Intracellular site of sucrose synthesis in leaves

Bird, I. F.; Cornelius, M. J.; Keys, A. J.; Whittingham, C. P.

doi:10.1016/s0031-9422(00)91267-6

Cited by 98 publications

(25 citation statements)

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“…The close association between the intercellular distribution of M6PR and that of Rubisco is also in accord with the proposed role of M6PR in assimilating newly fixed carbon that is being exported from the plastids. SPS plays a parallel role in the cytosolic SUC synthetic pathway (Bird et al, 1974;Robinson and Walker, 1979;Usuda and Edwards, 1980;Stitt et al, 1987), but as yet there are no reports of immunocytochemical localization of this enzyme. Such reports are considered to be essential if a protein is to be incontrovertibly identified as cytosolic (Herman, 1988), and they would also help resolve the apparent differences in bundle sheath and mesophyll distributions of SPS in C4 species (Stitt et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

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Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

1993

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991 64-4238 (V.R.F.)Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens 1.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (MCPR), and immunocytochemical techniques, we have resolved both the intercellular and intracellular sites of mannitol synthesis. In leaves, MCPR was found only in cells containing ribulose-l,5-bisphosphate carboxylase/oxygenase. MCPR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by MCPR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that MCPR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of MCPR is, therefore, in the assimilation of carbon being exported from the chloroplast, and i t seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.The acyclic sugar alcohols (polyols) are second only to the sugars as principal products of carbon assimilation and account for an estimated 30% of global primary production (Bieleski, 1982). In a11 polyol-producing higher plants studied to date, Suc is also a major photoassimilate. In some species, sucrosyl-oligosaccharides (eg. raffinose) are found in the phloem sap in addition to the polyol and SUC. The nature of primary production and transport in higher plant polyol producers raises fundamental questions as to why such an apparently complex system has evolved when Suc alone performs adequately in many other species. Questions are also raised as to how carbon partitioning into polyols and other primary products is regulated at a biochemical, cellular, and tissue level. Celery (Apium graveolens L.) is a fine model in which to study such questions, because mature leaves

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Section: Discussionmentioning

confidence: 99%

“…There is substantial evidence to indicate that Suc synthesis occurs in the cytosol (Bird et al, 1974;Robinson and Walker, 1979;Usuda and Edwards, 1980;Stitt et al, 1987, and refs. therein), although until the early 1970s it was believed that the site of synthesis was the chloroplast (Lewis, 1984;Stitt et al, 1987).…”

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Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

1993

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“…Some of its kinetic properties have been postulated to be pertinent to its regulation in vivo (19, 23). However, very little has been published on the kinetic properties of the leaf sucrose-P synthase, even though the enzyme activity has been detected in various leaves (2,6,9,13,16,17,20).The following describes a partial purification of the spinach leaf sucrose-P synthase and its preliminary kinetic characterization. Emphasis of the studies were directed to determining possible regulatory modes of the enzymic activity.…”

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confidence: 99%

“…The sucrose biosynthetic enzymes, UDPglucose pyrophosphorylase and sucrose-P phosphatase, are located almost exclusively in the cytosol (2,21). Synthesis of sucrose in the leaf, therefore, would be dependent on the photosynthetic products, 3-P-glycerate and triose-P, formed in the chloroplast during photosynthesis that are able to permeate the chloroplast membrane (5,11,12).…”

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Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase

Amir

Preiss²

1982

Plant Physiol.

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The spinach (Spinaca oeracca) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturatio curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At The biosynthesis of sucrose-P was first reported by Leloir and Cardini (14) to occur via transfer of glucose from UDPglucose to fructose-6-P. Hawker and Hatch (10) and Hawker (7) subsequently reported the presence in plant tissues of a specific sucrose-6-P phosphatase. Earlier labeling studies (1, 3, 4) suggested that, in leaves, sucrose was synthesized from hexose phosphates and not from free sugars in the cell, and that implicated sucrose-P synthase as the enzyme responsible for sucrose synthesis in plants.The enzyme isolated from wheat germ has been highly purified and studied in great detail (18,23). Some of its kinetic properties have been postulated to be pertinent to its regulation in vivo (19, 23). However, very little has been published on the kinetic properties of the leaf sucrose-P synthase, even though the enzyme activity has been detected in various leaves (2,6,9,13,16,17,20).The following describes a partial purification of the spinach leaf sucrose-P synthase and its preliminary kinetic characterization. Emphasis of the studies were directed to determining possible regulatory modes of the enzymic activity. Hepes (pH 7.5); 20 mmi f-mercaptoethanol; 2 mm EDTA and 2% ethyleneglycol. The extraction was carried out in a vacuum flask under an atmosphere of N2. After the extract had been squeezed through a cheese cloth, it was centrifuged at 16,000g for 20 min, and the supernatant fluid was saved. This solution was poured onto a DEAE-cellulose column (2.5 x 20 cm), previously equilibrated with a buffer solution, containing 20 mm Hepes-NaOH (pH 7.5), 10%o ethyleneglycol, 2 mm Na2EDTA, and 5 mM f,-mercaptoethanol. The column was washed with about 200 ml of the above buffer, and the enzyme was eluted with a 500 ml linear NaCl gradient (0 to 0.5 M) in the same buffer. The fractions with the sucrose-P synthase activity were pooled and concentrated in an Amicon TCF 10 thin-channel cell with a PM-10 membrane. After concentration to about 5 ml, the enzyme fraction was diluted to 20 ml with the above DEAE cellulose equilibration buffer. The enzyme was frozen and stored in liquid N2.Enzyme Assays.Assay of Sucrose-P Synthase. The reaction mixture contained the following: 5 ,umol Hepes-NaOH (pH 7.5); 1.2 ,Amol fructose-6-P; 1.2 umol UDP-["4C]glucose (specific activity, 100,000 to 400,000 cpm/4mol); 0.2 mg BSA; 0.5 ,umol MgCl2; and enzyme; in a total volume of 0.125 ml. After 20 min at 37°C, the reaction was stopped by immersing the tubes in boiling water for 1 min. The sucrose-6-P formed was hydrolyzed to sucrose with E. coli alkaline phosphatase (0.1 unit/assay). After incubation for 30 min at 37°C, 0.4 ml of a 5% sucrose solution was added. The diluted reaction mixture was loaded onto a l-ml DEAE cellulose column and eluted with 5 ml H20...

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Photosynthesis Carbon Metabolism

Latzko

Kelly

Thirty Years of Photosynthesis 1974–2004

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Intracellular site of sucrose synthesis in leaves

Cited by 98 publications

References 37 publications

Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase

Photosynthesis Carbon Metabolism

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