Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4697-471, July 1985 Botany Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells (muldenzyme system/uridine diphosphate glucose/tonoplast) MARGARET THOM AND ANDREW MARETZKI Experiment Station, Hawaiian Sugar Planters' Association, Aiea, HI 9671 Communicated by Martin Gibbs, March 18, 1985 ABSTRACT Isolated vacuoles from sugarcane cells took up uridine diphosphate glucose (UDP-Glc) from the surrounding medium at a rapid rate. After a 7-min incubation of vacuoles with UDP-[14C]Glc, sucrose and sucrose phosphate were identified in the vacuole extract. UDP-Glc in the incubation medium was converted to hexose phosphates, sucrose, and glucose, with very little UDP-Glc remaining. Fructose 6- phosphate was not required for UDP-Glc uptake nor was [14C~fructose 6-phosphate taken up even in the presence of UDP-Glc. Glucose 6-phosphate and glucose 1-phosphate also were not taken up into vacuoles. UDP-Glc uptake showed saturation kinetics with a Km of.7 mm and a V.,. of 11.1 nmol/min per 16 vacuoles. The optimum ph for UDP-Glc uptake was between 6.5 and 7.. Uptake of UDP-Glc could be inhibited by p-chloromercuriphenylsulfonic acid, UDP, and GDP, and to a lesser extent by carbonyl cyanide m- chlorophenylhydrazone. The UDP-Glc binding site was specific for UDP-Glc; adenosine diphosphate glucose was not taken up, and guanosine diphosphate glucose did not compete with UDP-Glc for the binding site. The results suggest that sucrose transfer into vacuoles from sugarcane is via a group translocation mechanism, probably involving five tonoplast-bound enzymes. Plant cells can achieve homeostasis by concentrating energyrich molecules in discrete compartments and sequestering them there for release upon metabolic demand. Such is the case for sucrose, a relatively inert molecule, which upon hydrolysis can be returned immediately to the mainstream of intermediary metabolism. In sugarcane sink tissue, sucrose released from the translocation stream into the intercellular space requires cleavage to reducing sugars before being taken up by parenchyma cells. Sugar in excess of glycolytic demands undergoes intracellular resynthesis to sucrose. The process by which high concentrations of sucrose can accumulate in vacuoles of sugarcane stalk parenchyma has been a focus of interest for physiologists concerned with turnover and fate of photosynthetic products in sink tissue (1). Clearly, active transport mechanisms across membranes constitute a facet of the process, but demands for large expenditures of energy to facilitate accumulation of substances in metabolically quiescent compartments of stalk cells is incompatible with the simultaneous vigorous growth typical of sugarcane stalks. ATPase-dependent, proton-linked transport on the plasmalemma constitutes the principal mechanism for uptake of hexoses into the cytoplasmic compartment of sucrosestoring sugarcane cells. Indeed, evidence is overwhelming that, at the plasmalemma, an electrochemical gradient accounts for uptake of disaccharides as well as monosaccharides in plants (2). The situation at the tonoplast is less clear. Uptake of sucrose has been demonstrated with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. vacuoles isolated from Beta vulgaris, and the mechanism appears to conform with the requirements for a protonmotive transport system (3). However, sucrose transport into vacuoles isolated from barley mesophyll protoplasts appears to be via facilitated diffusion (4). In Saccharum sp., an ATPase-linked proton antiport system accounts for glucose uptake (5). Isolated vacuoles and vacuoles in situ have comparable uptake rates for glucose. However, sucrose uptake in isolated vacuoles was only 1% of the in situ rate. The sucrose uptake rate was low compared to that of glucose, a result inconsistent with relative abundance of these sugars in the vacuole (6). Damage to the transport site for sucrose during the isolation procedure was invoked as a possible explanation. An alternative explanation is that there may be no damage to the transport site, but vacuoles are inherently unable to recognize and transport sucrose. The present investigation with isolated vacuoles from cell cultures was initiated to clarify the role of sucrose phosphate (Suc-P) in sucrose accumulation across the tonoplast. The suggestion had been made a number of years ago that sucrose phosphatase might be the key to maintenance of a steep sucrose gradient between cytoplasmic and vacuolar compartments (1). An active, enzymatically promoted transfer of sucrose into the vacuole was proposed via cleavage of the phosphate moiety from the molecule after Suc-P was cytoplasmically synthesized from uridine diphosphate glucose (UDP-Glc) and fructose 6-phosphate (Fru-6-P) by the action of Suc-P synthase. A central role for Suc-P in vacuolar accumulation of sucrose would require hydrolysis by sucrose phosphatase either before or after being taken up. The present finding that Suc-P was not taken up by vacuole preparations independently confirms similar results recently obtained by J. Hawker and G. Smith (personal communication) and clears the way to search for uptake characteristics of precursors further removed from the sucrose molecule. Vectorial enzymatic group translocation by which active transport of organic molecules is facilitated across membranes was demonstrated in microorganisms (7) and animals (8) a number of years ago but has never been shown to function in plants. Results suggestive of a similar mechanism for sucrose accumulation in vacuoles from Vitus sp. pericarp protoplasts, however, were obtained recently from a compartmental efflux analysis of sugars (9). A series of membrane-bound reactions, culminating in the vectorial release of Suc-P inside the vacuoles, was postulated to account for the data. We now present evidence that group translocation is the primary mechanism by which sucrose is deposited in sugar- Abbreviations: Suc-P, sucrose phosphate; UDP-, GDP-, and ADP-Glc, UDP, GDP, and ADP glucose; Fru-6-P, fructose 6- phosphate; 3-O-MeGlc, 3-O-methylglucose; Glc-6-P, glucose 6- phosphate; Glc-1-P, glucose 1-phosphate; UDP-Gal, UDP galactose; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PCMBS, p- chloromercuriphenylsulfonic acid. 4697

4698 Botany: Thom and Maretzki cane vacuoles and that the process is specifically dependent on uptake and binding of UDP-Glc and only UDP-Glc. MATERIALS AND METHODS Plant Material. Sugarcane cell suspensions (a subclone of Saccharum sp. hybrid H5-729) were grown in White's inorganic salt mixture supplemented with yeast extract, arginine, sucrose, vitamins, and 2,4-dichlorophenoxyacetic acid (1). Cells were harvested 9-11 days after subculture. Chemicals. UDP-[14C (U)]Glc, ADP-[14C (U)]Glc, UDP galactose as UDP-[14C (U)]Gal, 3-O-methylglucose as 3-- Me[14C (U)]Glc, [14C (U)]glucose, and [14C (U)]fructose were obtained from New England Nuclear. Enzymes and organic chemicals were from Sigma. Protoplast and Vacuole Isolation. Protoplasts were isolated from suspension cultures as described (11) and resuspended in White's basal salt medium containing.5 M mannitol at ph 5.6 (medium A). Vacuoles were isolated from protoplasts by using a modification of the method described by Lorz et al. (12). Briefly, the protoplast suspension was layered over a cushion of 12% Ficoll made up in medium A and centrifuged for 6 min in a Beckman SW 41 rotor at 4, rpm. Vacuoles were recovered at the /12% Ficoll interface and washed three times in medium A by centrifugation at 5 x g. Vacuole numbers were determined by counting an appropriate dilution in a Sedgewick-Rafter counting chamber. Uptake Measurements. Vacuoles were resuspended in medium A. The ph was adjusted to 5.7 for the earlier experiments and to 6.5 for later experiments. Uptake was measured in a 5-ml volume in 25-ml Erlenmeyer flasks on a rotary shaker (75 rpm) at 25 C. Uptake was initiated by the addition of vacuoles. Aliquots (1 ml) were withdrawn at intervals (usually after 1-, 3-, 5-, and 7-min incubations). Vacuoles were separated from incubation medium by centrifugation through silicone oil (13). Radioactivity in the vacuole pellet and an aliquot of the supernatant was determined by scintillation spectrometry. In all experiments, uptake rates were calculated from the slope ofthe time course curve. Products of uptake into vacuoles and in the incubation medium were separated by paper chromatography in 1- butanol/acetic acid/h2, 4:1:5 (vol/vol), upper phase. Chromatograms were cut into 1-cm sections, and the radioactivity was determined for each section. Preparation of Labeled Sugar Phosphates. Radioactive Suc-P was prepared from [14C]Fru-6-P and UDP-Glc in the presence of Suc-P synthase. Suc-P synthase was purified from wheat germ by using a modification of the method described by Harbron et al. (14). The protein precipitating between 26% and 36% (NH4)2SO4 was dialyzed and placed over a column of AH-Sepharose 4B. The column was washed with.5 M N-(2-acetoamido)2-iminodiacetic acid (ADA)/ KOH buffer (ph 6.9), and Suc-P synthase was eluted with 1. M NaCl. The enzyme was purified '6-fold and was free of acid phosphatase and invertase. After [14C]Fru-6-P and UDP-Glc were incubated with the purified enzyme as described by Hawker and Hatch (15), the reaction mixture was streaked on Whatman 3 MM paper and developed twice in 4:1:5 1-butanol/acetic acid/h2, upper phase. The radioactive sugar phosphate band was eluted with 5% ethanol and concentrated under vacuum. The Suc-P sample was contaminated with sucrose (9%) and Fru-6-P (<2%). Labeled Fru-6-P, glucose 6-phosphate (Glc-6-P), and glucose 1-phosphate (Glc-1-P) were prepared enzymatically from the free hexose. The reaction mixtures were purified by paper chromatography as described above. No free sugars were present in any of the preparations. Proc. NatL Acad ScL USA 82 (1985) RESULTS Uptake of Sucrose and Its Metabolic Precursors. The uptake of sucrose and its metabolic precursors into isolated vacuoles is shown in Table 1. The uptake of UDP-Glc was 1 times higher than the glucose uptake rate, regardless of whether ATP and UTP were added to the glucose incubation medium. None of the sugar phosphates, alone or in combination with other added substances that could promote the biosynthetic transfer of substrate to sucrose, was taken up. This was surprising, particularly in the case of Fru-6-P incubated in the presence of UDP-Glc, since this combination provided the substrates for Suc-P synthase and since UDP-Glc was readily taken up. The failure of Suc-P to be taken up by vacuoles implies that sucrose phosphatase is not critical for sucrose transfer across the tonoplast. No extravacuolar hydrolysis was detected after a 7-min incubation with sugar phosphates. The uptake rates of glucose and 3--MeGlc, a nonmetabolizable analog of glucose in sugarcane cells, were similar. This is evidence that sucrose transfer into isolated vacuoles is not dependent on membrane-associated metabolism of free glucose. Sucrose was taken up by isolated vacuoles, but at a rate appreciably lower than that for glucose. Metabolic Products of UDP-Glc Uptake. The metabolites of UDP-Glc taken up by isolated vacuoles were determined in the vacuoles and in the incubation medium after removal of vacuoles. A UDP-Glc preparation uniformly labeled in the glucose moiety was supplied to vacuoles so that any sugar resulting from its metabolism would be labeled (Table 2). Vacuoles incubated with UDP-Glc for 7 min and extracted with 7% ethanol retained 5% of the radioactivity in the insoluble residue. Sugars in the soluble extract chromatographically distributed between phosphates (43%), free sucrose (49%), and glucose (8%). Upon hydrolysis of the extract with alkaline phosphatase, an additional 24% of the presumed sugar phosphates migrated as sucrose and, to a small extent, as glucose. Almost 7o of the soluble radioactivity after hydrolysis could be accounted for by sucrose. The nature of the alkaline phosphatase-resistant substance is not known. It could be that the alkaline phosphatase reaction did not go to completion. Table 1. Uptake of sucrose and its metabolic precursors Labeled Uptake rate, compound Addition nmol/min per 16 vacuoles UDP-Glc -.68 UDP-Glc Fru-6-P.67 Glucose.6 Glucose ATP + UTP + Mg2+.6 3-O-MeGlc.5 Fructose -.3 Fructose ATP + UTP + Mg2+.2 Sucrose.2 Sucrose ATP + Mg2+.2 Suc-P Suc-P ATP + Mg2+ Glc-6-P Glc-6-P Fru-6-P + UTP + Mg2+ Glc-1-P Glc-1-P Fru-6-P + UTP + Mg2+ Fru-6-P Fru-6-P UDP-Glc Uptake was measured in S ml of medium A adjusted to ph 5.6 and containing 5,uM substrate. Uptake was initiated by the addition of vacuoles (".5 x 16 vacuoles per ml of incubation medium). Samples were withdrawn at intervals, and the uptake rates were determined from the slope of the time course curve. Concentration of added Fru-6-P and UDP-Glc was 5 AtM, ofatp and UTP was 1 AM, and of Mg2" was 2 AM.

Botany: Thom and Maretzki Table 2. Percentage distribution of radioactivity in vacuoles and in incubation medium Vacuoles Medium Before After Before After hydrolysis hydrolysis hydrolysis hydrolysis Sugar phosphates/ UDP-Glc 42.9 18.9 73.1 2.4 Sucrose 48.9 69.8 23. 28.8 Glucose 8.1 11.3 4. 3.4 Fructose 2.2 Vacuoles were incubated in medium A adjusted to ph 5.6 and Uptake was terminated after a containing 5 um UDP-[14C]Glc. 7-min incubation. Vacuole extract and incubation medium were separated by chromatography before and after hydrolysis with insoluble alkaline phosphatase. Chromatograms were cut into 1-cm sections, and the distribution of radioactivity was determined. After a 7-min incubation of the vacuole preparation in 5 AM UDP-Glc, a large proportion (i.e., 6%) of the initial radioactivity remained in the medium. However, 23% of the radioactivity cochromatographed with sucrose and only 4% with glucose. Nearly 2% of the presumed phosphorylated pool resisted phosphatase hydrolysis; hence, only a small amount of UDP-Glc remained in the medium after a 7-min incubation. In contrast to the intravacuolar extract, most of the sugar phosphates were of hexose rather than sucrose origin. These results suggest that UDP-Glc is readily metabolized in the presence of isolated vacuoles. The possibility that UDP-Glc metabolism occurs at the outer surface of the vacuole with release of the products into the medium and subsequent synthesis of products into sucrose is disallowed since none of the products added alone or in combination caused sucrose to accumulate inside the vacuole (Table 1). The same situation holds true for cytoplasmic contaminants in the vacuole preparation. The mechanism must involve binding of UDP-Glc to the cytoplasmic surface of the tonoplast and directional release of products, culminating in sucrose transfer into the vacuole. The process is presented in the proposed scheme shown in Fig. 1. Suc-P is probably the initial product deposited in vacuoles and is later hydrolyzed to sucrose by sucrose phosphatase. Fate of the released phosphate cannot be determined at this time, but it is most likely exported from the vacuoles, since the amount of sucrose found in sugarcane cells shows no correlation with cellular phosphate concentration (16). Proc. NatL Acad Sci USA 82 (1985) 4699 Table 3. Uptake of nucleotide sugars Uptake rate, Substrate nmol/min per 16 vacuoles UDP-Glc.737 UDP-Gal.32 ADP-Glc Uptake was measured as described in Table 1. Nucleotide sugar concentration was 5,uM. Specificity of the UDP-GIc Binding Site. The binding site on the tonoplast was specific for UDP-Glc (Table 3). ADP-Glc was not taken up, and UDP-Gal was taken up at 5% of the UDP-Glc uptake rate. GDP glucose (GDP-Glc), added at a concentration 1 times higher than UDP-Glc, did not inhibit UDP-Glc uptake (results not shown). These results suggest that the group translocator for sucrose has a specific requirement for uridine sugar nucleotide and that galactose will not substitute for glucose in the molecule. Kinetics of UDP-GIc Uptake. The uptake of UDP-Glc showed saturation kinetics (Fig. 2) with a Km of.7 mm and a V.. of 11.1 nmol/min per 16 vacuoles. Since this is merely the first step prior to a series of interconversions that UDP-Glc must undergo on or in the membrane, the measured values may not represent rates of sucrose transfer. UDP-Glc serves as a substrate for Suc-P synthase and is also the source for Fru-6-P required to complete sucrose transfer; therefore, the maximum velocity for sucrose transfer would be only half of the calculated value-i.e., 5.5 nmol/min per 16 vacuoles. This value is considerably higher than the in situ rate of.2 nmol/min per 16 vacuoles found previously (6). The previ- 1.5-1...5. 1 1 1/S Sucrose + P sd liinside Suc-P E C / 61 Glu-6-P Fru-6-P I + Tonoplost Glu-`!-P -* UDPG UDPG -I O Outside FIG. 1. Proposed scheme of sucrose transfer into vacuoles. UDPG, UDP-Glc. 1 2 3 4 5 6 UDP-GIc, mm FIG. 2. Concentration dependence of UDP-Glc uptake. Uptake was measured in 5 ml of medium A adjusted to ph 6.5. (Inset) V, velocity; S, substrate.

47 Botany: Thomand Maretzki co E 8-7- ID 3-2- / 5 6 7 8 ph FIG. 3. ph dependence of UDP-Glc uptake. Uptake was measured in S ml of medium A adjusted to the appropriate ph. UDP-Glc concentration was 2 AtM. ous report was based on cultures in which the cytoplasmic UDP-Glc concentration was less than.15 mm-a value at which uptake would have been far below maximum velocity. ph Dependence of UDP-Glc Uptake. The cytoplasmic ph of sugarcane cells is near 7 (17). The optimum ph for UDP-Glc uptake by the vacuole preparation was between 6.5 and 7 (Fig. 3). This is circumstantial evidence that cytoplasmic ph is not limiting for UDP-Glc uptake. Potential Inhibitors of UDP-Glc Uptake. The sulflhydryl inhibitor, p-chloromercuriphenylsulfonic acid (PCMBS), and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), were added to vacuole suspensions at zero time. PCMBS completely prevented uptake of UDP-Glc, whereas CCCP at.5 mm permitted 46% of the normal UDP-Glc uptake rate (Table 4). PCMBS acts as a specific inhibitor of the sucrose carrier in Vicia faba (18) and Beta vulgaris (19), although these are proton symport systems. Partial inhibition by high concentrations of CCCP is not consistent with a clear-cut electrogenic transport mechanism involving proton transfer, but CCCP may be acting as a sulfhydryl inhibitor. Both UDP and GDP were effective inhibitors of UDP-Glc uptake (Table 4). These compounds could be acting as Table 4. Effect of inhibitors on UDP-Glc uptake Inhibitor Concentration, Uptake rate, added mm nmol/min per 16vacuoles None 4.86 PCMBS.5 CCCP.5 2.22 CCCP.5 3.3 UDP 2..9 GDP 2..3 Uptake was measured in 5 ml of medium A adjusted to ph 6.5 and containing 2 AuM UDP-['4C]Glc and inhibitor. Uptake was initiated by the addition ofvacuoles (-.5 x 16 vacuoles per ml of incubation medium). Samples were withdrawn at intervals, and the uptake rates were determined from the slope of the time course curve. Proc. NatL Acad Sd USA 82 (1985) allosteric inhibitors of a reaction within the membrane or could compete with UDP-Glc for a common binding site on the tonoplast. The failure of GDP-Glc to act as a competitor of UDP-Glc uptake (results not shown) makes the second hypothesis unlikely. DISCUSSION Results presented here suggest existence of a multienzyme system in or on the tonoplast of sugarcane cells for the vacuolar storage of sucrose. This system appears to represent the principal mechanism by which sucrose accumulates. UDP-Glc was the only molecule that triggered the process of sucrose transfer into the vacuole. This molecule uniquely provided both substrates (UDP-Glc as well as Fru-6-P) for the formation of Suc-P. To do so, several enzymes must be involved, each ofthem attached to or buried within the matrix ofthe membrane, At least two enzymes are candidates forthe conversion of UDP-Glc to Glc-i-P. UDP-Glc phosphorylase would form UDP, whereas UDP-Glc pyrophosphorylase would result in UTP. Phosphoglucose isomerase, phosphoglucomutase, and Suc-P synthase are usually associated with the cytosol. The appearance of both fructose and glucose phosphates in the medium when UDP-Glc was incubated with vacuoles for a few minutes is circumstantial evidence that these enzymes are also tonoplast-bound. The fact that none of the hexose phosphates was taken up by vacuoles is proof that the binding sites for these compounds are not exposed to the cytoplasmic surface. The final enzyme needed for sucrose deposition in vacuoles, sucrose phosphatase, may be a tonoplast-bound enzyme (inner surface). The series of enzymes necessary for this vectorial group translocator to function may span the membrane. Evidences for sidedness of the reactions involved are: (i) the apparent optimum uptake of UDP-Glc at cytoplasmic ph, (it) the inhibition of UDP-Glc uptake by PCMBS, a nonpenetrating sulfhydryl inhibitor, (fig) the predominant release of hexose phosphates to the exterior of the vacuole, and (iv) the predominant release of Suc-P and sucrose to the interior of the vacuole. The partial release into the medium of hexose phosphates generated from UDP-Glc by vacuole preparations stands in contrast to lack of hexose phosphate utilization for sucrose formation when these substances are supplied to the vacuole incubation medium together with UDP-Glc. This contradiction could have a number of explanations. (t) Sucrose phosphate synthesis may be the limiting reaction within the enzyme complex, and diffusion of sugar phosphates from the medium cannot compete with similar molecules derived from UDP-Glc and already bound to reaction sites within the complex. This would imply a configurational orientation of the group translocator that makes it unable to accept free sugar phosphates. (il) The passage of sugar phosphates through the tonoplast is unidirectionally determined by lipophilic charges within the membrane. (iii) Sugar phosphates in the medium originate from broken vacuoles. The data presented cannot distinguish among these alternatives. A picture, similar to the one proposed by Brown and Coombe (9), in which the membrane becomes a central dynamic vehicle for the formation as well as the transfer of sucrose is presented. However, in the case of grape pericarp vacuoles, cytoplasmic contamination could account for the results since synthesis of sucrose from glucose would require ATP, UTP, and Mg2, substances not available in isolated vacuole preparations. Kinetically, the driving force for the process is determined by the overall equilibrium of the individual reaction segments favoring the release of sucrose inside the vacuole. Glasziou and Gaylor (1) postulated that sucrose uptake into vacuoles is mediated by sucrose phosphatase and the hydrolysis of Suc-P is an integral part of the

Botany: Thom and Maretzki transfer system. Evidence presented here suggests that Suc-P is hydrolyzed after its entry into vacuoles. To what extent and how the system maintains a thermodynamic equilibrium remains one of the many questions to which answers must now be sought. In all, five enzymes in the tonoplast are postulated to participate in the formation and transfer of sucrose into vacuoles. Membrane group translocators that involve more than two enzymes are presently not recognized (2), although enzyme complexes of this magnitude are not considered unusual (21). A membrane matrix may be conducive to maintenance ofcorrect orientation of a multienzyme structure, so that molecular channeling of substrates and products through a highly organized complex becomes feasible. Nothing is presently known about the genesis of a membrane that can independently function in the catalysis of a reaction sequence. Sugarcane is a plant efficient in sucrose storage, balancing this process against vigorous growth. Consequently, the plant must have evolved growth and storage systems that make energetically efficient use of available photosynthate. We suggest that group translocation through a membrane, involving a number of enzymes, none directly dependent on ATP, may provide a system that optimizes the available energy supply. In addition, the evolution of a membrane that is essentially impermeable to a molecule such as sucrose would mean that less energy is required to retain the accumulated substance against a concentration gradient. It is unlikely that sugarcane is unique in the plant kingdom in having a group translocator for the process of vacuolar loading. Published with the approval of the Director as paper no. 586 in the Journal Series of the Experiment Station, Hawaiian Sugar Planters' Association. Proc. Natd Acad Sc. USA 82 (1985) 471 1. Glasziou, K. T. & Gayler, K. R. (1972) Bot. Rev. 38, 471-49. 2. Reinhold, L. & Kaplan, A. (1984) Annu. Rev. Plant Physiol. 35, 45-83. 3. Doll, S., Rodier, F. & Willenbrink, J. (1979) Planta 144, 47-411. 4. Kaiser, G. & Heber, U. (1984) Planta 161, 562-568. 5. Thom, M. & Komor, E. (1984) FEBS Lett. 173, 1-4. 6. Thom, M., Komor, E. & Maretzki, A. (1982) Plant Physiol. 69, 132-1325. 7. Kornberg, H. L. (1973) Proc. R. Soc. London Ser. B 18, 15-123. 8. Quinlan, D. C., Li, C. C. & Hochstadt, T. (1976) J. Supramol. Struct. 4, 427-439. 9. Brown, S. C. & Coombe, B. G. (1984) Physiol. Veg. 22, 231-24. 1. Nickell, L. G. & Maretzki, A. (1969) Physiol. Plant. 22, 117-125. 11. Thom, M., Maretzki, A. & Komor, E. (1982) Plant Physiol. 69, 1315-1319. 12. LUrz, H., Harms, C. T. & Potrykus, I. (1976) Biochim. Physiol. Pflanz. 169, 617-62. 13. Werden, K., Heldt, H. W. & Geller, G. (1972) Biochim. Biophys. Acta 283, 43-441. 14. Harbron, S., Foyer, C. & Walker, D. (1981) Arch. Biochem. Biophys. 212, 237-246. 15. Hawker, J. S. & Hatch, M. D. (1975) Methods Enzymol. 42, 341-347. 16. Thom, M., Maretzki, A., Komor, E. & Sakai, W. S. (1981) Plant Cell Tissue Otgan Culture 1, 3-14. 17. Komor, E., Thom, M. & Maretzki, A. (1981) Planta 153, 181-192. 18. Delrot, S., Despeghel, J. P. & Bonnemain, J. L. (198) Planta 149, 144-148. 19. Giaquinta, R. T. (1976) Plant Physiol. 57, 872-875. 2. Lo, T. C. Y. (1977) J. Supramol. Struct. 7, 463-48. 21. Welch, G. R. (1977) Prog. Biophys. Mol. Biol. 32, 13-191.