ADP-glucose Synthesis Research Articles

Editing of the starch synthase IIa gene led to transcriptomic and metabolomic changes and high amylose starch in barley

In this study, a range of barley allelic mutants lost ADPG binding structure of starch synthase IIa (SSIIa) were created through targeted mutagenesis of SSIIa by RNA-guided Cas9. The transcriptomic and qRT-PCR results showed the increased mRNA expression of HvGBSSI and the decreased HvSSIIa and HvSBEI levels in ssIIa mutant grains, which were consistent with the expressions of GBSSI, SSS and SBE enzymatic activities, respectively. However, the increased expressions of HvSSI cannot effectively compensate for the loss of HvSSIIa. The metabolic pathway analysis showed that the mutation of SSIIa led to increased ADP-glucose synthesis in barley grains. The ssIIa mutant grains had two and six times amylose, and RS contents in control grains, respectively, and significantly changed starch structure and functions compared to the controls. No metabolite changes could compensate for the decrease of starch biosynthesis in the ssIIa null mutant.

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria.

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.

Role of the inter‐subunit surface interaction in the regulation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens

ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) is a key regulatory enzyme involves in the starch and glycogen synthesis for plants and bacteria respectively, which catalyzes the synthesis of ADP‐glucose from glucose 1‐phosphate (Glc1P) and ATP. ADP‐Glc PPase from Agrobacterium tumefaciens is allosterically regulated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). Previously, it was observed that removal of a disulfide bridge between the α‐subunits of the potato tuber enzyme resulted in a more active enzyme [1], indicating that inter‐subunit interactions are important in the allosteric mechanism [2]. By studying the structure of the ADP‐Glc PPase enzyme of Agrobacterium tumefaciens, it comes to light that D141 is interacting with the R11 of the opposing subunit [3], which was suggested to be involved in activation by pyruvate [4]. To prove our hypothesis that D141 and R11 residues are involved in regulation, we made three single mutants: D141A, D141R, R11D, and the double mutant R11D‐D141R. D141A exhibited reduced Vmax values in the absence of activators (~4‐fold), reduced apparent affinity for Fru6P (~2.5‐fold), and lower apparent affinity for ATP (~3‐fold). In the presence of Fru6P, D141A exhibited only ~4.3% of the wild‐type activity, and affinity for ATP was not affected as well indicating a virtual insensitivity to Fru6P. When D141R was compared to wild‐type enzyme, the activity lowered ~3600‐fold. For D141R the apparent affinity for ATP was decreased in the presence of Fru6P. R11D was similarly active as the wild‐type, while it is completely insensitive to both activators Fru6P and Pyr. The double mutant R11D‐D141R was showing decreased activity (~95‐fold) in compared to wild type, and insensitive to both activators. Our results suggest that the functional role of the D141 and R11 residues is to interacting with the opposing subunit and enabling the regulation of the ADP‐Glc PPase.Support or Funding InformationNational Science Foundation

On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.

Transcriptional coordination and abscisic acid mediated regulation of sucrose transport and sucrose-to-starch metabolism related genes during grain filling in wheat (Triticum aestivum L.)

Combining physiological, molecular and biochemical approaches, this study investigated the transcriptional coordination and abscisic acid (ABA) mediated regulation of genes involved in sucrose import and its conversion to starch during grain filling in wheat. Sucrose import appears to be mediated by seed localized TaSUT1, mainly TaSUT1D, while sucrose cleavage by TaSuSy2. Temporal overlapping of the transcriptional activation of AGPL1 and AGPS1a that encode AGPase with that of the above genes suggests their significance in the synthesis of ADP-glucose; TaAGPL1A and TaAGPL1D contributing the majority of AGPL1 transcripts. ABA induced repressions of TaSUT1, TaSuSy2, TaAGPL1 and TaAGPS1a imply that ABA negatively regulates sucrose import into the endosperm and its subsequent metabolism to ADP-glucose, the substrate for starch synthesis. The formations of amyloses and amylopectin from ADP-glucose appear to be mediated by specific members of GBSS, and SS, SBE and DBE gene families, and the ABA-induced transcriptional change in most of these genes implies that ABA regulates amylose and amylopectin synthesis. The findings provide insights into the molecular mechanisms underlying the coordination and ABA mediated regulation of sucrose transport into the developing endosperm and its subsequent metabolism to starch during grain filling in wheat.

HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM-AGP pathway.

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

Intracellular Glycogen Assays

Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen breakdown is mediated by glycogen phosphorylase (GlgP) and debranching enzyme (GlgX), which catalyze the sequential phosphorolysis of α-1,4-glucosyl linkages in the glucan chain from the non-reducing ends and debranching of the limit dextrins generated by GlgP, respectively. An increasing number of studies have revealed the involvement of glycogen metabolism in a multitude of physiological functions in some prokaryotes beyond the function of synthesizing energy storage compounds. Lactobacillus acidophilus NCFM was the first probiotic lactic acid bacterium demonstrated to possess a functional glycogen biosynthesis pathway that is involved in its growth, bile tolerance and complex carbohydrate metabolism (Goh and Klaenhammer, 2013). The following qualitative (for detection of intracellular glycogen) and quantitative (for measurement of intracellular glycogen content) intracellular glycogen assay protocols for Lactobacillus acidophilus (L. acidophilus) were modified from previous works (Govons et al., 1969; Law et al., 1995; Parrou and Francois, 1997) and should be applicable to other lactic acid bacteria as well as most microorganisms.

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.

Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002

ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2–4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.

Reply to Smith et al.: No evidence to challenge the current paradigm on starch and cellulose biosynthesis involving sucrose synthase activity

The generally accepted view that sucrose synthase (SUS) activity is involved in the production of cellulose and starch biosynthesis (1 and references contained therein) has been challenged (2). Using a method for assaying SUS activity in the synthetic direction (conversion of UDP-glucose into sucrose), Barratt et al. (2) reported an almost total absence of SUS activity in sus1/sus2/sus3/sus4 Arabidopsis mutant stems and the presence of WT levels of starch and cellulose content in this organ. Bieniawska et al. (3) reported that SUS activities in WT Arabidopsis leaves were too low to account for the starch accumulation rate occurring during illumination. Barratt et al. (2) thus concluded that SUS is not required for starch and cellulose biosynthesis in Arabidopsis. However, in our study (1), we used a method for assaying SUS activity in the sucrose cleavage direction (UDP-glucose and ADP-glucose synthesis) and found that SUS activity in WT Arabidopsis leaves was approximately 10-fold higher than that reported by Bieniawska et al. (3), greatly exceeding the minimum needed to support the normal rate of starch accumulation during illumination. Furthermore, we found that SUS activities in the insoluble and soluble fractions of sus1/sus2/sus3/sus4 stems were approximately 10- and 100-fold higher, respectively, than those reported by Barratt et al. (2). Most importantly, we also found that SUS activity in the leaves and stems of the sus1/sus2/sus3/sus4 mutants was approximately 85% of those of WT plants, thus concluding that (i) most of SUS activity in Arabidopsis leaves and stems is not attributable to SUS1–4 and (ii) SUS activity in sus1/sus2/sus3/sus4 mutants is sufficient to support normal cellulose and starch biosynthesis. As discussed in our paper (1), we ascribed such marked differences between our results and those of Barratt et al. (2) and Bieniawska et al. (3) to the inadequate method and conditions for assaying SUS activity used by these investigators rather than to differences in the assay temperature. In their letter, Smith et al. (4) surmise that the method for assaying SUS activity should not distract attention when formulating conclusions about the role of SUS in starch and cellulose metabolism, and asserted that we ignored their immunolocalization studies of SUS1–6. However, we consider that the use of an inadequate method for assaying SUS activity is not a minor issue, because conclusions by Barratt et al. (2) and Bieniawska et al. (3) were strongly based on the use of such methods. Studies of SUS1–6 location conducted by Barratt et al. (2) and Bieniawska et al. (3) were carried out assuming the yet to be demonstrated conceptual idea that SUS1–6 are the sole proteins with SUS activity in Arabidopsis. Therefore, as indicated in our paper (1), further endeavors based on the production of plants totally lacking SUS activity and on the use of adequate SUS activity assay method(s) will be necessary to investigate the involvement of SUS activity in starch and cellulose metabolism. In conclusion, in our opinion, no pressing biological evidence has been presented by Barratt et al. (2) to challenge the current paradigm on cellulose and starch metabolism involving SUS activity. In this context, we must emphasize that Angeles-Nunez and Tiessen (5) have shown that SUS2 and SUS3 are required for channeling carbon toward ADP-glucose and starch in Arabidopsis seeds.

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124-13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1-4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING ( Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from <15 to 117% of wild type (WT), and that the kinetic properties of the enzyme were altered in several lines, indicating a role for the substituted amino acid residues in catalysis or substrate binding. These results validate the concept of using such a platform for efficient high-throughput screening of very large populations of mutants, natural accessions or introgression lines. AGPase was estimated to have a flux control coefficient of 0.20, indicating that the enzyme exerted only modest control over the rate of starch synthesis in plants grown under short day conditions (8 h light/16 h dark) with an irradiance of 150 μmol quanta m −2 s −1. Redox activation of the enzyme, via reduction of the intermolecular disulphide bridge between the two small subunits, was increased in several lines. This was sometimes, but not always, associated with a decrease in the abundance of the APS1 protein. In conclusion, the TILLING technique was used to generate an allelic series of aps1 mutants in A. thaliana that revealed new insights into the multi-layered regulation of AGPase. These mutants offer some advantages over the available loss-of-function mutants, e.g. adg1, for investigating the effects of subtle changes in the enzyme's activity on the rate of starch synthesis.

Studies of the Kinetic Mechanism of Maize Endosperm ADP-Glucose Pyrophosphorylase Uncovered Complex Regulatory Properties

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.

The Debate on the Pathway of Starch Synthesis: A Closer Look at Low-Starch Mutants Lacking Plastidial Phosphoglucomutase Supports the Chloroplast-Localized Pathway

ADPglucose (ADPGlc) is the substrate for starch synthesis in the plastids of higher plants. The glucosyl moiety is used by starch synthases to elongate the glucans that comprise starch. Recently, there has been renewed debate about the ADPGlc synthesis, with the widely accepted or classical pathway

The evolution of the starch biosynthetic pathway in cereals and other grasses

In most species, the precursor for starch synthesis, ADPglucose, is made exclusively in the plastids by the enzyme ADPglucose pyrophosphorylase (AGPase). However, in the endosperm of grasses, including the economically important cereals, ADPglucose is also made in the cytosol via a cytosolic form of AGPase. Cytosolic ADPglucose is imported into plastids for starch synthesis via an ADPglucose/ADP antiporter (ADPglucose transporter) in the plastid envelope. The genes encoding the two subunits of cytosolic AGPase and the ADPglucose transporter are unique to grasses. In this review, the evolutionary origins of this unique endosperm pathway of ADPglucose synthesis and its functional significance are discussed. It is proposed that the genes encoding the pathway originated from a whole-genome-duplication event in an early ancestor of the grasses.

Developmental analysis of maize endosperm proteome suggests a pivotal role for pyruvate orthophosphate dikinase.

Although the morphological steps of maize (Zea mays) endosperm development are well described, very little is known concerning the coordinated accumulation of the numerous proteins involved. Here, we present a proteomic study of maize endosperm development. The accumulation pattern of 409 proteins at seven developmental stages was examined. Hierarchical clustering analysis allowed four main developmental profiles to be recognized. Comprehensive investigation of the functions associated with clusters resulted in a consistent picture of the developmental coordination of cellular processes. Early stages, devoted to cellularization, cell division, and cell wall deposition, corresponded to maximal expression of actin, tubulins, and cell organization proteins, of respiration metabolism (glycolysis and tricarboxylic acid cycle), and of protection against reactive oxygen species. An important protein turnover, which is likely associated with the switch from growth and differentiation to storage, was also suggested from the high amount of proteases. A relative increase of abundance of the glycolytic enzymes compared to tricarboxylic acid enzymes is consistent with the recent demonstration of anoxic conditions during starch accumulation in the endosperm. The specific late-stage accumulation of the pyruvate orthophosphate dikinase may suggest a critical role of this enzyme in the starch-protein balance through inorganic pyrophosphate-dependent restriction of ADP-glucose synthesis in addition to its usually reported influence on the alanine-aromatic amino acid synthesis balance.

New enzymes, new pathways and an alternative view on starch biosynthesis in both photosynthetic and heterotrophic tissues of plants

Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor in the reaction catalyzed by starch synthase, the mechanism of starch biosynthesis in both leaves and heterotrophic organs has generally been considered to be an unidirectional process wherein ADPG pyrophosphorylase (AGPase) exclusively catalyzes the synthesis of ADPG and acts as the major limiting step of the gluconeogenic process. There is however mounting evidence that ADPG linked to starch biosynthesis is produced de novo in the cytosol by means of sucrose synthase (SuSy). In this review we show and discuss the numerous pitfalls of the ‘classic’ view of starch biosynthesis. In addition, we describe many overlooked aspects of both ADPG and starch metabolism. With the overall data we propose an ‘alternative’ model of starch biosynthesis, applicable to both photosynthetic and heterotrophic tissues, according to which both sucrose and starch biosynthetic processes are tightly interconnected by means of an ADPG synthesizing SuSy activity. According to this new view, starch metabolism embodies catabolic and anabolic reactions taking place simultaneously in which AGPase plays a vital role in the scavenging of starch breakdown products.

Sucrose Synthase Controls Both Intracellular ADP Glucose Levels and Transitory Starch Biosynthesis in Source Leaves

The prevailing model on transitory starch biosynthesis in source leaves assumes that the plastidial ADPglucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule, ADPG. However, recent investigations have shown that ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol. This finding is consistent with the occurrence of an 'alternative' gluconeogenic pathway wherein sucrose synthase (SuSy) is involved in the production of ADPG in the cytosol, whereas both plastidial phosphoglucomutase (pPGM) and AGP play a prime role in the scavenging of starch breakdown products. To test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato wild-type (WT) leaves with those of the starch-deficient mutants with reduced pPGM and AGP. These analyses provided evidence against the 'classical' model of starch biosynthesis, since ADPG levels in all the starch-deficient lines were normal compared with WT plants. Whether or not SuSy is involved in the synthesis of ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and SuSy-antisensed transgenic leaves. Importantly, SuSy-overexpressing leaves exhibited a large increase of both ADPG and starch levels compared with WT leaves, whereas SuSy-antisensed leaves accumulated low amounts of both ADPG and starch. These findings show that (i) ADPG produced by SuSy is linked to starch biosynthesis; (ii) SuSy exerts a strong control on the starch biosynthetic process; and (iii) SuSy, but not AGP, controls the production of ADPG accumulating in source leaves.