Planta (1984)161 : 562-568

P l a n t a 9 Springer-Verlag 1984

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts* Georg Kaiser and Ulrich Heber Lehrstuhl Botanik I der Universit/it, Mittlerer Dallenbergweg 64, D-8700 Wiirzburg, Federal Republic of Germany

Abstract. Sucrose transport has been investigated

in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K m of the carrier for sucrose uptake was 21 raM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg z+ present, nor by protonophores and valinomycin (with K + present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vacuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars. K e y words: Hordeum (sucrose transport, vacuole) - Protoplast - Sucrose (partitioning and transport)

- Tonoplast - Vacuole. * Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday Abbreviations." Hepes = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; pCMBS =p-chloromercuribenzene sulfonate

Introduction

In photosynthesis, the transport metabolites dihydroxyacetone phosphate and phosphoglycerate are transferred from the chloroplasts to the cytosol of mesophyll cells by the phosphate translocator of the chloroplast envelope (Heldt and Rapley 1970; Fliege et al. 1978). Outside the chloroplasts these metabolites are converted into secondary products of photosynthesis such as sucrose. Sucrose represents the main part of assimilated carbon in mesophyll protoplasts of wheat, spinach, and barley (Giersch et al. 1980; Kaiser et al. 1982). In kinetic work with spinach and wheat protoplasts it has been shown that after 10 min 14CO2fixation, about 90% of the total soluble label can be found outside the chloroplasts. Export of photosynthetic products from the protoplasts into the medium was insignificant (Giersch etal. 1980; Kaiser et al. 1979). If newly synthesized sucrose were retained in the cytosol of leaf cell protoplasts, calculation based on commonly observed rates of photosynthesis reveals that its accumulation would decrease water potential and consequently result in swelling of the cytosol. This swelling would severely disturb cytosolic metabolism by metabolite dilution within less than 1 h of efficient photosynthesis. It was therefore concluded that sucrose and possibly other soluble photosynthetic products were transported from the cytosol into the vacuole of isolated protoplasts. Indeed, in kinetic work with assimilating barley mesophyll protoplasts from which vacuoles were rapidly isolated, it has recently been shown that the transfer of photosynthetic products from the cytosol across the tonoplast into the vacuoles is a rapid process which may approach the rate of synthesis (Kaiser et al. 1982). Predominant among

G. Kaiser and U. Heber: Sucrose transport into vacuoles

imported products was sucrose, but transfer of malate, citrate, glutamate, glutamine, and alanine was also significant. Phosphate esters appeared to be excluded from the vacuoles. The ability to store sucrose inside the vacuoles is not restricted to isolated protoplasts which cannot export assimilates. Giaquinta (1978) has postulated that young ( = sink) leaves store part of the imported sucrose in the vacuoles of the mesophyll cells. Fisher and Outlaw (1979) demonstrated the existence of two distinct sucrose pools in the cytosol and vacuole of palisade parenchyma cells of Viciafaba leaves. Barley leaves store both sucrose and starch during the day as demonstrated by Gordon et al. (1980). Gerhardt and Heldt (1984) recently reported on a 40-fold increase in vacuolar sucrose in spinach leaves during the day. In order to understand intracellular sucrose transport, we have measured the transfer of sucrose across the tonoplast of isolated barley vacuoles. Material and methods Preparation of vacuoles. The preparation of protoplasts from young barley leaves has been described previously (Kaiser et al. 1982). Vacuoles were isolated from the protoplasts and purified by a modification of the method published by Martinoia et al. (1981). Protoplasts corresponding to 1 nag chlorophyll were suspended in 6 ml of a medium containing 0.4 M sorbitol, 1 mM MgC1/, 1 mM MnC12, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaC1, 30 mM KC1, 0.5 mM KH2PO4, 30 mM 4-(2-hydroxyethyl)-l-priperazineethanesulfonic acid (Hepes) (pH 7.8), 0.1% bovine serum albumin (w/v), 0.1% polyvinylpyrrotidone (w/v, 10 kD), and 12.5% osmotically adjusted Percoll (Deutsche Pharmacia, Freiburg, FRG). Resulting suspensions were squeezed at 273 K through a long needle (10 cm long, 0.7 mm diameter). By this treatment, the plasmalemma of about 30-50% of the protoplasts was ruptured, but most of the large central vacuoles which were released remained intact as shown by phase-contrast microscopy. After transfer into centrifuge tubes (step 1), lysates (6 ml) were overlayered with 5 ml medium (as above) which contained 10 gg neutral red ml 1 instead of Percoll (step 2), and with 2 ml medium (as above) without Percoll and neutral red but with 0.4 M glycinebetaine instead of 0.4 M sorbitol, and with 30 mM 2-(N-morpholino)ethanesulfonie acid (Mes) (pH 5.6) instead of 30 mM Hepes (pH 7.8) (step 3). The vacuoles were purified by flotation (2 min, 100g, followed by 2 rain ll00g) at 277 K. Because of the replacement of sorbitol (MW 182.17) by glycinebetaine (MW 117.2) (Wyn Jones et al. 1977), the stained vacuoles accumulated at the bottom of step 3 and could easily be removed from the gradient. Phases 2 and 3 were sucked off, and the protoplasts which had remained intact during the first lysis were again squeezed through the needle and purified as described above. After addition of osmotically adjusted Percoll (final concentration 8%), the collected vacuoles were transferred into a centrifuge tube and overlayered with 2 ml of step-3 medium as described above. After a second flotation (2 min, 650g) the

563 purified vacuoles were removed from the gradient and stored on ice. The yield of purified vacuoles was 25-35%. The total time needed for isolation and purification was about 1.5 h from the beginning of protoplast lysis.

Uptake experiments. Aliquots containing about l0 s vacuoles were incubated in a medium based on step-3 medium as described above but containing 30% osmotically adjusted Percoll to which [14C]sucrose and solutes such as ATP, ionophores etc. were added as indicated in the legends of Figs. and Tables. Incubation was carried out at pH 5.6 and 295 K unless stated otherwise. Sucrose uptake was terminated at the times indicated by silicone-oil centrifugation (Werdan et al. 1972) using phenylmethylsilicone-oil AR 200, density 1.04 g cm 3 (Waeker, Miinchen, FRG), in a Beckman Microfuge B (Mfinchen, FRG) for 15 s at about 9700g. The kinetics of [14C]sucrose uptake were determined by scintillation spectrometry. Assays. Acid phosphatases were assayed by the method of Leigh and Walker (1980). Initially, they were assumed to be exclusively localized in the vacuoles. For quantification of purified vacuoles, enzyme activities were therefore measured in the vacuolar fraction and compared with the enzyme activity in the protoplast suspension from which the vacuoles had been isolated. Since the cell number of the protoplast suspension was known, the number of vacuoles could be calculated under the assumption that only one vacuole can be isolated per protoplast (Kaiser et al. 1982). After the main part of this work was completed, it was realized that only about 75% of the activity of acid phosphatases are localized in the large central vacuole. Sucrose uptake rates calculated under the assumption of a 100% localization are therefore orerestimates. They were corrected for the initial error. Marker enzymes for different cell constituents were measured as described before, and contaminations were found to be as low as previously reported (Kaiser et al. 1982). Endogenous vacuolar oligosaccharides (predominantly sucrose) were measured by determining glucose levels before and after acidic hydrolysis (1 N HCI, 1 h, 373 K) spectrophotometrically at 610 nm by the glucose oxidase (EC 1.1.3.4)-Periodmethod (Test Combination Glucose, No 124 028, Boehringer, Mannheim, FRG). The sucrose concentration was calculated from the differences in vacuolar glucose content before and after hydrolysis. The vacuolar content of maltose and higher soluble 1,4-glucans was measured as glucose after hydrolysis by ~-l,4-amyloglucosidase (EC 3.2.1.) (Bergmeyer 1970). It was found to be negligible. Chlorophyll was determined according to Arnon (1949).

Results and discussion

Uptake of [14C]sucrose into isolated barley vacuoles was pH-dependent. Maximum uptake was observed at pH 5.6 (Fig. 1). Above pH 7.5, uptake was decreased by about 50%. A pH optimum of about 5.5 has also been found by Thorn et al. (1982) for sugar uptake into isolated sugarcane vacuoles and by Guy et al. (1979) for the transport of the D-glucose analogue 3-O-methylglucose into pea vacuoles. Decreased

564

G. Kaiser and U. Heber: Sucrose transport into vacuoles

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Fig. 1. pH-Dependence of [14C]sucrose uptake by isolated vacuoles (1 mM; 0.72 MBq mmol-t), Incubation at 295 K for 4 min. At pH-values above 7.0 Mes was replaced by Hepes. 100% = 37.5 pmol sucrose (105 vacuoles)- 1 (4 min)- 1

transport at p H 7 is surprising as the cytosolic pH is thought to be close to neutrality in vivo (Smith and Raven 1979). Figure 2 shows that the rate of [14C]sucrose transport into isolated vacuoles was dependent on the sugar concentration in the incubation medium. Transfer was found to be linear over a wide range of concentrations (100 ~tM-100 m M ) for at least 25 min. Because of contaminations caused mainly by residual medium which was carried with the vacuoles through the silicone oil, a significant amount of radioactive sucrose was found in the vacuolar fraction even after the shortest incubation times. Extrapolation to zero incubation time served to determine this contamination, which was then subtracted from measured values. Corrected uptake data are shown in Fig. 3. Isolated barley vacuoles exhibited saturation kinetics for [14C]sucrose uptake. Saturation was observed at about 100 raM, half-saturation close to 20 m M (Fig. 3A, B). Uptake in the presence of 100 m M external sucrose caused an increase in the concentration of vacuolar sucrose of only 4.6 m M within the measuring time of 4 min. Initial sucrose concentrations in the vacuoles were very low (below 4 raM) because endogenous sugar was lost by efflux from the vacuoles during storage for about 1 h before the experiment. Therefore, the observed saturation was not caused by concentration equilibration between vacuolar space and medium. The Vm, x of transport as revealed by a Lineweaver-Burk plot was 120 nmol sucrose (105 vacuoles)- a h-1. 105 Protoplasts contained about 7-10 gg chlorophyll. If each protoplast yielded one

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Fig. 2. Time course of [14C]sucrose uptake by isolated vacuoles at different external concentrations. Incubation at pH 5.6, 295 K. The concentration of glycinebetaine was decreased when sucrose concentrations were increased to avoid osmotic effects pH 5.6; 295 K; incubetion 4 m/n

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Fig. 3. A Concentration dependence of [14C]sucrose uptake by isolated vacuoles. Half-saturation of uptake was close to 20 mM. For details see text. B Lineweaver-Burk plot of the reciprocal rate of sucrose uptake versus reciprocal sucrose concentration

vacuole only (as is very likely, see Kaiser et al. 1982), a transfer capacity of 120 nmol (105 vacuoles) -1 h -1 would correspond to 1 2 - 1 7 g m o l sucrose transfer m g - t chlorophyll h - 1 or the transfer into the vacuole of about 180 patoms carbon mg-1 chlorophyll h-1. In comparison, rates of light-saturated photosynthesis of leaves in air are usually not much higher than 100gmol CO2 reduced m g - 1 chlorophyll h - 1 Sucrose transport into the vacuoles of intact protoplasts in vivo was observed to be 15 nmol (105 protoplasts) -1 h -1 at a calculated cytosolic sucrose concentration of about 6 m M (Fig. 7 and

G. Kaiser and U. Heber: Sucrose transport into vacuoles Table 1. Sucrose uptake by isolated vacuoles. Means of three experiments are shown. Vacuoles were incubated with 1 mM [l*C]sucrose (0.72 MBq mmo1-1) at pH 5.6 and 295 K for 12 min

Control ATP (2 mM) ATP (4 raM) MgATP (2 raM) MgATP (4 raM) MgATP (2-4 mM) + NH4-molybdate (5 mM)

Sucrose [nmol (10 s vacuoles)- 1]

% of control

0.31 0.32 0.32 0.31 0.32 0.32

100 103 103 100 103 103

Table 2 in Kaiser et al. 1982). This compares with a transfer rate of about 28 nmol sucrose per 105 isolated vacuoles in vitro at an external sucrose concentration of 6 m M (Fig. 3). In the in-vitro experiment, the vacuolar sucrose concentration was likely to be lower than in the in-vivo experiment. This may account for the faster transport in vitro. It is apparent fiom the data that the sucrose transport system of the vacuoles had not been impaired during the isolation of the vacuoles. A low-affinity transport system for s u c r o s e ( K m 22 mM) has also been described for the tonoplast Table 2. Sucrose uptake by isolated vacuoles. Means of three experiments. Valinomycin was tested in the presence of 30 and 130 mM K +. Vacuoles were incubated in the presence of 1 mM [14C]sucrose (0.72 MBq mmo1-1) at pH 5.6 and 295 K for 12 rain; ~g=electric potential difference

565

of isolated red-beet vacuoles (Willenbrink and Doll 1979). Regarding the question of whether or not sucrose transport is energy-dependent, Table 1 shows that sucrose uptake was not stimulated by ATP or pyrophosphate, with or without Mg 2+ added. Since unspecific vacuolar phosphatases originating from broken vacuoles rapidly hydrolyzed ATP and pyrophosphate, ammonium molybdate was added to inhibit substrate degradation. Inhibition was highly effective (data not shown), but even in the presence of molybdate, sucrose uptake was not influenced by ATP or pyrophosphate. This was true at slightly acidic (pH 5.6) and at neutral pH. Attempts to decrease transport by suppressing the membrane potential were unsuccessful. Different concentrations and combinations of nigericin, valinomycin, gramicidin, and the highly effective uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), agents known to degrade ion gradients, which are potential energy sources, failed to decrease sucrose transport (Table 2). This indicates that, contrary to reports published earlier on other plant material (Doll et al. 1979; Guy et al. 1979; Thorn et al. 1982) sucrose transfer into isolated barley vacuoles is not energy-dependent. Table 3 shows data on the specificity of sucrose uptake. The experiments were carried out accord-

Control Nigericin (10 -6 M) CCCP (10 -6 M)

Valinomycin (10 -6 M) Val.+Nig. (each 10 6 M) Val_+Nig. (each 3• M) Gramicidin (10 -6 M)

Main effect on

Sucrose [nmol (105 vacuoles) 1]

% of control

A pH A pH A ~g A pH, A ~ ApH, A ~ A pH, A ~

0.54 0.55 0.63 0.55 0.48 0.56 0.51

100 102 117 102 89 104 94

Table 3. Competitive inhibition of su-

Numbers of C-atoms

crose transport into isolated vacuoles by various sugars. Means of three experiments. The concentration of glycinebetaine was decreased corresponding to increased sugar concentrations to avoid osmotic effects. Vacuoles were incubated at pH 5.6 and 293 K for 12 rain

Sucrose [nmol (105 vacuoles) - 1 h - 1]

% of uptake observed with 100 mM SUCrOSe

Sucrose (100 raM) Sucrose (10 mM) Sucrose (10 mM)+xylose (90 raM) Sucrose (10 raM)+ glucose (90 raM) Sucrose (10 m M ) + fructose (90 raM) Sucrose (10 raM)+ lactose (90 raM) Sucrose (10 raM)+ maltose (90 raM) Sucrose (10 raM)+raffinose (90 mM) Sucrose (10 raM)+ sorbitol (90 raM)

12 12 5 6 6 12 12 18 6

143 29 17 22 18 18 14 13 30

100 20.3 11.9 15.4 12.6 12.6 9.8 9.1 21.0

566

ing to Heldt and Rapley (1970). With 100 m M [14C]sucrose, uptake into the vacuoles was satu 2 rated. The corresponding rate was 143 nmol (105 vacuoles) -1 h -1. With 10 m M p4C]sucrose and 90 m M of another sugar, uptake should be 1/10 of this rate, i.e. 14 nmol (105 vacuoles) -* h -~, if the other (unlabelled) sugar were fully competitive with sucrose. If it were not competitive, p4C]sucrose uptake should be as high in the absence as in the presence of the unlabelled sugar, i.e. 29 nmol (10 s vacuoles)- 1 h - a. Table 3 shows that the rate of sucrose uptake decreased in the presence of various sugars, indicating competition with sucrose. Powerful competitors were maltose and raffinose which were fully competitive with sucrose. The other sugars listed in Table 3 were less effective as inhibitors of sucrose uptake than maltose and raffinose. Sorbitol, which is very slowly taken up by isolated vacuoles (data not shown), had no effect on sucrose uptake, i.e. was not competitive. Possibly, it enters the vacuoles by diffusion, not catalysis. In vacuoles of red beet, raffinose was also found to be the most effective inhibitor of sucrose uptake (Willenbrink and Doll 1979). Saturation of transport (Fig. 3 A) and competition with transfer (Table 3) are customary criteria for carrier-mediated transport. Chemical protein modification should affect transport if the carrier is a protein. Of the inhibitors investigated, only an SH-group modifier was effective. Sucrose transport was completely inhibited by 1 m M p-chloromercuribenzene sulfonate (pCMBS) after 20 min incubation of the vacuoles with the inhibitor (data not shown). Whereas in the experiments of Figs. 1-3 sucrose influx into the vacuoles was measured, the experiment of Fig. 4 describes effiux. Vacuoles were first loaded with [*4C]sucrose and then transferred to three different media. One contained 1 m M pCMBS, another 50 m M unlabelled sucrose and the third was free of sucrose. After different incubation times, retention of radioactivity in the vacuoles was measured. Labelled sucrose was rapidly lost from the vacuoles only when external sucrose was absent. In the presence of external sucrose, effiux of radioactivity decreased. This effect can be more readily explained by the decrease in the specific activity of internal sucrose brought about by the influx of external unlabelled sucrose than by a competition between external and internal sucrose for binding Sites of the carrier. When preloaded vacuoles were transferred to a medium containing pCMBS, no efflux of label was apparent from 4 to 12 min after the transfer.

G. Kaiser and U. Heber: Sucrose transport into vacuoles

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Fig. 4. Efflux of [l~C]sucrose from isolated vacuoles. The suspensions had been preloaded with 1 mM [14C]sucrose (0.72 MBq mmo1-1) for 30 min at pH 5.6, 295 K, and were then purified by silicone-oil centrifugation (see Material and methods). The vacuoles were resuspended, pCMBS or unlabelled sucrose was added to the samples, and incubations were terminated at the times indicated by a second silicone-oil centrifugation. o - - o control; n - - n + 1 mM pCMBS; z x - - ~ + 50 mM unlabelled sucrose (pCMBS and sucrose added at zero time)

The data clearly show that sucrose can be readily transported into and out of the vacuoles. Very probably, the same carrier catalyses transport in both directions. No evidence for energization is available in either transport direction. In previous work, predarkened green mesophyll protoplasts were illuminated in the presence of 14CO2 for 10 min. Subsequently, the protoplasts were lysed. Since the content of label in the liberated vacuoles remained largely c o n s t a n t for 15 min, it had been concluded that isolated vacuoles retained labelled material effectively. As t h e data of Fig. 4 show, this view cannot be extended to sucrose (which is not significantly labelled in the vacuole of barley mesophyll protoplasts 12 min after the transition from dark to light; Kaiser et al. 1982). Boller and Alibert (1983) have recently reported that in protoplasts from Melilotus alba the bulk of newly fixed neutral photosynthates remained in the cytosol. A sucrose gradient between cytosol and vacuole was calculated to be in the range of 70-140 m M after 60-120 min photosynthesis. These data indicate that sucrose transport into the vacuoles of mesophyll cells from Melilotus is much slower than transport in barley. However, the data could also be explained on the assumption that during the time required for isolating the vacuoles considerable sucrose had been lost from the vacuoles.

The role of the barley vacuole in sucrose partitioning. The sucrose content of barley or spinach leaves increases during the daily illumination period

G. Kaiser and U. Heber: Sucrose transport into vacuoles ( G o r d o n et al. 1980, 1982; G e r h a r d t a n d H e l d t 1984). T h e m a j o r p a r t o f the sucrose r e m a i n i n g in the l e a f is t e m p o r a r i l y stored in the vacuoles o f the m e s o p h y l l cells. This is c o n c l u d e d f r o m w o r k with barley m e s o p h y l l p r o t o p l a s t s (Kaiser et al. 1982) a n d n o n a q u e o u s fractions p r e p a r e d f r o m spinach leaves ( G e r h a r d t a n d H e l d t 1984). Relatively little is k n o w n a b o u t the s y s t e m responsible for p h l o e m loading o f sucrose. A p p a r e n t K m values o f a b o u t 16 m M sucrose h a v e been p u b l i s h e d for leaf slices o f B e t a vulgaris ( G i a q u i n t a 1977), isolated m i n o r vein nets o f t o b a c c o leaves ( C a t a l d o 1974), a n d for t r a n s l o c a t i o n o f sucrose f r o m s u g a r - b e e t leaves ( S o v o n i c k et al. 1974). H o w e v e r , because o f unstirred layers at the t r a n s p o r t sites, these values p r o b a b l y reflect u p p e r limits. T h e y m a y indicate t h a t at low cytosolic sucrose c o n c e n t r a t i o n s e x p o r t o f assimilated sucrose is f a v o r e d o v e r s e q u e s t r a t i o n in the vacuole. Still, as the sucrose c o n t e n t rises in the cytosol during p h o t o s y n t h e s i s (because the affinity o f the l o a d i n g system for sucrose is low), transfer o f sucrose into the v a c u o l e increases also. Flux o f sucrose into the vacuole minimizes efflux o f w a t e r i n t o the cytosol, a c o n s e q u e n c e o f the lowering o f the cytosolic w a t e r potential, which w o u l d o c c u r during the a c c u m u l a t i o n o f p h o t o s y n t h e t i c p r o d ucts in the cytosol. W a t e r flux into the cytosol w o u l d lead to dilution o f metabolites. I n chloroplasts, dilution o f s t r o m a l solutes caused b y influx o f w a t e r has been s h o w n to inhibit p h o t o s y n t h e s i s m e t a b o l i s m (Kaiser et al. 1981 a, b). I n h i b i t i o n was reversible as long as r u p t u r e o f the c h l o r o p l a s t env e l o p e was avoided. O u r d a t a give no evidence o f energization o f sucrose t r a n s p o r t . It is conceivable t h a t the isolation o f the vacuoles has led to the u n c o u p l i n g o f the sucrose carrier f r o m an energy source. H o w ever, it should be n o t e d t h a t there is no r e a s o n to p o s t u l a t e energization. Because o f the large volu m e o f the vacuole, passive t r a n s p o r t by facilitated diffusion is sufficient to satisfy a storage f u n c t i o n o f the vacuole. Active uphill t r a n s p o r t w o u l d n o t only c o n s u m e energy, it w o u l d also result in w a t e r u p t a k e by the vacuole, i.e. in d e h y d r a t i o n o f the cytosol. Passive t r a n s p o r t w o u l d not require c o m plex r e g u l a t i o n to r e s p o n d to loss o f sucrose f r o m the cytosol in the d a r k by reversing the direction o f t r a n s p o r t . T h e vacuole has thus n o t only a storage b u t also a buffer function. We are grateful to Dr. E. Martinoia for critical discussions. This work was supported by the Deutsche Forschungsgemeinschaft.

567

References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Bergmeyer, H.U. (1970) In: Methoden der enzymatischen Analyse, pp. 39(~397, Bergmeyer, H.U., ed., 2nd edn. Verlag Chemie, Weinheim/Bergstrage Boller, T., Alibert, G. (1983) Photosynthesis in protoplasts from Meli]otus alba: distribution of products between vacuole and cytosol. Z. Pflanzenphysiol. 110, 231-238 Cataldo, D.A. (1974) Vein loading: the role of the symplast in intracellular transport of carbohydrate between the mesophyll and minor veins of tobacco leaves. Plant Physiol. 53, 912-917 Doll, S., Rodier, F., Willenbrink, J. (1979) Accumulation of sucrose in vacuoles isolated from red beet tissue. Planta 144, 407-411 Fisher, D.B., Outlaw, W.H., Jr. (1979) Sucrose compartimentation in the palisade parenchyma of Viciafaba. Plant Physiol. 64, 481--483 Fliege, R., F1/igge, U.I., Werdan, K., Heldt, H.W. (1978) Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochim. Biophys. Acta 502, 232-247 Gerhardt, R., Heldt, H.W. (1984) Measurement of subcellular metabolite levels in leaves by fractionation of freeze stopped material in nonaequeous media. Plant Physiol. (in press) Giaquinta, R. (1977) Phloem loading of sucrose, pH dependence and selectivity. Plant Physiol. 59, 750-755 Giaquinta, R. (1978) Source and sink leaf metabolism in regulation to phloem translocation. Carbon partitioning and enzymology. Plant Physiol. 61,380-385 Giersch, C., Heber, U., Kaiser, G., Walker, D.A., Robinson, S.P. (1980) Intracellular metabolite gradients and flow of carbon during photosynthesis of leaf protoplasts. Arch. Biochem. Biophys. 205, 246-259 Gordon, A.J., Ryle, G.J.A., Webb, G. (1980) The relationship between sucrose and starch during dark export from leaves of uniculm barley. J. Exp. Bot. 31,845-850 Gordon, A.J., Ryle, G.J.A., Mitchell, D.F., Powell, C.E. (1982) The dynamics of carbon supply from leaves of barley plants grown in long or short days. J. Exp. Bot. 33, 241 250 Guy, M., Reinhold, L., Michaeli, D. (1979) Direct evidence for a sugar transport mechanism in isolated vacuoles. Plant Physiol. 64, 61-64 Heldt, H.W., Rapley, L. (1970) Specific transport of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate and of dicarboxylates across the inner membrane of spinach chloroplasts. FEBS Lett. 10, 143-148 Kaiser, G., Martinoia, E., Wiemken, A. (1982) Rapid appearance of photosynthetic products in the vacuoles isolated from barley mesophyll protoplasts by a new fast method. Z. Pflanzenphysiol. 107, 103-113 Kaiser, W.M., Kaiser, G., Prachuab, P.K., Wildman, S.G., Heber, U. (1981 a) Photosynthesis under osmotic stress. Inhibition of photosynthesis of intact chloroplasts, protoplasts, and leaf slices at high osmotic potentials. Planta 153, 416~422 Kaiser, W.M., Kaiser, G., Sch6ner, S., Neimanis, S. (1981 b) Photosynthesis under osmotic stress. Differential recovery of photosynthetic activities of stroma enzymes, intact chloroplasts, protoplasts, and leaf slices after exposure to high solute concentration. Planta 153, 430-435 Kaiser, W.M., Paul, J.S., Bassham, J.A. (1979) Release of photosynthates from mesophyll cells in vitro and in vivo. Z. Pflanzenphysiol. 94, 377-385

568 Leigh, R.A., Walker, R.R. (1980) ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgar& L.). Planta 150, 222-229 Martinoia, E., Heck, U., Wiemken, A. (1981) Vacuoles as storage compartments for nitrate in barley leaves. Nature (London) 289, 292-294 Smith, F.A., Raven, J.A. (1979) Intracellular pH and its regulation. Annu. Rev. Plant. Physiol. 30, 28%311 Sovonick, S.A., Geiger, D.A., Fellows, R.J. (1974) Evidence for active phloem loading in the minor veins of sugar beet. Plant Physiol. 54, 886-891 Thorn, M., Komor, E., Maretzki, A. (1982) Vacuoles from sugarcane suspension cultures. II. Characterization of sugar uptake. Plant Physiol. 69, 1320-1325

Erratum Planta (1984) 160, 41-51, paper by M. Iino, W.R. Briggs, E. Sch/ifer: Phytochrome-mediated phototropism in maize seedling shoots. In the legend to Fig. 1 it should read "with a fixed fluence rate of either 3.2 or 7.0 gmolm -2 s -1'' (instead of 0.32 or O.7).

G. Kaiser and U. Heber: Sucrose transport into vacuoles Werdan, K., Heldt, H.W., Geller, G. (1972) Accumulation of bicarbonate in intact chloroplasts following a pH gradient. Biochim. Biophys. Acta 283, 430M41 Willenbrink, J., Doll, S. (1979) Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specifity of the sucrose uptake system. Planta 147, 159-162 Wyn Jones, R.G., Storey, R., Leigh, R.A., Ahmad, N., Pollard, A. (1977) A hypothesis on osmotic osmoregulation. In: Regulation of cell membrane activity in plants, pp. 121-136, Marr6, E., Ciferri, O., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Received 25 January; accepted 8 March 1984