Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis Milagros Rodrı´guez-Lo´ pez, Edurne Baroja-Ferna´ ndez, Aitor Zandueta-Criado, and Javier Pozueta-Romero* Instituto de Agrobiotecnologı´ay Recursos Naturales, Universidad Pu´blica de Navarra ͞Consejo Superior de Investigaciones Cientı´ficas,Carretera de Mutilva s͞n, Mutilva Baja, 31192 Navarra, Spain Communicated by Andre´T. Jagendorf, Cornell University, Ithaca, NY, April 13, 2000 (received for review November 28, 1999) A distinct phosphodiesterasic activity (EC 3.1.4) was found in both their possible occurrence in several plant species. As a result, we mono- and dicotyledonous plants that catalyzes the hydrolytic have now found a phosphodiesterasic activity that catalyzes the breakdown of ADPglucose (ADPG) to produce equimolar amounts hydrolytic breakdown of ADPG. In this paper, we report of glucose-1-phosphate and AMP. The enzyme responsible for this the subcellular localization and biochemical characterization of activity, referred to as ADPG pyrophosphatase (AGPPase), was the enzyme responsible for this activity, referred to as ADPG purified over 1,100-fold from barley leaves and subjected to pyrophosphatase (AGPPase).† Based on the results presented in biochemical characterization. The calculated Keq؅ (modified equi- this work using different plant sources, we discuss that AGPPase librium constant) value for the ADPG hydrolytic reaction at pH 7.0 may be involved in controlling the intracellular levels of ADPG and 25°C is 110, and its standard-state free-energy change value linked to starch biosynthesis. kJ). Kinetic analyses showed 4.18 ؍ G؅)is؊2.9 kcal͞mol (1 kcal⌬) that, although AGPPase can hydrolyze several low-molecular Materials and Methods weight phosphodiester bond-containing compounds, ADPG Plant Material. Barley (Hordeum vulgare cv. Scarlett) and wheat ؍ proved to be the best substrate (Km 0.5 mM). Pi and phosphor- (Triticum aestivum cv. Marius) plants were grown in the field. ylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, Seeds at different developmental stages and leaves were har- NADP؉, and AMP are inhibitors of AGPPase. Subcellular localiza- vested and stored at Ϫ80°C until used. Bell pepper (Capsicum tion studies revealed that AGPPase is localized exclusively in the annuum cv. Yolo Wonder), tomato (Lycopersicon sculentum cv. plastidial compartment of cultured cells of sycamore (Acer pseu- Ailsa Craig), potato (Solanum tuberosum cv. De´sire´e), and doplatanus L.), whereas it occurs both inside and outside the Arabidopsis thaliana (cv. Columbia and Adg1) were grown in plastid in barley endosperm. In this paper, evidence is presented greenhouse conditions. Protoplasts and amyloplasts from the that shows that AGPPase, whose activity declines concomitantly suspension-cultured cells of sycamore (Acer pseudoplatanus L.) with the accumulation of starch during development of sink were obtained as described by Pozueta-Romero et al. (6). organs, competes with starch synthase (ADPG:1,4-␣-D-glucan 4-␣- Amyloplasts from young barley endosperms were isolated as PLANT BIOLOGY D-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly block- described by Thorbjornsen et al. (7). ing the starch biosynthesis. Protein Extraction and Purification. Unless otherwise indicated, all lthough the pyrophosphorolytic reactions leading to the steps were carried out at 4°C. For small-scale extractions, plant Aproduction of gluconeogenic intermediates such as ADP- tissues were homogenized with 3-fold of extraction buffer (50 glucose (ADPG) and UDPglucose (UDPG) are readily revers- mM Mes, pH 6.0͞1 mM EDTA͞2 mM DTT) with a Waring ible, they mainly proceed toward the direction of nucleotide Blender, and were filtered through four layers of Miracloth and sugar synthesis (1). On the other hand, plant enzymes that desalted by ultrafiltration on Centricon YM-10 (Amicon, Bed- irreversibly cleave nucleotide sugars have been described that ford, MA). For purification of AGPPase, 600 ml of homogenate may effectively interrupt the flow of glycosyl moieties toward the obtained from 200 g of young barley leaves were centrifuged at biosynthesis of end products such as starch, cell wall polysac- 100,000 ϫ g for 30 min, and the supernatant was adjusted to 50% charides, or sucrose (2). It is conceivable that in conjunction with (NH4)2SO4. The precipitate obtained after 30 min of centrifu- ADPG pyrophosphorylase (AGPase; EC 2.7.7.27), UDPG py- gation at 30,000 ϫ g (at 20°C) was resuspended in 520 ml of 50 rophosphorylase, sucrose synthase, and starch synthase, among mM Mes, pH 4.2͞1 mM EDTA͞2 mM DTT, heated in a water others, these enzymes will participate in controlling the levels of bath at 62°C for 20 min, cooled on ice, and centrifuged at nucleotide sugars engaged in starch formation. Among a few 30,000 ϫ g for 20 min. Proteins from the supernatant (520 ml) enzymes reported to date that can hydrolyze nucleotide sugars were precipitated with 50% (NH4)2SO4 and resuspended in 5.7 in plants, ADPG phosphorylase is shown to catalyze the phos- ml of extraction buffer. The sample was then subjected to gel phorolytic breakdown of ADPG (3). UDPG phosphorylase is filtration on a Superdex 200 column (Pharmacia LKB) preequili- ͞ known to split UDPG in the presence of Pi, and its activities are brated with 50 mM Mes, pH 6.0 150 mM NaCl. The elution was greatly stimulated by some signal metabolites, indicating that it carried out with the same buffer at a flow rate of 0.3 ml͞min, and may play an important role in the control of photosynthate partitioning (4). ⌬ Ј Based on experimental grounds, Pozueta-Romero et al. (5) Abbreviations: G , standard-state free-energy change; ADPG, ADPglucose; AGPase, ADPG ͞ pyrophosphorylase; AGPPase, ADPG pyrophosphatase; G1P, glucose-1-phosphate; 3-PGA, have proposed the operation of synthesis breakdown metabolic 3-phosphoglycerate; PNPP, p-nitrophenyl phosphate; UDPG, UDPglucose. cycles controlling the rate of starch formation. According to this *To whom reprint requests should addressed. E-mail: javier.pozueta@ unavarra.es. hypothesis, the balance between enzymatic activities catalyzing †AGPPase also can be referred to as adenosine diphosphate glucose phosphodiesterase. the synthesis of gluconeogenic intermediates and those activities The publication costs of this article were defrayed in part by page charge payment. This catalyzing their breakdown can determine the net rate of starch article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. synthesis. Because of the possibility that activities hydrolyzing §1734 solely to indicate this fact. ADPG, the universal starch precursor, may exist in plants that Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.120168097. regulate the metabolism of this polyglucan, we have explored Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.120168097 PNAS ͉ July 18, 2000 ͉ vol. 97 ͉ no. 15 ͉ 8705–8710 Downloaded by guest on October 1, 2021 150-␮l fractions were collected. The purified enzyme prepara- Table 1. ADPG hydrolyzing enzyme activities in higher plants tion was subjected to electrophoresis in nondenaturating 7% Specific activity, milliunits͞ acrylamide gels at pH 4.3, which were prepared as described by mg of protein Reisfeld et al. (8). The native molecular mass of AGPPase was determined from ϩADPGϩ Plant tissues ϩADPG ϩADPGϩ PP PP ϩ3-PGA a plot of Kav (partition coefficient) versus log molecular mass of i i protein standards. Protein content was measured by the Brad- Monocotyledonous ford method using the Bio-Rad prepared reagent. Barley leaf 113.7 Ϯ 3.5 72.2 Ϯ 0.7 103.8 Ϯ 5.0 Barley leaf endosperm, 2.0 Ϯ 0.2 15.9 Ϯ 1.2 21.8 Ϯ 0.3 Enzyme Assays. Unless otherwise indicated, all enzymatic reac- 30 days after pollination tions were performed at 37°C. Measurements of AGPase and Wheat leaf 22.4 Ϯ 2.5 15.1 Ϯ 0.9 50.8 Ϯ 0.6 AGPPase activities were performed by using the two-step spec- Wheat leaf endosperm, 1.7 Ϯ 0.2 48.3 Ϯ 2.6 56.4 Ϯ 1.4 trophotometric determination of glucose-1-phosphate (G1P) 30 days after pollination described by Sowokinos (9). In step one, the reaction mixture for Dicotyledonous AGPPase contained 50 mM Mes (pH 6.0), the specified amount A. thaliana leaf, Wt 5.2 Ϯ 0.6 5.1 Ϯ 0.8 42.1 Ϯ 3.3 ␮ of ADPG and protein extract, in a total volume of 50 l. For A. thaliana leaf, Adg 5.9 Ϯ 1.7 6.5 Ϯ 1.3 7.2 Ϯ 0.2 AGPase assays, the reaction mixture contained 50 mM Hepes Pepper leaf 5.0 Ϯ 0.6 7.3 Ϯ 1.0 24.8 Ϯ 0.8 (pH 7.5), 5 mM MgCl2,3mMPPi, and 10 mM 3-phosphoglyc- Tomato leaf 5.6 Ϯ 0.5 16.1 Ϯ 0.1 82.4 Ϯ 6.9 erate (3-PGA). All assays were run with minus ADPG blanks. Sycamore cultured cells 16.5 Ϯ 7.2 30.6 Ϯ 5.4 137.4 Ϯ 39.8 After 20 min of incubation, the reaction was stopped by boiling Ϯ in a dry bath for 2 min. In step two, G1P was determined Data are presented as the mean SD obtained from five independent spectrophotometrically in a 300-␮l mixture containing 50 mM experiments. Details of enzyme assay methods to measure G1P production are described in Material and Methods. Hepes (pH 7.0), 1 mM EDTA, 2 mM MgCl2, 15 mM KCl, 0.6 mM NADϩ, 1 unit each of phosphoglucomutase and glucose-6- phosphate dehydrogenase from Leuconostoc mesenteroides, and dissociation constants (K ) as well as the pattern of inhibition ␮ i 30 l of the step-one reaction. After 20 min of incubation, the were determined from Dixon plots and Lineweaver–Burk plots. NADH production was monitored at 340 nm by using a Multi- skan EX spectrophotometer (Labsystems, Chicago).