ADP-glucose Pyrophosphorylase Activity Research Articles

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

Production of high‐starch, low‐glucose potatoes through over‐expression of the metabolic regulator SnRK1

Transgenic potato (Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene (SnRK1) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%-167% compared with that in the wild-type. Glucose levels were decreased, at 17%-56% of the levels of the wild-type, and the starch content showed an increase of 23%-30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, alpha-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%-60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.

Genetic modification of cassava for enhanced starch production

To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava (Manihot esculenta Crantz). Cassava has one of the highest rates of CO(2) fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source-sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.

Combined Transcript and Metabolite Profiling of Arabidopsis Leaves Reveals Fundamental Effects of the Thiol-Disulfide Status on Plant Metabolism

In this study, we used gas chromatography-mass spectrometry analysis in combination with flux analysis and the Affymetrix ATH1 GeneChip to survey the metabolome and transcriptome of Arabidopsis (Arabidopsis thaliana) leaves in response to manipulation of the thiol-disulfide status. Feeding low concentrations of the sulfhydryl reagent dithiothreitol for 1 h at the end of the dark period led to posttranslational redox activation of ADP-glucose pyrophosphorylase and major alterations in leaf carbon partitioning, including an increased flux into major respiratory pathways, starch, cell wall, and amino acid synthesis, and a reduced flux to sucrose. This was accompanied by a decrease in the levels of hexose phosphates, while metabolites in the second half of the tricarboxylic acid cycle and various amino acids increased, indicating a stimulation of anaplerotic fluxes reliant on alpha-ketoglutarate. There was also an increase in shikimate as a precursor of secondary plant products and marked changes in the levels of the minor sugars involved in ascorbate synthesis and cell wall metabolism. Transcript profiling revealed a relatively small number of changes in the levels of transcripts coding for components of redox regulation, transport processes, and cell wall, protein, and amino acid metabolism, while there were no major alterations in transcript levels coding for enzymes involved in central metabolic pathways. These results provide a global picture of the effect of redox and reveal the utility of transcript and metabolite profiling as systemic strategies to uncover the occurrence of redox modulation in vivo.

Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase

Trehalose is the most widespread disaccharide in nature, occurring in bacteria, fungi, insects, and plants. Its precursor, trehalose 6-phosphate (T6P), is also indispensable for the regulation of sugar utilization and growth, but the sites of action are largely unresolved. Here we use genetic and biochemical approaches to investigate whether T6P acts to regulate starch synthesis in plastids of higher plants. Feeding of trehalose to Arabidopsis leaves led to stimulation of starch synthesis within 30 min, accompanied by activation of ADP-glucose pyrophosphorylase (AGPase) via posttranslational redox modification. The response resembled sucrose but not glucose feeding and depended on the expression of SNF1-related kinase. We also analyzed transgenic Arabidopsis plants with T6P levels increased by expression of T6P synthase or decreased by expression of T6P phosphatase (TPP) in the cytosol. Compared with wild type, leaves of T6P synthase-expressing plants had increased redox activation of AGPase and increased starch, whereas TPP-expressing plants showed the opposite. Moreover, TPP expression prevented the increase in AGPase activation in response to sucrose or trehalose feeding. Incubation of intact isolated chloroplasts with 100 muM T6P significantly and specifically increased reductive activation of AGPase within 15 min. Results provide evidence that T6P is synthesized in the cytosol and acts on plastidial metabolism by promoting thioredoxin-mediated redox transfer to AGPase in response to cytosolic sugar levels, thereby allowing starch synthesis to be regulated independently of light. The discovery informs about the evolution of plant metabolism and how chloroplasts of prokaryotic origin use an intermediate of the ancient trehalose pathway to report the metabolic status of the cytosol.

Effect of sucrose on activity of starch synthesis enzymes in rice ears in culture

Grain filling of rice (Oryza sativa L.) is thought to be affected not only by the supply of carbohydrate from source organs, but also by sink activity, including the activities of enzymes related in starch synthesis. To examine the effect of translocated carbohydrate on sink activity, we cultured detached ears (cut 7 days after flowering) in a solution containing sucrose at various concentrations, glutamine and vitamins. Grain weights 16 days after the start of ear culture increased with increasing sucrose concentration up to 50–70 g sucrose l−1, but not above this range, probably because of low water potential. Activities of ADP glucose pyrophosphorylase, soluble starch synthase and sucrose synthase rapidly decreased in grains grown without sucrose, but increased with increasing sucrose concentration up to 50–70 g l−1. Expression of mRNAs for these enzymes and for sucrose transporter was also generally increased by sucrose at 50–70 g l−1. Though there was a good correlation between activity and expression of mRNA for sucrose synthase, the correlation for other enzymes was not very good. Thus, we suggested that the sink activity of grains in rice ears, and therefore the grain filling, was affected by the supply of carbohydrate from source organs, and that it was also owing to factors other than transcriptional regulation.

Effects of long-term soil drought on photosynthesis and carbohydrate metabolism in mature robusta coffee ( Coffea canephora Pierre var. kouillou) leaves

Four clones of robusta coffee representing drought-tolerant (14 and 120) and drought-sensitive (46 and 109A) genotypes were submitted to a slowly imposed water deficit. Sampling and measurements were performed when predawn leaf water potential ( Ψ pd) approximately −2.0 and −3.0 MPa was reached. Regardless of the clone evaluated, drought led to sharper decreases in stomatal conductance than in photosynthesis, which was accompanied by significant declines in internal to ambient CO 2 concentration ratio. Little or no effect of drought on chlorophyll a fluorescence parameters was observed. Regardless of the stress intensity, starch decreased remarkably. This was not accompanied by significant changes in concentration of soluble sugars, with the exception of clone 120 in which a rise in sucrose and hexose concentrations was found when Ψ pd reached −3.0 MPa. At Ψ pd = −2.0 MPa, activity of acid invertase increased only in clone 120; at Ψ pd = −3.0 MPa, it increased in clones 14, 46 and 120, while activity of sucrose synthase declined, but only in clone 109A. Drought-induced decrease in ADP-glucose pyrophosphorylase activity was found only in clones 14 and 46, irrespective of stress intensity. At Ψ pd = −3.0 MPa, maximal extractable and activation state of sucrose-phosphate synthase (SPS) decreased in all clones with the exception of clone 120, in which SPS activity was maintained in parallel to a rising activity of fructose-1,6-bisphosphatase. Changes in SPS activity could neither be explained by the CO 2 decrease linked to stomatal closure nor by differences in leaf water status.

Positional cues for the starch/lipid balance in maize kernels and resource partitioning to the embryo

This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect assimilate partitioning and ATP distribution within the kernel. These questions were addressed through combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis. Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2 concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation (approximately 3.8 microm), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for peripheral localization of the protein and lipid storing structures in maize kernels.

Roles of isoamylase and ADP‐glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.

Temporal and Spatial Variations of Carbohydrate Content in Rice Leaf Sheath and Their Varietal Differences

In rice plant, carbohydrates accumulated in leaf sheaths before heading are translocated to grain and affect yield formation greatly. To clarify the intrinsic mechanism of carbohydrate metabolism in the leaf sheath, we investigated the temporal and spatial variations of carbohydrate metabolism in the third leaf sheath counted from the top and their varietal differences. The results revealed that the amount of carbohydrate decreased from the base to the tip of the leaf sheath, irrespective of variety and developmental stage. However, the proportion of starch content in the basal one-fifth of the leaf sheath to that in the whole sheath varied from 35% to 60% with the variety. Comparing the activities of enzymes related to starch metabolism at the base, middle and tip of the leaf sheath in IR65598-112-2 (New plant type) with those in Nipponbare, the activities of ADP-glucose pyrophosphorylase, branching enzyme and granule-bound starch synthase (GBSS) showed varietal differences. Particularly, the activity of GBSS may play an important role in the varietal difference in spatial variation of starch content in the leaf sheath. In IR65598-112-2, the sucrose content in the leaf sheath was extremely high, suggesting that sucrose may be one of the carbohydrate reserves in this line.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves.

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg-1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

CO2 Assimilation, Photosynthetic Enzymes, and Carbohydrates of `Concord' Grape Leaves in Response to Iron Supply

Own-rooted 1-year-old `Concord' grapevines (Vitis labruscana Bailey) were fertigated twice weekly for 11 weeks with 1, 10, 20, 50, or 100 μm iron (Fe) from ferric ethylenediamine di (o-hydroxyphenylacetic) acid (Fe-EDDHA) in a complete nutrient solution. As Fe supply increased, leaf total Fe content did not show a significant change, whereas active Fe (extracted by 2,2′-dipyridyl) content increased curvilinearly. Chlorophyll (Chl) content increased as Fe supply increased, with a greater response at the lower Fe rates. Chl a: b ratio remained relatively constant over the range of Fe supply, except for a slight increase at the lowest Fe treatment. Both CO2 assimilation and stomatal conductance increased curvilinearly with increasing leaf active Fe, whereas intercellular CO2 concentrations decreased linearly. Activities of key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase (FBPase), and a key enzyme in sucrose synthesis, cytosolic FBPase, all increased linearly with increasing leaf active Fe. No significant difference was found in the activities of ADP-glucose pyrophosphorylase (AGPase) and sucrose phosphate synthase (SPS) of leaves between the lowest and the highest Fe treatments, whereas slightly lower activities of AGPase and SPS were observed in the other three Fe treatments. Content of 3-phosphoglycerate (PGA) increased curvilinearly with increasing leaf active Fe, whereas glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and the ratio of G6P: F6P remained unchanged over the range of Fe supply. Concentrations of glucose, fructose, sucrose, starch, and total nonstructural carbohydrates (TNC) at both dusk and predawn increased with increasing leaf active Fe. Concentrations of starch and TNC at any given leaf active Fe content were higher at dusk than at predawn, but both glucose and fructose showed the opposite trend. No difference in sucrose concentration was found at dusk or predawn. The export of carbon from starch breakdown during the night, calculated as the difference between dusk and predawn measurements, increased as leaf active Fe content increased. The ratio of starch to sucrose at both dusk and predawn also increased with increasing leaf active Fe. In conclusion, Fe limitation reduces the activities of Rubisco and other photosynthetic enzymes, and hence CO2 assimilation capacity. Fe-deficient grapevines have lower concentrations of nonstructural carbohydrates in source leaves and, therefore, are source limited.

Adjustment of diurnal starch turnover to short days: depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period.

A larger proportion of the fixed carbon is retained as starch in the leaf in short days, providing a larger store to support metabolism and carbon export during the long night. The mechanisms that facilitate this adjustment of the sink-source balance are unknown. Starchless pgm mutants were analysed to discover responses that are triggered when diurnal starch turnover is disturbed. Sugars accumulated to high levels during the day, and fell to very low levels by the middle of the night. Sugars rose rapidly in the roots and rosette after illumination, and decreased later in the light period. Global transcript profiling revealed only small differences between pgm and Col0 at the end of the day but large differences at the end of the night, when pgm resembled Col0 after a 4-6 h prolongation of the night and many genes required for biosynthesis and growth were repressed [Plant J. 37 (2004) 914]. It is concluded that transient sugar depletion at the end of the night inhibits carbon utilization at the start of the ensuing light period. A second set of experiments investigated the stimulation of starch synthesis in response to short days in wild-type Col0. In short days, sugars were very low in the roots and rosette at the end of the dark period, and after illumination accumulated rapidly in both organs to levels that were higher than in long days. The response resembles pgm, except that carbohydrate accumulated in the leaf as starch instead of sugars. A similar response was found after transfer from long to short days. Inclusion of sugar in the rooting medium attenuated the stimulation of starch synthesis. Post-translational activation of ADP-glucose pyrophosphorylase (AGPase) was increased in pgm, and in Col0 in short days. It is concluded that starch synthesis is stimulated in short day conditions because sugar depletion at the end of the night triggers a temporary inhibition of growth and carbohydrate utilization in the first part of the light period, leading to transient accumulation of sugar and activation of AGPase.

CO2 Assimilation, Photosynthetic Enzymes, and Carbohydrates of Grape Leaves (Vitis labrusca L. cv. Concord) in Response to Iron Supply

Own-rooted one-year-old `Concord' grapevines were fertigated twice weekly for 11 weeks with 1, 10, 20, 50, OR 100 μmol iron (Fe) from ferric ethylenediamine di (o-hydroxyphenylacetic) acid in a complete nutrient solution. As Fe supply increased, leaf total Fe content did not change, whereas active Fe (extracted by 2, 2'-dipyridyl) and total chlorophyll content increased curvilinearly. CO2 assimilation and stomatal conductance increased curvilinearly with increasing active Fe, whereas intercellular CO2 concentrations decreased linearly. Activities of key Calvin cycle enzymes, Rubisco, NADP-glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, stromal fructose-1,6-bisphosphatase (FBPase), and a key enzyme in sucrose synthesis, cytosolic FBPase, all increased linearly with increasing active Fe. No difference was found in the activities of ADP-glucose pyrophosphorylase and sucrose phosphate synthase of leaves between the lowest and the highest treatments, whereas slightly lower activities were observed in the middle Fe treatments. Content of 3-phosphoglycerate increased curvilinearly with increased active Fe, whereas glucose-6-phosphate and fructose-6-phosphate did not change. Glucose, fructose, sucrose, starch, and total non-structural carbohydrates at both dusk and pre-dawn increased with increasing active Fe. Carbon export from starch breakdown during the night, calculated as the difference between dusk and predawn levels, increased as active Fe increased. In conclusion, Fe limitation reduces the activities of Rubisco and other photosynthetic enzymes, and hence CO2 assimilation capacity. Fe-deficient grapevines have lower concentrations of non-structural carbohydrates in source leaves, and therefore, are source limited.

The maize Sh2r6hs ADP-glucose pyrophosphorylase (AGP) large subunit confers enhanced AGP properties in transgenic wheat ( Triticum aestivum)

The rate of cereal seed starch biosynthesis is determined largely by the activity of starch biosynthetic enzymes within the endosperm. The heterotetrameric allosteric enzyme ADP-glucose pyrophosphorylase (AGP) controls the first committed step in starch synthesis, and is inhibited by phosphate (P i) and activated by 3-PGA. An altered AGP large subunit ( Sh2r6hs) from maize containing two alterations relative to the wild-type maize Sh2 sequence was introduced into wheat. The rev6 alteration confers reduced P i inhibition and decreased heat stability in maize and the hs alteration has been shown to increase AGP subunit interactions and enzyme stability in Escherichia coli. We have previously shown that low-level expression of Sh2r6hs under control of the maize Sh2 promoter increases yield in wheat and rice. Our objective here was to examine how Sh2r6hs affects AGP activity and yield in wheat and whether further yield increases were possible with greater Sh2r6hs expression. We therefore developed an additional population of transgenic wheat expressing SH2R6HS under control of the endosperm specific wheat Glutenin ( Glu) promoter. Yield increases in high-expressing Glu promoter Sh2r6hs lines were comparable to those with the Sh2 promoter despite the fact that the Glu promoter confers ∼20 times higher levels of Sh2r6hs expression than the Sh2 promoter and five times more SH2R6HS accumulation versus native wheat large AGP subunits. However, the high expression allowed endosperm AGP kinetic analysis. The I 0.5 of wheat AGP was increased from 2.0 to 4.1 mM P i by SH2R6HS expression and the A 0.5 was decreased from 0.82 to 0.31 mM 3-PGA. Two properties of Sh2r6hs AGP were identified that indicate greater subunit stability: wheat AGP small subunit levels were higher without variation in small subunit transcript levels, and in vitro activity was twice as great as untransformed AGP activity after heat treatment. Both r6 and hs alterations act independently and synergistically to modify AGP allosteric-regulation, increase AGP subunit stability, and increase yield in plants expressing Sh2r6hs.

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.

Changes in Photosynthesis and Carbohydrate Metabolism in Mature Apple Leaves in Response to Whole Plant Source-Sink Manipulation

Photosynthesis and carbohydrate metabolism in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] source leaves were monitored during a 7-day period after source-sink manipulations by girdling or partial defoliation treatments. In the girdling treatment, sorbitol, sucrose, glucose, and starch accumulated in leaves, and net photosynthetic rates (Pn) at 350 μL·L-1 CO2 decreased during a 7-day period. Pn measured at 1000 μL·L-1 [CO2] was also decreased but the changes were less. Stomatal conductance and intracellular CO2 concentration decreased markedly in leaves of girdled shoots. When shoots were partially defoliated, starch and glucose concentrations in remaining source leaves declined steadily during the 7-day study period. Sorbitol and sucrose concentrations decreased during the first 2 days after defoliation, then increased the following 5 days. Pn of the remaining leaves measured at ambient and elevated CO2 levels were enhanced markedly. Aldose-6-phosphate reductase activity in source leaves increased markedly from 27.5 to 39.2 μmol·h-1·g-1 fresh weight (FW) after partial defoliation but remained unchanged in leaves after girdling. Selective and maximum sucrose phosphate synthase (SPS) activities increased following partial defoliation and decreased following girdling. ADP-glucose pyrophosphorylase activity remained relatively unchanged in the partial defoliation treatments but increased markedly in the girdled-shoot leaves. These results suggested that girdling-induced photosynthetic inhibition is mainly due to stomatal limitation, however, the photosynthesis enhancement by partial defoliation may be due primarily to acceleration of photosynthetic capacity per se. These studies showed that the metabolism of sorbitol, sucrose and starch, three photosynthetic end products in mature apple leaves, was coordinately regulated in source leaves in response to source-sink manipulations.

Effect of Sucrose Solution Injection at Different Growth Stages on Production and ADP-glucose Pyrophosphorylase Activity of Roots in Sweet Potato, Ipomoea batatas Lam. Cultivars.

Effect of Sucrose Solution Injection at Different Growth Stages on Production and ADP-glucose Pyrophosphorylase Activity of Roots in Sweet Potato, Ipomoea batatas Lam. Cultivars.

Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase.

In this work we test the hypothesis that yield of rice ( Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGP large subunit sequence ( Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat stability, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10-15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.