Oxidation of Imported or Endogenous Carbohydrates by Isolated Chloroplasts from Green Pepper Fruits

Recently, we demonstrated that intact chloroplasts isolated from green pepper (Capsicum annum L.) fruits use exogenous glucose-6-phosphate (Glc-6-P) as the most efficient precursor for starch biosynthesis (O. Batz, R. Scheibe, H.E. Neuhaus [1995] Planta 196: 50-57). Here we demonstrate that these chloroplasts transport this hexose phosphate in counter-exchange for orthophosphate. By measuring the release of 14CO2 from [1-14C]Glc-6-P, we show that isolated fruit chloroplasts also use exogenous Glc-6-P as a substrate for the oxidative pentose-phosphate pathway. The rate of decarboxylation appears to be linear with time and is significantly reduced in the presence of Triton X-100, indicating that the reaction is dependent on plastid integrity. Pyruvate has been identified as a positive effector for flux through the oxidative pentose-phosphate pathway. However, the highest rates of Glc-6-P-driven oxidative pentosephosphate pathway activity are achieved in the presence of nitrite, 2-oxoglutarate, and glutamine, indicating a strong interaction between nitrogen metabolism and this pathway. In addition, we show that carbohydrates liberated during starch mobilization are used as substrates for the oxidative pentose-phosphate pathway. Orthophosphate was found to act as an activator for the observed 14CO2 release from carbohydrates formerly bound as starch. In this context, we demonstrate that exogenous Glc-6-P competes with endogenous carbohydrates. A possible interaction between exogenous and endogenous carbohydrates is discussed with respect to altered levels of carbohydrates during fruit development.     

Glucose-1-phosphate transport into protoplasts and chloroplasts from leaves of Arabidopsis

Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.     

Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation

We isolated pgi1-1, an Arabidopsis mutant with a decreased plastid phospho-glucose (Glc) isomerase activity. While pgi1-1 mutant has a deficiency in leaf starch synthesis, it accumulates starch in root cap cells. It has been shown that a plastid transporter for hexose phosphate transports cytosolic Glc-6-P into plastids and expresses restricted mainly to the heterotrophic tissues. The decreased starch content in leaves of the pgi1-1 mutant indicates that cytosolic Glc-6-P cannot be efficiently transported into chloroplasts to complement the mutant's deficiency in chloroplastic phospho-Glc isomerase activity for starch synthesis. We cloned the Arabidopsis PGI1 gene and showed that it encodes the plastid phospho-Glc isomerase. The pgi1-1 allele was found to have a single nucleotide substitution, causing a Ser to Phe transition. While the flowering times of the Arabidopsis starch-deficient mutants pgi1, pgm1, and adg1 were similar to that of the wild type under long-day conditions, it was significantly delayed under short-day conditions. The pleiotropic phenotype of late flowering conferred by these starch metabolic mutations suggests that carbohydrate metabolism plays an important role in floral initiation.     

Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos

Measurements of metabolic fluxes in whole embryos and isolated plastids have revealed major changes in the pathways of carbon utilization during cotyledon filling by oilseed rape (Brassica napus L.) embryos. In the early cotyledon stage (stage A), embryos used sucrose (Suc) predominantly for starch synthesis. Plastids isolated from these embryos imported glucose-6-phosphate (Glc-6-P) and partitioned it to starch and fatty acids synthesis and to the oxidative pentose phosphate pathway in the ratio of 2:1:1 on a hexose basis. Of the substrates tested, Glc-6-P gave the highest rates of fatty acid synthesis by the plastids and pyruvate was used weakly. By the mid- to late-cotyledon stage (stage C), oil accumulation by the embryos was rapid, as was their utilization of Suc for oil synthesis in vitro. Plastids from C-stage embryos differed markedly from those of stage-A embryos: (a) pyruvate uptake and utilization for fatty acid synthesis increased by respectively 18- and 25-fold; (b) Glc-6-P partitioning was predominantly to the oxidative pentose phosphate pathway (respective ratios of 1:1:3); and (c) the rate of plastidial fatty acid synthesis more than doubled. This increased rate of fatty synthesis was dependent upon the increase in pyruvate uptake and was mediated through the induction of a saturable transporter activity.     

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.     

Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters.

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.     

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-¹⁴C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-¹⁴C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [³³P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.     

Competitive inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate

In plant cells, the reversible isomerization between fructose 6-phosphate (Fru6P) and glucose 6-phosphate (Glc6P) is catalyzed by a cytosolic and a chloroplastic isoenzyme of phosphoglucose isomerase (PGI, EC 5.3.1.9). The extractable activities of both PGI isoenzymes are in large excess compared with the flux required for product synthesis, but the measured Glu6P/Fru6P ratio in illuminated chloroplasts and in whole leaves is always displaced from equilibrium. Cytosolic (PGI 2) and stromal (PGI 1) isoenzymes were purified from spinach leaves and used to investigate the possibility of metabolic regulation at this step. Several metabolites were found to inhibit PGI, but within the physiological concentration range, only erythrose 4-phosphate (Ery4P) inhibited significantly. The inhibition was competitive, with K_i values below 10 μM for PGI 2 and 1. The physiological significance of the inhibition of PGI by Ery4P was assessed in isolated intact spinach chloroplasts. We conclude that, in vivo, this inhibition is probably responsible for the observed displacement from equilibrium in the chloroplasts, but limits the carbon flow towards starch synthesis only when Fru6P is low. In contrast, the inhibition by Ery4P is unlikely to play any role in the cytosolic carbon metabolism because both Fru6P concentration and PGI activity, are much higher than in the chloroplast stroma.     

Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.     

Comparison of the metabolic properties of plastids isolated from developing leaves or embryos of Brassica napus L

Plastids isolated from developing leaves and embryos of oilseed rape (Brassica napus L.) were incubated with substrates in the light or the dark, with or without exogenous ATP. Incorporation of HCO^-3, and carbon from a range of substrates into fatty acids and/or starch by leaf chloroplasts was absolutely light-dependent and was unaffected by provision of ATP. Incorporation of HCO^-3 into fatty acids and/or starch by embryo plastids was also light-dependent. However, the light-dependent rates attained, when expressed on a comparable basis, were less than 32% of those from Glc6P (plus ATP), which was the most effective substrate for starch and fatty acid synthesis. In the light alone the rates of carbon incorporation from Glc6P, pyruvate and acetate into fatty acids, and from Glc6P into starch by embryo plastids were less than 27% of the respective ATP-dependent (dark) rates. Light had no effect on these ATP-dependent rates of synthesis by embryo plastids. While transporter activities for both glucose and Glc6P were present in embryo plastids, leaf chloroplasts did not have the latter activity. It is concluded that light at in vivo levels can contribute energy to carbon metabolism in embryo plastids. However, this contribution is likely to be small and these plastids are therefore largely dependent upon interaction with the cytosol for the ATP, reducing power and carbon precursors that are required for maximal rates of starch and fatty acid synthesis.     

Unidirectional transport of orthophosphate across the envelope of isolated cauliflower-bud amyloplasts

Isolated amyloplasts from cauliflower (Brassica oleracea L. var botrytis) buds are able to export orthophosphate unidirectionally into the incubation medium. This orthophosphate transport appears to be protein-mediated, as indicated by the following observations: (i) low temperature and the presence of inhibitors of protein-mediated transport reduced the rate of orthophosphate export, and (ii) the rate of orthophosphate export became saturated with rising internal substrate concentrations. Micromolar concentrations of 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid inhibited the rate of unidirectional orthophosphate export, thus indicating the involvement of the amyloplastic glucose-6-phosphate (Glc6P)translocator in the unidirectional export of orthophosphate. The effect of rising concentrations of orthophosphate upon the activity of ADP glucose pyrophosphorylase in desalted extracts was determined. Orthophosphate given in concentrations similar to those measured in the amyloplastic stroma under conditions of steady-state rates of Glc6P-dependent starch synthesis inhibited the activity of ADP-glucose pyrophosphorylase significantly. However, even under strong limiting substrate conditions the residual activity was sufficient to catalyze the flux of carbon into starch. The maximal rates of orthophosphate transport (in the counter-exchange mode) by isolated spinach (Spinacia oleracea L.) chloroplasts and by isolated cauliflower-bud amyloplasts were also determined. These rates were compared with the maximal rates of undirectional orthophosphate export by these plastids. From these measurements we can conclude that, compared with spinach chloroplasts, isolated amyloplasts of cauliflower exhibit a fivefold greater ratio of unidirectional orthophosphate transport to maximal rate of orthophosphate transport in the counter-exchange mode compared to spinach chloroplasts. The determined rate of maximal unidirectional orthophosphate export is sufficient to catalyze the release of additional inorganic phosphate liberated in the amyloplastic stroma during the process of Glc6P-dependent starch synthesis.     

Photosynthetic carbon metabolism of isolated corn chloroplasts

Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO₂ per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of ¹⁴C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO₂ at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.     

Maltose metabolism by pea chloroplasts

The aim of this work was to investigate the origin of maltose formed during starch breakdown in the dark by chloroplasts of Pisum sativum. The maximum catalytic activities of maltose phosphorylase and maltase in pea leaves were shown to be low, relative to those of enzymes known to be involved in starch breakdown. Fractionation of pea leaves indicated that the chloroplasts lack maltase but have enough maltose phosphorylase to synthesize the amounts of maltose formed when isolated chloroplasts breakdown starch. The absence of exogenous phosphate markedly reduced starch breakdown and maltose accumulation by isolated chloroplasts. When [¹⁴C]glucose was supplied to chloroplasts that were breaking down starch in the dark, maltose was labelled and most of the label was in the glucose moeity. It is suggested that maltose phosphorylase, using glucose-1-phosphate formed from starch by α-glucan phosphorylase, is responsible for, at least some of, the synthesis of maltose during starch breakdown by pea chloroplasts in vitro.     

A possible rate-limiting function of chloroplast hexosemonophosphate isomerase in starch synthesis of leaves

Levels of fructose 6-phosphate and glucose 6-phosphate were measured in chloroplasts which had been isolated non-aqueously from leaves of various plants. a large decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate in the light indicated considerable displacement of the hexosephosphate isomerase reaction from equilibrium in leaves of spinach and red beet which were photosynthesizing at high rates. The decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate was correlated with an increase in the chloroplastic level of 3-phosphoglyceric acid, which proved to be a competitive inhibitor of chloroplast hexosephosphate isomerase. Other metabolites, especially the product of the reaction, glucose 6-phosphate, and ions in concentrations as present in the stroma under natural conditions, cause a further reduction in the rate of the forward reaction of the hexosemonophosphate isomerase. When the concentration of O₂ in air was decreased from 21 to 2%, both the rate of leaf photosynthesis and the ratio of glucose 6-phosphate to fructose 6-phosphate increased, whereas the concentration of 3-phosphoglyceric acid and starch synthesis decreased. The results are explained in terms of activation of ADPglucose pyrophosphorylase and of inhibition of hexosephosphate isomerase by 3-phosphoglyceric acid. Hexosephosphate isomerase appears to assume a rate-limiting function in starch synthesis in the light when ADPglucose pyrophosphorylase is activated.     

Phosphate Translocator of Isolated Guard-Cell Chloroplasts from Pisum sativum L. Transports Glucose-6-Phosphate

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.     

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.