Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.

Starch Synthesis in Potato Tubers Is Regulated by Post-Translational Redox Modification of ADP-Glucose Pyrophosphorylase: A Novel Regulatory Mechanism Linking Starch Synthesis to the Sucrose Supply

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It was recently shown that potato (Solanum tuberosum) tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular Cys bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farre, P. Geigenberger [2002] Plant Cell 14: 2191-2213). We show here that AGPase is also subject to posttranslational regulation in leaves of pea (Pisum sativum), potato, and Arabidopsis. Conversion is accompanied by an increase in activity, which involves changes in the kinetic properties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer when preilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves via the petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced in the starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPB monomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novel posttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels in the leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recycling of phosphate during photosynthetic carbon fixation and sucrose synthesis.

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ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other Plant species

The key regulatory step in starch biosynthesis is catalyzed by the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase). In leaf and storage tissue, the enzyme catalyzes the synthesis of ADP-glucose from glucose-l-phosphate and ATP. Using heterologous probes from maize, two sets (B and S) of cDNA clones encoding potato AGPase were isolated from a tuber-specific cDNA library. Sequence analysis revealed homology to other plant and bacterial sequences. Transcript sizes are 1.9 kb (AGPase B) and 2.1 kb (AGPase S). Northern blot experiments show that the two genes differ in their expression patterns in different organs. Furthermore, one of the genes (AGPase S) is strongly inducible by metabolizable carbohydrates (e.g. sucrose) at the RNA level. The accumulation of AGPase S mRNA was always found to be accompanied by an increase in starch content. This suggests a link between aGPase S expression and the status of a tissue as either a sink for or a source of carbohydrates. By contrast, expression of AGPase B is much less variable under various experimental conditions.

One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose.

In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of α-1,4-glucosidic chains. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). Generally, this enzyme is allosterically regulated by intermediates of the major carbon assimilatory pathway in the respective organism. In oxygenic photosynthesizers, ADPGlc PPase is mainly regulated by 3-phosphoglycerate (activator) and inorganic orthophosphate (inhibitor), interacting in four different patterns. Recent reports have shown that in higher plants, some of the enzymes could also be redox regulated. In eukaryotes, the enzyme is a heterotetramer comprised of two distinct subunits, a catalytic and a modulatory subunit. The latter has been proposed as related to variations in regulation of the enzyme in different plant tissues. Random and site-directed mutagenesis experiments of conserved amino acids revealed important residues for catalysis and regulation. Prediction of the ADPGlc PPase secondary structure suggests that it shares a common folding pattern to other sugar-nucleotide pyrophosphorylases, and they evolved from a common ancestor.

ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis.

Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally. In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These observations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Ds-induced sh2 allele conditions at least two transcripts in the endosperm.

Identification and molecular characterization of shrunken-2 cDNA clones of maize.

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C3-NH2). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg2+ are reported. The enzyme Vmax is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.

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Joseph R. Sowokinos

Papers

Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES.