Comparison of Starch and ADP-Glucose Pyrophosphorylase Levels in Nonembryogenic Cells and Developing Embryos from Induced Carrot Cultures

Cultures of carrot (Daucus carota L.) in a medium without added 2,4-dichlorophenoxyacetic acid were separated into fractions of embryos at different stages of development (large globular and heart, torpedo, and germinating) and nonembryogenic cells. The average starch content per cell in these fractions was similar. However, due to the smaller sizes of the cells of the embryos relative to the nonembryogenic cells, starch content per weight of tissue was higher in the embryos. The ADP-glucose pyrophosphorylase activity per cell in the nonembryogenic cells was double that of the embryo cells. Furthermore, the ratio of ADP-glucose pyrophosphorylase to starch was over 2-fold higher in the nonembryogenic cells, indicating that starch content is not simply determined by ADP-glucose pyrophosphorylase levels. ADP-glucose pyrophosphorylase activity of all culture fractions was directly proportional to the level of a single 50 kilodalton polypeptide detected by immunoblot analysis, using antiserum raised to the purified spinach leaf enzyme. In the same immunoblot analysis, novel polypeptides of 63 and 100 kilodalton were detected in embryos but were absent from nonembryogenic cells. This is one of the few reported examples of specific proteins which differentially accumulate in embryos and nonembryogenic cells.     

Pyrophosphorylases in Solanum tuberosum: II. CATALYTIC PROPERTIES AND REGULATION OF ADP-GLUCOSE AND UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITIES IN POTATOES

Pyrophosphorylytic kinetic constants (S_0.5, V_max) of partially purified UDP-glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S_0.5 of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the V_max were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A_0.5 = 4.5 × 10⁻⁶ molar). The S_0.5 for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P-glycerate while the V_max was increased nearly 4-fold. The magnitude of 3-P-glycerate stimulation was dependent upon the integrity of key sulfhydryl groups (−SH) and pH. Oxidation or blockage of −SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I_0.5 = 8.8 × 10⁻⁵. molar). This inhibition was reversed with 3-P-glycerate (1.2 × 10⁻⁴ molar), resulting in an increased I_0.5 value of 1.5 × 10⁻³ molar. Likewise, orthophosphate (7.5 × 10⁻⁴ molar) caused a decrease in the activation efficiency of 3-P-glycerate (A_0.5 from 4.5 × 10⁻⁶ molar to 6.7 × 10⁻⁵ molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of α-glucan biosynthesis in Solanum tuberosum is discussed.     

Pyrophosphorylases in Solanum tuberosum: I. Changes in ADP-Glucose and UDP-Glucose Pyrophosphorylase Activities Associated with Starch Biosynthesis during Tuberization, Maturation, and Storage of Potatoes

Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.     

Starch Synthesizing Enzymes in Chloroplasts of Developing Leaves of Spinach (Spinacea oleracea L.)

In experiments in which RuDP carboxylase activity was used asa marker for the integrity of isolated chloroplasts, more than90% of the starch synthase activity and more than 80% of theADP-glucose pyrophosphorylase activity of spinach leaves wasfound to be located in chloroplasts. Less than 2% of the UDP-glucosepyrophosphorylase was associated with chloroplasts. The activityof starch synthase per chloroplast remained almost constantduring plastid replication in developing leaves on whole plantsand in leaf discs cultured for 7 d on agar under different lightconditions. The ADP-glucose pyrophosphorylase activity of chloroplastsincreased during leaf development and was much lower in dark-growntissues. The results suggest that the synthesis of starch iscontrolled by the synthesis of ADP-glucose pyrophosphorylaseas well as by the previously known control of activity by metabolitessuch as 3-phosphoglyceric acid and inorganic phosphate.     

Enzymic and substrate basis for the anaplerotic step in guard cells

From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K_mpep of the guard cell enzyme is 60 μm at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270μm (=2.2 × 10⁻¹⁵ moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells.     

Taxonomic survey for the presence of ribulose-1,5-bisphosphate carboxylase activity in guard cells

Guard cell pairs were dissected from freeze-dried leaves of plants representing 15 families, including monocots, dicots, and pteridophytes. All three major photosynthetic carbon pathways (C₂, C₄, and Crassulacean acid metabolism) were represented. These individual guard cell pairs were assayed quantitatively for ribulose-1,5-bisphosphate carboxylase specific activity. Assay sensitivity averaged 0.08 picomoles of ribulose-P₂ dependent P-glycerate formation (i.e. 100-fold more sensitive than required to detect the activity present in a single Vicia faba mesophyll cell). The calculated specific activities for guard cells and mesophyll cells averaged 4 and 472 millimoles per kilogram dry weight per hour, respectively. For all species surveyed, (a) the enzyme activity calculated for guard cells was below the detection limit of the assay, or (b) the specific activity (weight or cell basis) calculated for guard cells was less than 1% of the specific activity calculated for adjacent mesophyll cells. Based on this survey, the generalization is made that the photosynthetic carbon reduction pathway is absent, or virtually so, in guard cell chloroplasts.     

Adenosine Diphosphate-Glucose Pyrophosphorylase Control of Starch Accumulation in Rust-infected Wheat Leaves

The variation in starch content in healthy and Puccinia striiformsi-infected wheat leaves was measured from 5 to 15 days after inoculation. The starch content of diseased leaves relative to healthy leaves decreased from 5 to 9 days, increased from 9 to 12 days to twice that of healthy leaves, and decreased from 12 to 15 days after inoculation. Electron micrographs of plant tissues indicated that the starch accumulated in the chloroplasts of host cells adjacent to fungal hyphae. Variations in sugar phosphates, ATP, and inorganic phosphate were measured during the infection process. ADP-glucose pyrophosphorylase was extracted and partially purified from healthy and diseased leaves. When proportionate concentrations of sugar phosphates and inorganic phosphate found in healthy and diseased leaves during the infection process were placed in the assay mixture, ADP-glucose pyrophosphorylase activity was similar to the pattern of starch accumulation and was almost the inverse of the variation observed in inorganic phosphate in diseased leaves during the infection process. A mechanism to explain the accumulation of starch is presented and discussed. This mechanism is based on the regulation of ADP-glucose pyrophosphorylase by changes in effector molecule concentrations during the infection process. Reasons for these changes are presented.     

Stomatal Responses to CO(2) in Paphiopedilum and Phragmipedium: Role of the Guard Cell Chloroplast

A role of the guard cell chloroplasts in the CO₂ response of stomata was investigated through a comparison of the leaf gas exchange characteristics of two closely related orchids: Paphiopedilum harrisianum, which lacks guard cell chloroplasts and Phragmipedium longifolium, which has chlorophyllous guard cells. Leaves of both species had an apparent quantum yield for assimilation of about 0.05, with photosynthesis saturating at 0.300 to 0.400 millimoles per square meter per second. CO₂ curves were obtained by measuring steady-state assimilation and stomatal conductance under 0.180 or 0.053 millimoles per square meter per second white light, or darkness, at 0 to 400 microliters per liter ambient CO₂. The response of assimilation to changes in CO₂ was similar in the two species, but the response of conductance was consistently weaker in Paphiopedilum than in Phragmipedium. The data suggest involvement of guard cell chloroplasts in the stomatal response to CO₂ and in the coupling of assimilation and conductance in the intact leaf.     

Characteristics of plastids responsible for starch synthesis in developing pea embryos

The nature of the starch-synthesising plastids in developing pea (Pisum sativum L.) embryos has been investigated. Chlorophyll and starch were distributed throughout the cotyledon during development. Chlorophyll content increased initially, then showed little change up to the point of drying out of the embryo. Starch content per embryo increased dramatically throughout development. The chlorophyll content per unit volume was highest on the outer edge of the cotyledon, while the starch content was highest on inner face. Nycodenz gradients, which fractionated mechanically-prepared plastids according to their starch content, failed to achieve any significant separation of plastids rich in starch and ADP-glucose pyrophosphorylase from those rich in chlorophyll and a Calvin-cycle marker enzyme, NADP-glyceraldehyde-3-phosphate dehydrogenase. However, material that was not sufficiently dense to enter the gradients was enriched in activity of the Calvin-cycle marker enzyme relative to that of ADP-glucose pyrophosphorylase. Nomarski and epi-fluorescence microscopy showed that intact, isolated plastids, including those with very large starch grains, invariably contained chlorophyll in stromal structures peripheral to the starch grain. We suggest that the starch-storing plastids of developing pea embryos are derived directly from chloroplasts, and retain chloroplast-like characteristics throughout their development. Developing pea embryos also contain chloroplasts which store little or no starch. These are probably located primarily on the outer edge of the cotyledons where there is sufficient light for photosynthesis at some stages of development.     

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.     

Regulation of ADPGlucose Synthesis in Guard Cells of Commelina communis

The activator specificity of the ADPglucose pyrophosphorylase from Commelina communis guard cells is the same as observed for the mesophyll cell enzyme. 3-Phosphoglycerate was found to be the most effective activator. Fifty per cent of maximal stimulation was observed at about 100 micromolar. Inorganic phosphate was found to be a potent inhibitor giving 50% inhibition at 0.3 millimolar. These results are discussed with respect to regulation of starch synthesis in guard cells.     

Pea seed triose phosphate isomerase

Triose phosphate isomerase was purified ca 250-fold from pea seed extracts. The k_m for D-glyceraldehyde-3-P was 0.44 mM and for dihydroxyacetone-P, 0.88 mM. P-enolpyruvate, 2-P-glycerate, 3-P-glycerate and 2-P-glycolate were strongly inhibitory. Pi and arsenate also inhibited pea seed triose phosphate isomerase.     

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.     

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis in higher plants. A 3.2-kb promoter of the large subunit gene of the AGPase from potato has been isolated and its activity analyzed in transgenic potato and tobacco plants using a promoter-beta-glucuronidase fusion system. The promoter was active in various starch-containing cells, including guard cells, tuber parenchyma cells, and the starch sheath layer of stems and petioles. No expression was observed in mesophyll cells. Analysis of various promoter derivatives showed that with respect to expression in petioles and stems, essential elements must be located in the 5' distal region of the promoter, whereas elements important for expression in tuber parenchyma cells are located in an internal fragment comprising nucleotides from positions -500 to -1200. Finally, a 0.3-kb 5' proximal promoter fragment was identified that was sufficient to obtain exclusive expression in guard cells of transgenic potato and tobacco plants. The implications of our observations are discussed with respect to starch synthesis in various tissues and the use of the newly identified promoter as a tool for stomatal biology.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.