanger (Zeiss), xenon lamp (Zeiss XBO 75), photometer (Zeiss 01), microprocessor (MSP 201, Zeiss) and an IBM computer (PS/2), was used to determine fluorescence intensity at the excitation ratio 485/ 436nm. Emission wavelengths at 510-550nm were monitored. All measurements were performed with a Planneofluar X 40/0.75 objective (Zeiss) for phase contrast. Adjustments of signals and noise were made automatically. By means of ratio microscopy, the effect of different dye concentrations were eliminated (Bright et al., 1987).
Statistical analyses The values presented are the means of duplicate samples of four or five independent experiments. In the determination of intracellular pH, 10 replications each from five independent protoplast batchs were used. For the comparison of mean values between sucrose and/ or ABA treatments and controls, the t-test was used.
protoplasts after 10 min in light were included in some experiments. PCMBS, however, did not change the rates of Oz evolution nor 0 2 consumption (not shown). In contrast to the absence of effects on light induced Oz evolution, ABA inhibited 14 C-sucrose synthesis (Fig. 1a). All protoplast suspensions incubated in light in an ABA medium without sucrose accumulated up to 25 o/o less 14C-sucrose than the control samples without ABA. The highest inhibiting effects were found at 10-7 mol/L ABA. The inhibition by ABA was partly reversed by adding sucrose to the ABA medium (Fig. 1 b), although an exception to this was the combination of 10- 9 mol/L ABA and 5 mmol/L sucrose, in which there was a 30 o/o decrease in sucrose synthesis. There was a distinct stimulating effect of ABA in the incubating medium on the effiux of sucrose from the protoplasts (Fig. 2 a), and this effect was dependent on the presence of sucrose in the medium (Fig. 2 b). Without sucrose in the medium, the effiux of newly synthesized 14C-sucrose into the surrounding medium increased at all ABA concentrations tested and peaked at 10-7 mol/L ABA, at which the sucrose effiux was about 180 o/o higher compared with the control (Fig. 2a). The effects of ABA varied in the presence of sucrose at various concentrations (Fig. 2 b). At 10- 6 mol!L ABA, the effiux of 14C-sucrose was not clearly affected at all external sucrose concentrations. In the presence of 10- 9 -10-7 mol/L ABA, the combined action with sucrose was most prominent. Effiux of 14C-sucrose into the surrounding medium increased steadily with the decrease of ABA concentration in the medium and the highest effiuxes were found at 10- 9 mol/L ABA. The ex-
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Our previous investigation (Opaskornkul et al., 1994) showed that exogenous sucrose (0-20 mmol/L) had no effect on 0 2 evolution nor 0 2 consumption. In our present investigation, no effect on the rate of 0 2 evolution nor 0 2 consumption by sucrose (0, 1, 5, 20 mmol/L) and ABA (lo- 9 to 10- mol/L) at various combinations of concentrations in the medium was observed (not shown). Also a direct application of sucrose and ABA to the medium of photosynthesizing protop lasts did not change the rates of 0 2 evolution nor 0 2 consumption. To rule out that PCMBS purchased from Sigma might contain contaminating levels of Hg2+, which might in turn have a pronounced effect on the respiration of the pea protoplasts, control tests were performed regarding the effects of PCMBS. A direct application of PCMBS at a final concentration of 100 Jlmol/L to the medium of photosynthesizing
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ABA concentration in the medium (moi/L) Fig. 1: Change of 14C-sucrose synthesized in pea mesophyll protoplasts during photosynthesis for 20 min in a medium containing H 14 C0 3- and different combinations of sucrose and ABA concentrations. (a) at 0 mmol!L sucrose, (b) at 1-20 mmol!L sucrose. Amount of 14C-sucrose at 0 mmol!L sucrose and 0 mol/L ABA = 100%.
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served at 1 mmol/L sucrose (Table 2). Increasing concentrations of sucrose in the medium caused a successively lesser decline of the [H+] in the protoplasts. Control tests were performed regarding the effects on cytosolic [H+] by sugars other than sucrose. In contrast to the effect of sucrose, the cytosolic [H+] increased after addition of glucose or fructose (Table 3). Addition of PCMBS into the
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Fig. 2: Change of 14C-sucrose efflux from pea mesophyll protoplasts during photosynthesis for 15 min in a medium containing H 14C0 3and different combinations of sucrose and ABA concentrations. (a) at 0 mmol!L sucrose, (b) at 1-20 mmol!L sucrose. Amount of 14C-sucrose at 0 mmol!L sucrose and 0 mol/L ABA= 100 o/o.
ternal sucrose concentration was also important for the efflux rate. The higher the sucrose concentration the more the effect on the efflux of 14C-sucrose at 10- 9 mol/L ABA. Compared with the control, the efflux of 14C-sucrose in the presence of 10- 9 mol!L ABA was 135% to 290% at 1 and 20 mmol/L external sucrose, respectively. The cytosolic concentration of H+ in the protoplasts was estimated by the use of BCECF. Without any addition, there was no change in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a pea protoplast (not shown), and it remained stable for at least 10 min at experimental temperature. The addition of different concentrations of ABA into a medium without sucrose caused a transient increase in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a pea protoplast. This reflects an increase in cytosolic pH (i.e. [H+] in the cytosol decreased, Fig. 3); thereafter, the fluorescence ratio stabilized. The largest decrease of [H+] was about 12% and was most pronounced at 10- 8 mol!L ABA (Table 1). On the other hand, addition of 100 Jlmol!L PCMBS after the addition of ABA caused a 10 % increase in cytosolic [H+]. To investi§ate whether the effect of ABA on the decrease of cytosolic [H ] was due to a general acidification, HCl at the corresponding concentration was used instead of ABA. This treatment did not change the cytosolic [H+] in the protoplast significantly (data not shown). Addition of different concentrations of sucrose into a medium without ABA also caused a transient increase in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a protoplast, reflecting an increase in cytosolic pH ([H+] decreased). The largest decrease of [H+], about 10%, was ob-
ABA llf6 M-rCMRS 100 mM-
Fig. 3: Effects of ABA at various concentrations or of PCMBS on the BCECF fluorescence intensity ratio (485/436 nm) with time in a pea mesophyll protoplast. The figure shows a representative trace. Table 1: Immediate changes in the cytosolic concentration of H+ in pea protoplasts upon addition of ABA and PCMBS with and without prior addition of sucrose. All values ± SE are means of 5 X 10 replicates. Sucrose concentration (mmol!L)
0
ABA concentration (mmol!L)
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Changes in [H+] (o/o)
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ABA Effect on Sucrose and Protons Distribution samples with hexoses did not cause a transient change of cytosolic [H+]. Control tests were also performed using a nonreducing disaccharide cellobiose. The cytosolic [H+] remained unchanged after addition of this sugar (Table 3). Addition of PCMBS into the samples with cellobiose did not cause a transient change of cytosolic [H +]. In the presence of both ABA and sucrose at different concentrations, the effect of ABA on cyrosolic pH varied (Table 9 mol/L ABA, subsequent addi2). With an incubation of tion of 1, 5 and 20 mmol/L sucrose to the medium caused a transient decrease in cyrosolic [H+]. The most pronounced decrease of [H+], about 11 %, took place in the presence of 5 mmol/L sucrose at 10- 9 mol/L ABA.
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Table 3: Immediate changes in the cytosolic concentration of H+ in pea protoplasts upon addition of ABA and PCMBS with and without prior addition of different kinds of sugars. All values ± SE are means of7 replicates. Sugar concentration (mmol/L) glucose
1 mmol!L
ABA concentration (mol!L)
5 mmoi/L
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Fig. 4: Effects of sucrose at various concentrations or PCMBS on the BCECF fluorescence intensity ratio (485/436 nm) with time in a pea mesophyll protoplast with prior addition of 10- 9 mol!L ABA. The figure shows a representative trace.
Except for samples with 1mmol/L sucrose, sucrose in combinations with ABA higher than 10- 9 mol/L caused a transient increase in cyrosolic [H+] (Table 2). The largest increase of [H+], around 13%, was obtained at 20 mmol/L sucrose 7 mol/L ABA. At all ABA concentrations tested, and PCMBS showed a transient increase in cytosolic [H+]. The maximal increase of internal [H+] upon addition of PCMBS was about 25 %, obtained after addition of 20 mmol/L sucrose in combination with 10- 9 mol/L ABA (Table 2 and Fig. 4).
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Table 2: Immediate changes in the cytosolic concentration of H + in pea protoplasts upon addition of sucrose and PCMBS with or without prior addition of ABA at different concentrations. All values ± SE are means of 5 X 10 replicates. ABA concentration (mol!L)
451
Discussion
The results from our previous investigation (Opaskornkul et al., 1994) supported the concept of an apoplastic phloem loading of sucrose in pea leaves. It also demonstrated that the sucrose efflux from the mesophyll cells is influenced by the apoplastic sucrose concentration. In the present paper, the results suggest that the mechanism responsible for export of sucrose from the mesophyll cells into apoplastic space is a sucrose/H+ cotransport and this mechanism is substantially influenced by various factors in the apoplastic space, such as the sucrose concentration and the ABA concentrations.
452
CHANA 0PASKORNKUL, SYLVIA LINDBERG,
and ]AN-ERIC TILLBERG
In the experiments with application of exogenous sucrose but without ABA (Table 2), the decrease in cytosolic [H+) was more pronounced at I-5 mmol/L sucrose than at 20 mmol/L sucrose. These concentrations of sucrose had the strongest stimulating effects on the efflux of newly synthesized sucrose from the pea mesophyll cells against a concentration gradient according to our previous work. The coincidence of the two events thus indicates a sucrose/H+ cotransport. At sucrose concentrations higher than 5 mmol/L, the change in cyrosolic [H+) was less evident (Table 2), although there was still some sucrose efflux. This may indicate that the export of sucrose into the apoplast occurred via other mechanisms than by a sucrose/H+ cotransport at sucrose concentrations more than 5 mmol/L. As there was also a sucrose efflux in the absence of external sucrose (Fig. 2A), but no change of cytosolic [H+) before the addition of sucrose (not shown), a simple diffusion is likely to be responsible for some part of the sucrose efflux. The cyrosolic [H+) increased after addition of glucose or fructose (Table 3). The lack of cytosolic [H+) decrease upon the addition of a non-reducing disaccharide cellobiose, glucose or fructose indicates that the decrease of cytosolic [H+) was induced mainly by sucrose, and it may also indicate that the pea mesophyll cells probably possess a hexose/H+ symport mechanism in the plasmalemma, which permits them to retrieve the hexoses from the apoplastic space. Cyrosolic [H+) increased as a result of uptake of hexoses into the cytosol. Mter the transition of the leaf tissue from sink to source stage, the mesophyll cells from several plant species lose their ability to take up hexoses from the apoplast (Quick and Schaffer, I996). In the protoplasts treated with various low concentrations of ABA, ABA induced cyrosolic alkalinization, resulting in a decrease of cyrosolic [H+) (Table I). Similar results were obtained in other plant organs as reported by Gehring et a!. (1990) and Van der Veen et a!. (1992). The same concentrations of ABA also have the most pronounced effects on the efflux of sucrose (Fig. 2 a). The coincidence of the two events supports the concept of a sucrose/H+ cotransport mechanism. ABA at these concentrations (below w-7 mol/L) seems to be optimal for promotinf the export of sucrose. At higher ABA concentrations (>10- mol/L), the decrease in the cytosolic [H+) as well as the efflux of sucrose were less than at ABA concentrations below w-7 mol/L. A decrease in the translocation of sucrose into the phloem caused by high ABA concentrations has been reported by Tietz et a!. (I98I), and this decrease in turn may be the result of the decline of the efflux of sucrose and H+ from the mesophyll cells. ABA at concentrations beyond w-7 mol/L might induce a stress condition to the mesophyll cells. It is not clear, however, how ABA might have an impact on sucrose efflux in both stress and non-stress conditions. PCMBS is a non-permeant potent inhibitor of sucrose/H+ symport. It inhibits phloem loading and blocks the efflux of sucrose into the apoplast, implying the impairment of apoplastic phloem loading (Van Bel, 1993). In the present study, PCMBS caused an increase of cytosolic [H+], after addition of the inhibitor to samples containing ABA or sucrose (Tables 1 and 2), but PCMBS failed to give similar results in the samples with hexoses (Table 3). This corroborates with the
concept of a sucrose/H+ symport. The concept of a sucrose/H+ symport at the plasmalemma was suggested by Giaguinta (1977) and confirmed by Bush (1989), based on data from experiments using plasmalemma vesicles from sugar beet leaves. No data have been previously reported on a mechanism for sucrose loading into the apoplastic space in pea plants. Part of the decrease of cyrosolic [H+) might also be the result of a sucrose/H+ antiport activity at the tonoplast membrane of the mesophyll cells (Bush, I992). This can occur when the sucrose previously stored in the vaculoes is released into the cytosol for export. Both ABA and sucrose are regulators of the sucrose/H+ symport, but by different mechanisms. ABA may be involved in many cytoplasmic events, including promotion of an increase in cytosolic [Ci+) and a decrease in cytosolic [K+) (Blatt and Thiel, I993). This leads to an increase in cytosolic [H+], which in turn may have a promoting effect on the sugar transport mechanism. The sucrose may directly affect the sugar transport protein itself, and it has been suggested that the apoplastic sucrose may provide the energy for vein loading in leaf tissue (Pitcher et a!., I99I). A combination of external ABA and sucrose resulted in a different impact on the sucrose efflux compared with the effects by ABA or sucrose added separately to the samples (Fi~. 2 b, Tables I and 2). The optimal combination was 10- mol/L ABA and 20 mmol/L sucrose. The high effectiveness at a lower ABA concentration (10- 9 mol/L) in this combined action may be due to the presence of sucrose. Sucrose might increase receptivity of the ABA binding protein at the plasma membrane; the stimulating effect of ABA, therefore, reaches the optimal condition at a lower concentration. It is known that the inhibition of photosynthesis caused by the stress hormone ABA in an intact leaf occurs via the effects of ABA on the closure of the stomata. The lack of direct effects of ABA on the photosynthetic rate in isolated mesophyll cells, however, has been reported for various plant species (Mawson et a!., 1981; Raschke and Hedrich, 1985). Photosynthesis in the protoplasts prepared for our experiment was not affected by ABA in any of the apoplastic sucrose concentrations tested (not shown). On the other hand, suppression by ABA of the 14 C-sucrose synthesis in the cell was very pronounced (Fig. I), whereas exogenous sucrose does not exert this effect, as shown in our previous study (Opaskornkul et a!., I994). It is not clear, however, how ABA interferes with the sucrose synthesis process, but it is probably due to effects on the enzyme activity of Rubisco by reduction of the carboxylase activity or increase of the oxygenase activity (Brenner and Cheikh, I995). Direct effects, however, of ABA on FBPase or SPS activities, which are the main regulators of the sucrose synthesis process, have not been reported. As abscisic acid-binding protein is thought to be plasma membrane-localized (Hahn, I996), it is unlikely that ABA would have a direct effect on the cytoplasmically-localized sucrose synthesis process. References M. R. and M. THIEL: Hormonal control of ion channel gating. Annu. Rev. Plant Physiol. 44, 543-567 (1993).
BLATT,
ABA Effect on Sucrose and Protons Distribution BRENNER, M. L. and N. CHEIKH: The role of hormones in photosynthate partitioning and seed filling. In: DAVIES, P. J. (ed.): Plant hormones, Physiology, Biochemistry and Molecular Biology, pp. 649-670. Kluwer Academic Publishers, The Netherlands (1995). BRIGHT, G. R., G. W FISHER, J. RoGOWSKA, and D. L. TAYLOR: Fluorescence ratio imaging microscopy: Temporal and spatial measurements of cytoplasmic pH. J. Cell Bioi. 104, 1019-1033
(1987).
BusH, D. R.: Proton-coupled sucrose transport in plasmalemma vesicles isolated from sugar beet (Beta vulgaris L. cv. Great western) leaves. Plant Physiol. 89, 1318-1323 (1989). BusH, D. R.: The proton-sucrose symport. Photosynthesis Research 32, 155-165 (1992). ELIASSON, L.: Effects of nutrients and light on growth and root formation in Pisum sativum cuttings. Physiol. Plant. 43, 13-18
(1978).
GEHRING, C. A., H. R. IRVING, and R. W. PARISH: Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells. Proc. Nat!. Acad. Sci. 87, 9645-9649 (1990). GIAQUINTA, R. T.: Possible role of pH gradient and membrane ATPase in the loading of sucrose into the sieve tube. Nature 267,
369-370 (1977).
- Phloem loading of sucrose. Annu. Rev. Plant Physiol. 34, 347-
387 (1983).
GILLESPIE, J. 1.: Microspectrofluorimetry. Seeing into the future of cell physiology. Microscopy Anal. 13, 17-20 (1989). HAHN, M. G.: Signal perception at the plasma membrane: binding proteins and receptors. In: SMALLWOOD, M., J. P. KNox, and D. J. BowLES (eds.): Membranes: Specialized Functions in Plants, pp. 103-126. Bios Scientific Publishers LTD, The UK (1996). HARTUNG, Wand J. W RADIN: Abscisic acid in the mesophyll apoplast and in the root xylem sap of water -stressed plants: the significance of pH Gradients. Current Topics in Pl. Biochem. and Physiol. 8, 110-124 (1989). LiNDBERG, S. and H. STRID: Aluminium induces rapid changes in the cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum). Physiol. Plant.
99, 405-414 (1997).
453
MAWSON, B. T., B. CoLMAN, and W R. CuMMINS: Abscisic acid and photosynthesis in isolated leaf mesophyll cell. Plant Physiol.
67, 233-236 (1981).
MoRRIS, D. A.: Hormonal regulation of source-sink relationships: An overview of potential control mechanisms. In: ZAMsKI, E. and A. A. ScHAFFER (eds.): Photoassimilate Distribution in Plants and Crops, Source-Sink Relationships, pp. 441-466. Marcel Dekker, Inc. USA (1996). 0PASKORNKUL, C., M. GREGER, and J.-E. TILLBERG: Effects of apoplastic sucrose on carbohydrate pools and sucrose effiux of mesophyll protoplasts of pea (Pisum sativum). Physiol. Plant. 90,
685-691 (1994).
PITCHER, L. H., J. DAIE, and R. E. WvsE: Does apoplastic sucrose induce the ability for sucrose loading in developing leaves of sugarbeet? Plant Cell Physiol. 32, 705-711 (1991). QmcK, W P. and A. A. ScHAFFER: Sucrose metabolism in sources and sinks. In: ZAMSKI, E. and A. A. ScHAFFER (eds.): Photoassimilate Distribution in Plants and Crops, Source-Sink Relationships, pp. 115-156. Marcel Dekker, Inc. USA (1996). RAscHKE, K. and R. HEDRICH: Simultaneous and independent effects of abscisic acid on stomata and the photosynthesic apparatus in the whole leaves. Planta 163, 105-118 (1985). SESTAK, Z., J. CATSKY and P. G. JARVIS: Determination of Chlorophyll a & b. In: Plant Photosynthetic Production, Manuals of Methods, pp. 672-698. Dr. W Junk N. V. Publishers, The Netherlands (1971). TIETZ, A., M. LuDEWIG, M. DINGKUHN, and K. DCiRFFLING: Effects of abscisic acid on the tranport of assimilates in barley. Planta 152, 557-561 (1981). TuRGEON, R. and D. U. BEEBE: The evidence for symplastic phloem loading. Plant Physiol. 96, 349-354 (1991). VAN BEL, A. J. E.: Strategies of Phloem loading. Annu. Rev. Plant Physiol. 44, 253-281 (1993). VANDERVEEN, R., S. HEIMOVAARA-DIJKSTRA, and M. WANG: Cytosolic alkalinization mediated by Abscisic acid is necessary, but not sufficient, for Abscisic acid-induced gene expression in Barley Aleurone protoplasts. Plant Physiol. 100, 699-705 (1992).
J Plant Physiol. Vol. 154. pp. 447-453 (1999)
Plan• Physioloay
http:/ /www.urbanfischer.de/journals/jpp
© 1999 URBAN & FISCHER
Effects of ABA on the Distribution of Sucrose and Protons Across the Plasmalemma of Pea Mesophyll Protoplasts. Suggesting a Sucrose/Proton Symport CHANA 0PASKORNKUL\ SYLVIA LINDBERG 2 ,
and }AN-ERIC TILLBERG 1
1
Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden
2
Department of Plant Physiology, Swedish University of Agricultural Sciences, Box 7047, S-750 07 Uppsala, Sweden
Received December 16, 1997 · Accepted July 15, 1998
Summary The effects of abscisic acid (10- 9 -10- 6 mol/L) were studied on light induced COrdependent 0 2 evolution, sucrose synthesis, efflux of sucrose and changes in cytosolic proton concentration in mesophyll protoplasts of Pisum sativum L. cv. Fenomen. Photosynthesis did not change with increased apoplastic ABA or sucrose concentrations although sucrose synthesis decreased in the presence of ABA. In the absence of apoplastic sucrose the sucrose efflux increased and was most pronounced at 10-7 mol/L ABA. The sucrose efflux also increased in the presence of ABA and sucrose together and was most pronounced at 10- 9 mol/L ABA and 20 mmol/L apoplastic sucrose. The cytosolic proton concentration decreased upon addition of ABA with or without sucrose, but increased upon addition of p-chloromercuribenzenesulfonic acid, a sucrose transport inhibitor. The cytosolic proton concentration decreased in the presence of 10- 9 mol/L ABA in combination with various apoplastic sucrose concentrations. In the presence of 10-7-10- 6 mol/L ABA in combination with apoplastic sucrose levels higher than 1 mmol/L, the cytosolic concentration of proton increases. The results indicate that there is a relationship between the external environment and the sucrose efflux and cytosolic proton concentration in pea mesophyll protoplasts and that there is a sucrose/ proton symport at the plasmalemma of pea mesophyll cells. They also show that sucrose increases the sensitivity to ABA in its action on sucrose efflux and cytosolic proton concentration.
Key words: ABA concentration, apoplastic space, cytosolic [W ], pea, photosynthesis, Pisum sativum, protoplast, sucrose concentration, sucrose efflux, sucrose/proton symport. Abbreviations: BCECF-AM = tetra[acetoxymethyl] ester of the fluorescent dye bis-carboxyethyl-carboxyfluorescein; PCMBS = p-chloromercuribenzenesulfonic acid. Introduction
Phloem loading is the transport of photosynthate from the mesophyll into the sieve tube-companion cell complex in the minor veins of the mature leaf. It provides the driving force for long-distance phloem translocation of photoassimilates, and thus it is an important determinant of the growth and development of the plant. Two theoretical concepts for phloem loading in source leaves are usually proposed: (1) symplastic loading via the continuity of the plasmodesmata
from mesophyll into the sieve tubes (Turgeon and Beebe, 1991), and (2) apoplastic loading of the sieve tubes, which requires the transport of photosynthates from the symplastic system of the mesophyll cells into the free apoplastic space, and subsequently energy-dependent, carrier-mediated transport into the sieve tube-companion cell complex (Bush, 1992). Although the apoplastic loading concept is still the commonly accepted hypothesis, in recent years there is a growing tendency to consider phloem loading as a process that may be exerted by different mechanisms in the various 0176-1617/99/154/447$ 12.00/0
448
CHANA 0PASKORNKUL, SYLVIA LINDBERG, and ]AN-ERic TILLBERG
plant species. Sugar-beet and pea are among the species being apoplastically loaded whereas, e.g. Coleus blumei is supposedly symplastically loaded. The different strategies probably have ecophysiological significance, as they are associated with specific environmental conditions (Van Bel, 1993). Efflux of sucrose from mesophyll cells into the free apoplastic space, in the vicinity of the conducting complex, is an inevitable step in the apoplastic loading concept. Changes of physical and chemical factors in the apoplastic space, such as sucrose and abscisic acid concentrations, may have important influence on both efflux of sucrose from the mesophyll cells and on the transport of sucrose into the sieve tube-companion cell complex. The estimated concentration of the apoplastic sucrose ranges between 0.1 and 5 mmol/L in several species and has been found to vary on a diurnal basis within a plant leaf. It has been suggested that apoplastic sucrose may play an important role in the induction of a putative phloemlocated sucrose carrier, leading to vein loading and export from the leaf (Giaquinta, 1983). In a previous report (Opaskornkul et al., 1994), we demonstrated that sucrose efflux from the mesophyll cells is influenced by the apoplastic sucrose concentration. ABA may be involved in phloem unloading of photoassimilates by regulating the sink strength in several sink tissues (Brenner and Cheikh, 1995). The endogenous concentration of ABA in the apoplast can increase a hundred fold very rapidly during stress (Hartung and Radin, 1989). Inhibition of photosynthesis by ABA in an intact leaf occurs via the impact of ABA on stomatal closure. However, the primary effect of ABA on phloem loading of sucrose is not clearly demonstrated (Morris, 1996). Despite the importance of sucrose efflux in the apoplastic loading hypothesis, no direct experimental evidence for such a mechanism has been shown, the preferential site of sucrose release from the mesophyll cells into the apoplastic space is also still unknown. Moreover, a study of the environmental conditions in the free space regarding the regulation of sucrose efflux has not been reported. To contribute to a better knowledge of an apoplastic loading mechanism and of the sucrose-efflux step from mesophyll cells adjacent to the minor vein, we investigated the efflux of newly-formed sucrose from the mesophyll protoplasts of source leaves under different conditions of an artificial apoplastic sucrose and ABA pool. We also investigated photosynthesis and sucrose synthesis as well as the cytosolic pH changes in the protoplasts upon addition of ABA and sucrose.
Materials and Methods Growth conditions Pea plants (Pisum sativum L. cv. Fenomen) were grown in an aerated Hoagland solution in a climate chamber (Eliasson, 1978). They were irradiated for 16 h with a PPFD of 300 Jlmol m- 2s-1 at 25 "C and RH 85 %, and kept in the dark for 8 h at 18 "C and RH 65%.
Protoplast isolation Protoplasts were isolated according to the method of Opaskornkul et al. (1994). The purified protoplast stock was kept on ice until
use (within 4 hours after isolation). The viability and activities of the protoplasts from the same preparation were studied before and after the experiments mentioned below by vital staining with Evans blue and by measurements of COrdependent 0 2 evolution in a photosynthetic-assay medium (see below).
Measurement ofphotosynthesis and respiration The C02-dependent 02 evolution and the 0 2 consumption of the protoplasts were followed in the presence of sucrose in combinations with ABA at different concentrations. Mesophyll protoplasts corresponding to 50-60 Jlg Chl mL -I were preincubated for 5 min in the dark at 25 "C in a photosynthetic-assay medium (0.4 mol!L sorbitol, 1 mmol!L CaC[z, 10 mmol!L NaHC0 3 , 1 mmol/L EDTA, 25 mmol/L Hepes-KOH, pH 7.8) containing ABA and/or sucrose. The final concentrations of sucrose in the medium were 1, 5 and 20 mmol!L and of ABA in the range of 10- 9 to 10- 6 mol!L. The photosynthetic experiments were carried out using a waterjacketed Clark-type oxygen electrode (Hansatech LTD, King's Lynn, UK) at 2TC and a PPFD of300J.imolm- 2 s-1, provided by a 250W-fiber optic lamp at the surface of the electrode cell. To prevent protoplast destruction, the magnetic stirring speed was set at a minimum while still maintaining a stable response. The vitality of the protoplasts was controlled afterwards. The respiration experiments were carried out in the same way but in darkness. Chlorophyll content was measured according to Sestak et al. (1971).
Measurement of 14C-sucrose synthesis The measurements of synthesis of 14C-sugars in protoplasts were carried out in the same photosynthetic-assay medium as mentioned above; however with the addition of 14C-NaHC0 3 (370 MBq mL - 1), the final concentration of NaHC0 3 was 5 mmol!L, final concentrations of sucrose ranged from 0 to 20 mmol!L, and ABA from 10- 9 to w- 6 mol!L. Each sample of 1 mL, corresponding to 100 Jlg Chl mL -I, was incubated for 20 min at 27"C in a shaking water bath at 30 strokes per min and was illuminated with incandescent spotlights from under the water bath, which provided a PPFD of 275 Jlmol m - 2s-1 at the sample level. At the end of the incubation period, the vials with the samples were immediately frozen in liquid nitrogen and kept at -18 "C until analysis. Methods for extraction and purification of sugars as well as determination of the content of 14C-sugars by HPLC and a scintillation counter technique were carried out according to Opaskornkul et al. (1994).
Determination of 14 C-sucrose efflux Protoplasts were suspended in a 100-mL Erlenmeyer-flask with photosynthetic assay medium (0.4 mol!L sorbitol, 2 mmol!L NaHC0 3, 1 mmol!L EDTA, 25 mmol/L Hepes-KOH, pH 7.8) and 14C-NaHC0 3 (740 MBq mL -I) to obtain a suspension of 10 mL corresponding to 100 Jlg Chl mL -I. After incubation for 5 min under the same conditions as above, the protoplast suspension was washed 2 X 3 min at 100 gn with ice-cold buffer. After the last centrifugation, the suspension was divided equally into 20 centrifuge tubes. The protoplasts in each tube were suspended in 1 mL photosyntheticassay medium containing ABA and/or sucrose. The final concentrations of sucrose and ABA in the medium ranged from 0-20 mmol! L and 10- 9 to w- 6 mol!L, respectively. Determination of 14C-sucrose efflux was according to Opaskornkul et al. (1994).
Determination ofintracellular pH With some modifications, determination of the cytosolic concentration of H+ was carried out according to the method of Lindberg
ABA Effect on Sucrose and Protons Distribution and Strid (1997). Mesophyll protoplasts were loaded with 8 Jlmoi!L tetra[acetoxymethyl] ester of the fluorescent dye bis-carboxyethylcarboxyfluorescein, BCECF-AM (Molecular Probes, Eugene, Ore., USA) at 4 'C in a dye loading buffer (0.5 moi!L sorbitol, 1 mmoi!L CaC1 2, 0.5 mmoi!L MgC1 2, 0.05 % PVP, 0.2% BSA, 5 mmoi!L MES-KOH, pH 5.5). The protoplast suspension was washed once with the same medium as above before dye loading. During loading, non-specific esterase activity of the cytoplasm is assumed to split the ester into an ion-binding carboxylate form, thereby changing its fluorescence excitation spectra (Gillespie, 1989). After 1 h of incubation in the loading medium, the protoplast suspension was washed once with the same medium; thereafter, the protoplast suspension was centrifuged at 100 gn for 3 min, and the supernatant was removed and replaced with a measuring buffer (0.5 mol/L sorbitol, 1 mmol/L CaClz, 5 mmol/L KCl, 0.05 % PVP, 0.5% BSA, 5 mmol!L Tris-HCl, pH 7). The dye-loaded protoplasts were kept at 4 'C until measurement. Fluorescence measurements were carried out on a single protoplast in the same buffer before and after application of ABA, sucrose or the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS; Sigma). Control tests were performed using glucose, fructose or a non-reducing disaccharide cellobiose. Micro slides were covered with poly-L-lysine (MW 150,000-300,000; Sigma) in order to attach protoplasts to their surface. Application of different solutions was done with a microsyringe and fluorescence measurements were performed at room temperature (20 ± 2 'C). An epi-fluorescence microscope (Axiovert 10; Zeiss, Oberkochen, Germany), supplied with an electromagnetic filter-exchanger (Zeiss), xenon lamp (Zeiss XBO 75), photometer (Zeiss 01), microprocessor (MSP 201, Zeiss) and an IBM computer (PS/2), was used to determine fluorescence intensity at the excitation ratio 485/ 436nm. Emission wavelengths at 510-550nm were monitored. All measurements were performed with a Planneofluar X 40/0.75 objective (Zeiss) for phase contrast. Adjustments of signals and noise were made automatically. By means of ratio microscopy, the effect of different dye concentrations were eliminated (Bright et al., 1987).
Statistical analyses The values presented are the means of duplicate samples of four or five independent experiments. In the determination of intracellular pH, 10 replications each from five independent protoplast batchs were used. For the comparison of mean values between sucrose and/ or ABA treatments and controls, the t-test was used.
protoplasts after 10 min in light were included in some experiments. PCMBS, however, did not change the rates of Oz evolution nor 0 2 consumption (not shown). In contrast to the absence of effects on light induced Oz evolution, ABA inhibited 14 C-sucrose synthesis (Fig. 1a). All protoplast suspensions incubated in light in an ABA medium without sucrose accumulated up to 25 o/o less 14C-sucrose than the control samples without ABA. The highest inhibiting effects were found at 10-7 mol/L ABA. The inhibition by ABA was partly reversed by adding sucrose to the ABA medium (Fig. 1 b), although an exception to this was the combination of 10- 9 mol/L ABA and 5 mmol/L sucrose, in which there was a 30 o/o decrease in sucrose synthesis. There was a distinct stimulating effect of ABA in the incubating medium on the effiux of sucrose from the protoplasts (Fig. 2 a), and this effect was dependent on the presence of sucrose in the medium (Fig. 2 b). Without sucrose in the medium, the effiux of newly synthesized 14C-sucrose into the surrounding medium increased at all ABA concentrations tested and peaked at 10-7 mol/L ABA, at which the sucrose effiux was about 180 o/o higher compared with the control (Fig. 2a). The effects of ABA varied in the presence of sucrose at various concentrations (Fig. 2 b). At 10- 6 mol!L ABA, the effiux of 14C-sucrose was not clearly affected at all external sucrose concentrations. In the presence of 10- 9 -10-7 mol/L ABA, the combined action with sucrose was most prominent. Effiux of 14C-sucrose into the surrounding medium increased steadily with the decrease of ABA concentration in the medium and the highest effiuxes were found at 10- 9 mol/L ABA. The ex-
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Our previous investigation (Opaskornkul et al., 1994) showed that exogenous sucrose (0-20 mmol/L) had no effect on 0 2 evolution nor 0 2 consumption. In our present investigation, no effect on the rate of 0 2 evolution nor 0 2 consumption by sucrose (0, 1, 5, 20 mmol/L) and ABA (lo- 9 to 10- mol/L) at various combinations of concentrations in the medium was observed (not shown). Also a direct application of sucrose and ABA to the medium of photosynthesizing protop lasts did not change the rates of 0 2 evolution nor 0 2 consumption. To rule out that PCMBS purchased from Sigma might contain contaminating levels of Hg2+, which might in turn have a pronounced effect on the respiration of the pea protoplasts, control tests were performed regarding the effects of PCMBS. A direct application of PCMBS at a final concentration of 100 Jlmol/L to the medium of photosynthesizing
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ABA concentration in the medium (moi/L) Fig. 1: Change of 14C-sucrose synthesized in pea mesophyll protoplasts during photosynthesis for 20 min in a medium containing H 14 C0 3- and different combinations of sucrose and ABA concentrations. (a) at 0 mmol!L sucrose, (b) at 1-20 mmol!L sucrose. Amount of 14C-sucrose at 0 mmol!L sucrose and 0 mol/L ABA = 100%.
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served at 1 mmol/L sucrose (Table 2). Increasing concentrations of sucrose in the medium caused a successively lesser decline of the [H+] in the protoplasts. Control tests were performed regarding the effects on cytosolic [H+] by sugars other than sucrose. In contrast to the effect of sucrose, the cytosolic [H+] increased after addition of glucose or fructose (Table 3). Addition of PCMBS into the
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ternal sucrose concentration was also important for the efflux rate. The higher the sucrose concentration the more the effect on the efflux of 14C-sucrose at 10- 9 mol/L ABA. Compared with the control, the efflux of 14C-sucrose in the presence of 10- 9 mol!L ABA was 135% to 290% at 1 and 20 mmol/L external sucrose, respectively. The cytosolic concentration of H+ in the protoplasts was estimated by the use of BCECF. Without any addition, there was no change in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a pea protoplast (not shown), and it remained stable for at least 10 min at experimental temperature. The addition of different concentrations of ABA into a medium without sucrose caused a transient increase in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a pea protoplast. This reflects an increase in cytosolic pH (i.e. [H+] in the cytosol decreased, Fig. 3); thereafter, the fluorescence ratio stabilized. The largest decrease of [H+] was about 12% and was most pronounced at 10- 8 mol!L ABA (Table 1). On the other hand, addition of 100 Jlmol!L PCMBS after the addition of ABA caused a 10 % increase in cytosolic [H+]. To investi§ate whether the effect of ABA on the decrease of cytosolic [H ] was due to a general acidification, HCl at the corresponding concentration was used instead of ABA. This treatment did not change the cytosolic [H+] in the protoplast significantly (data not shown). Addition of different concentrations of sucrose into a medium without ABA also caused a transient increase in the fluorescence ratio 485/436 nm of BCECF in the cytosol of a protoplast, reflecting an increase in cytosolic pH ([H+] decreased). The largest decrease of [H+], about 10%, was ob-
ABA llf6 M-rCMRS 100 mM-
Fig. 3: Effects of ABA at various concentrations or of PCMBS on the BCECF fluorescence intensity ratio (485/436 nm) with time in a pea mesophyll protoplast. The figure shows a representative trace. Table 1: Immediate changes in the cytosolic concentration of H+ in pea protoplasts upon addition of ABA and PCMBS with and without prior addition of sucrose. All values ± SE are means of 5 X 10 replicates. Sucrose concentration (mmol!L)
0
ABA concentration (mmol!L)
w-9 w-8 w-7 w-6
PCMBS 100 Jlmol!L
20
0 10-9 10-8
w-7 w-6
PCMBS 100 Jlmol!L
Changes in [H+] (o/o)
-8.2±0.5 -12.5±1.2 -10.3±1.2 -5.2±0.7 +10.2±1.1 -6.4±1.1 -17.2±0.4 -6.9±2.0 -0.9±0.5 -0.5±0.2 + 11.2± 1.7
ABA Effect on Sucrose and Protons Distribution samples with hexoses did not cause a transient change of cytosolic [H+]. Control tests were also performed using a nonreducing disaccharide cellobiose. The cytosolic [H+] remained unchanged after addition of this sugar (Table 3). Addition of PCMBS into the samples with cellobiose did not cause a transient change of cytosolic [H +]. In the presence of both ABA and sucrose at different concentrations, the effect of ABA on cyrosolic pH varied (Table 9 mol/L ABA, subsequent addi2). With an incubation of tion of 1, 5 and 20 mmol/L sucrose to the medium caused a transient decrease in cyrosolic [H+]. The most pronounced decrease of [H+], about 11 %, took place in the presence of 5 mmol/L sucrose at 10- 9 mol/L ABA.
Change of Fluorescence intensity ratio 485/436 nm
w-
~Sucrose
~
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sucrose concentration (mmol/L)
-10.5± 1.3 -7.5±1.2 -5.3±1.1 -1.5±1.0
5 20
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-5.7± 1.5 -7.8±1.6 -11.2± 1.3 -4.8±0.8 +25.5±2.1
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-4.5±1.1 +6.1±0.5 + 12.7± 1.3 +3.2±1.2
~mol!L
-6.2± 1.6 + 10.2±2.6 +7.5±1.9 + 7.9± 1.5
0 1
5 20 PCMBS 100 10-7
1
5 20 PCMBS 100 1
5 20 PCMBS 100
Table 3: Immediate changes in the cytosolic concentration of H+ in pea protoplasts upon addition of ABA and PCMBS with and without prior addition of different kinds of sugars. All values ± SE are means of7 replicates. Sugar concentration (mmol/L) glucose
1 mmol!L
ABA concentration (mol!L)
5 mmoi/L
PCMBS 100 cellobiose 1 mmol!L 5 mmol!L 10 mmol/L 10 mmol/L
~mol!L
+4.6±0.8 -0.6±0.5 -0.3±0.1
~mol!L
+0.5±0.2 +0.2±0.2 +0.4±0.5 -0.6±0.2 +0.2±0.1
0
0 0 0 10-9
PCMBS 100
--..
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-
Fig. 4: Effects of sucrose at various concentrations or PCMBS on the BCECF fluorescence intensity ratio (485/436 nm) with time in a pea mesophyll protoplast with prior addition of 10- 9 mol!L ABA. The figure shows a representative trace.
Except for samples with 1mmol/L sucrose, sucrose in combinations with ABA higher than 10- 9 mol/L caused a transient increase in cyrosolic [H+] (Table 2). The largest increase of [H+], around 13%, was obtained at 20 mmol/L sucrose 7 mol/L ABA. At all ABA concentrations tested, and PCMBS showed a transient increase in cytosolic [H+]. The maximal increase of internal [H+] upon addition of PCMBS was about 25 %, obtained after addition of 20 mmol/L sucrose in combination with 10- 9 mol/L ABA (Table 2 and Fig. 4).
w-
(%)
~mol!L
1o-')
100m~!
Changes in [H+] +5.2±0.6 +1.2±0.1 +0.2±0.2
0 10-')
PCMBS 100 fructose
PCMDS
(%)
1
10-9
Sucrose 20 mM -
Changes in [H+]
2
2 mM
~ ".]..
Table 2: Immediate changes in the cytosolic concentration of H + in pea protoplasts upon addition of sucrose and PCMBS with or without prior addition of ABA at different concentrations. All values ± SE are means of 5 X 10 replicates. ABA concentration (mol!L)
451
Discussion
The results from our previous investigation (Opaskornkul et al., 1994) supported the concept of an apoplastic phloem loading of sucrose in pea leaves. It also demonstrated that the sucrose efflux from the mesophyll cells is influenced by the apoplastic sucrose concentration. In the present paper, the results suggest that the mechanism responsible for export of sucrose from the mesophyll cells into apoplastic space is a sucrose/H+ cotransport and this mechanism is substantially influenced by various factors in the apoplastic space, such as the sucrose concentration and the ABA concentrations.
452
CHANA 0PASKORNKUL, SYLVIA LINDBERG,
and ]AN-ERIC TILLBERG
In the experiments with application of exogenous sucrose but without ABA (Table 2), the decrease in cytosolic [H+) was more pronounced at I-5 mmol/L sucrose than at 20 mmol/L sucrose. These concentrations of sucrose had the strongest stimulating effects on the efflux of newly synthesized sucrose from the pea mesophyll cells against a concentration gradient according to our previous work. The coincidence of the two events thus indicates a sucrose/H+ cotransport. At sucrose concentrations higher than 5 mmol/L, the change in cyrosolic [H+) was less evident (Table 2), although there was still some sucrose efflux. This may indicate that the export of sucrose into the apoplast occurred via other mechanisms than by a sucrose/H+ cotransport at sucrose concentrations more than 5 mmol/L. As there was also a sucrose efflux in the absence of external sucrose (Fig. 2A), but no change of cytosolic [H+) before the addition of sucrose (not shown), a simple diffusion is likely to be responsible for some part of the sucrose efflux. The cyrosolic [H+) increased after addition of glucose or fructose (Table 3). The lack of cytosolic [H+) decrease upon the addition of a non-reducing disaccharide cellobiose, glucose or fructose indicates that the decrease of cytosolic [H+) was induced mainly by sucrose, and it may also indicate that the pea mesophyll cells probably possess a hexose/H+ symport mechanism in the plasmalemma, which permits them to retrieve the hexoses from the apoplastic space. Cyrosolic [H+) increased as a result of uptake of hexoses into the cytosol. Mter the transition of the leaf tissue from sink to source stage, the mesophyll cells from several plant species lose their ability to take up hexoses from the apoplast (Quick and Schaffer, I996). In the protoplasts treated with various low concentrations of ABA, ABA induced cyrosolic alkalinization, resulting in a decrease of cyrosolic [H+) (Table I). Similar results were obtained in other plant organs as reported by Gehring et a!. (1990) and Van der Veen et a!. (1992). The same concentrations of ABA also have the most pronounced effects on the efflux of sucrose (Fig. 2 a). The coincidence of the two events supports the concept of a sucrose/H+ cotransport mechanism. ABA at these concentrations (below w-7 mol/L) seems to be optimal for promotinf the export of sucrose. At higher ABA concentrations (>10- mol/L), the decrease in the cytosolic [H+) as well as the efflux of sucrose were less than at ABA concentrations below w-7 mol/L. A decrease in the translocation of sucrose into the phloem caused by high ABA concentrations has been reported by Tietz et a!. (I98I), and this decrease in turn may be the result of the decline of the efflux of sucrose and H+ from the mesophyll cells. ABA at concentrations beyond w-7 mol/L might induce a stress condition to the mesophyll cells. It is not clear, however, how ABA might have an impact on sucrose efflux in both stress and non-stress conditions. PCMBS is a non-permeant potent inhibitor of sucrose/H+ symport. It inhibits phloem loading and blocks the efflux of sucrose into the apoplast, implying the impairment of apoplastic phloem loading (Van Bel, 1993). In the present study, PCMBS caused an increase of cytosolic [H+], after addition of the inhibitor to samples containing ABA or sucrose (Tables 1 and 2), but PCMBS failed to give similar results in the samples with hexoses (Table 3). This corroborates with the
concept of a sucrose/H+ symport. The concept of a sucrose/H+ symport at the plasmalemma was suggested by Giaguinta (1977) and confirmed by Bush (1989), based on data from experiments using plasmalemma vesicles from sugar beet leaves. No data have been previously reported on a mechanism for sucrose loading into the apoplastic space in pea plants. Part of the decrease of cyrosolic [H+) might also be the result of a sucrose/H+ antiport activity at the tonoplast membrane of the mesophyll cells (Bush, I992). This can occur when the sucrose previously stored in the vaculoes is released into the cytosol for export. Both ABA and sucrose are regulators of the sucrose/H+ symport, but by different mechanisms. ABA may be involved in many cytoplasmic events, including promotion of an increase in cytosolic [Ci+) and a decrease in cytosolic [K+) (Blatt and Thiel, I993). This leads to an increase in cytosolic [H+], which in turn may have a promoting effect on the sugar transport mechanism. The sucrose may directly affect the sugar transport protein itself, and it has been suggested that the apoplastic sucrose may provide the energy for vein loading in leaf tissue (Pitcher et a!., I99I). A combination of external ABA and sucrose resulted in a different impact on the sucrose efflux compared with the effects by ABA or sucrose added separately to the samples (Fi~. 2 b, Tables I and 2). The optimal combination was 10- mol/L ABA and 20 mmol/L sucrose. The high effectiveness at a lower ABA concentration (10- 9 mol/L) in this combined action may be due to the presence of sucrose. Sucrose might increase receptivity of the ABA binding protein at the plasma membrane; the stimulating effect of ABA, therefore, reaches the optimal condition at a lower concentration. It is known that the inhibition of photosynthesis caused by the stress hormone ABA in an intact leaf occurs via the effects of ABA on the closure of the stomata. The lack of direct effects of ABA on the photosynthetic rate in isolated mesophyll cells, however, has been reported for various plant species (Mawson et a!., 1981; Raschke and Hedrich, 1985). Photosynthesis in the protoplasts prepared for our experiment was not affected by ABA in any of the apoplastic sucrose concentrations tested (not shown). On the other hand, suppression by ABA of the 14 C-sucrose synthesis in the cell was very pronounced (Fig. I), whereas exogenous sucrose does not exert this effect, as shown in our previous study (Opaskornkul et a!., I994). It is not clear, however, how ABA interferes with the sucrose synthesis process, but it is probably due to effects on the enzyme activity of Rubisco by reduction of the carboxylase activity or increase of the oxygenase activity (Brenner and Cheikh, I995). Direct effects, however, of ABA on FBPase or SPS activities, which are the main regulators of the sucrose synthesis process, have not been reported. As abscisic acid-binding protein is thought to be plasma membrane-localized (Hahn, I996), it is unlikely that ABA would have a direct effect on the cytoplasmically-localized sucrose synthesis process. References M. R. and M. THIEL: Hormonal control of ion channel gating. Annu. Rev. Plant Physiol. 44, 543-567 (1993).
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