Plastidial localization of a potato 'Nudix' hydrolase of ADP-glucose linked to starch biosynthesis

Escherichia coli and potato (Solanum tuberosum) ADP-sugar pyrophosphatases (EcASPP and StASPP, respectively) are 'Nudix' hydrolases of the bacterial glycogen and starch precursor molecule, ADP-glucose (ADPG). We have previously shown that potato leaves expressing EcASPP either in the cytosol or in the chloroplast exhibited large reductions in the levels of starch, suggesting the occurrence of cytosolic and plastidial pools of ADPG linked to starch biosynthesis. In this work, we produced and characterized potato and Arabidopsis plants expressing EcASPP and StASPP fused with green fluorescent protein (GFP). Confocal fluorescence microscopy analyses of these plants confirmed that EcASPP-GFP has a cytosolic localization, whereas StASPP-GFP occurs in the plastid stroma. Both source leaves and potato tubers from EcASPP-GFP-expressing plants showed a large reduction of the levels of both ADPG and starch. In contrast, StASPP-GFP-expressing leaves and tubers exhibited reduced starch and normal ADPG contents when compared with control plants. With the exception of starch synthase in StASPP-GFP-expressing plants, no pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in EcASPP-GFP- and StASPP-GFP-expressing plants. The overall data (i) show that potato plants possess a plastidial ASPP that has access to ADPG linked to starch biosynthesis and (ii) are consistent with the occurrence of plastidic and cytosolic pools of ADPG linked to starch biosynthesis.     

Post-translational redox modification of ADP-glucose pyrophosphorylase in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis leaves

ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme comprising two small and two large subunits that catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism assumes that post-translational redox modification of AGP in response to light is a major determinant of fine regulation of transitory starch accumulation. According to this view, under oxidizing conditions occurring during the night the two AGP small subunits (APS1) are covalently linked via an intermolecular disulfide bridge that inactivates the protein, whereas under reducing conditions occurring during the day NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this work we have analyzed changes in the redox status of APS1 during dark-light transition in leaves of plants cultured under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli redox-insensitive AGP (GlgC) in the chloroplast. We also characterized aps1 plants expressing a redox-insensitive, mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when plants were cultured under photo-oxidative conditions. We also found that starch accumulation rates during the light in leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore, the pattern of starch accumulation during illumination in leaves of APS1mut-expressing aps1 mutants was similar to that of APS1-expressing aps1 mutants at any light intensity. The overall data demonstrate that post-translational redox modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.     

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.     

A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin.     

Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves

Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.     

Cloning, expression and characterization of a Nudix hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis thaliana

'Nudix' hydrolases are widely distributed nucleotide pyrophosphatases that possess a conserved GX5EX7REUXEEXGU motif where U is usually isoleucine, leucine or valine. Among them, Escherichia coli ADP-sugar pyrophosphatase (ASPP) has been shown to catalyze the hydrolytic breakdown of ADP-glucose linked to bacterial glycogen biosynthesis. Comparisons of the 31 different Nudix-encoding sequences of the Arabidopsis genome with those coding for known bacterial and mammalian ASPPs identified one sequence possessing important divergences in the Nudix motif that, once expressed in E. coli, produced a protein with ASPP activity. This protein, designated as AtASPP, shares strong homology with hypothetical rice and potato proteins, indicating that ASPPs are widely distributed in both mono- and dicotyledonous plants. As a first step to test the possible involvement of plant ASPPs in regulating the intracellular levels of ADP-glucose linked to starch biosynthesis, we produced and characterized AtASPP-overexpressing Arabidopsis plants. Source leaves from these plants exhibited a large reduction in the levels of both ADP-glucose and starch, indicating that plant ASPPs catalyze the hydrolytic breakdown of a sizable pool of ADP-glucose linked to starch biosynthesis. No pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in AtASPP-overexpressing leaves. The overall information provides the first evidence for the existence of plant Nudix hydrolases that have access to an intracellular pool of ADP-glucose linked to starch biosynthesis.     

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.     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

Expression of a wheat ADP-glucose pyrophosphorylase gene during development of normal and water-stress-affected anthers

In wheat (Triticum aestivum L.), water deficit during meiosis in the microspore mother cells (MMCs) induces pollen abortion, resulting in the failure of fertilization and a reduction in grain set. In stressed plants, meiosis in MMCs proceeds normally but subsequent pollen development is arrested. Unlike normal pollen grains, which accumulate starch during the late maturation phase, stress-affected anthers contain pollen grains with little or no starch. Stress also alters the normal distribution of starch in the anther wall and connective tissue. To determine how starch biosynthesis is regulated within the developing anthers of stressed plants, we studied the expression of ADP-glucose pyrophosphorylase (AGP), which catalyzes the rate limiting step of starch biosynthesis. Two partial-length cDNAs corresponding to the large subunit of AGP were amplified by RT-PCR from anther RNA, and used as probes to monitor AGP expression in developing anthers of normal and water-stressed plants. These clones, WAL1 and WAL2, had identical deduced amino acid sequences and shared 96% sequence identity at the nucleic acid level. In normal anthers, AGP expression was biphasic, indicating that AGP expression is required for starch biosynthesis both during meiosis and later during pollen maturation. AGP expression in stressed anthers was not affected during the first phase of starch accumulation, but was strongly inhibited during the second phase. We conclude from these results that the reduced starch deposition later in the development of stressed pollen could be the result of a lower expression of AGP. However, this inhibition of AGP expression is unlikely to be the primary cause of male sterility because anatomical symptoms of pollen abortion are observed prior to the time when AGP expression is inhibited.     

Trehalose induces the ADP-glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis

In Arabidopsis, genes encoding functional enzymes for the synthesis and degradation of trehalose have been detected recently. In this study we analyzed how trehalose affects the metabolism and development of Arabidopsis seedlings. Exogenously applied trehalose (25 mM) strongly reduced the elongation of the roots and, concomitantly, induced a strong accumulation of starch in the shoots, whereas the contents of soluble sugars were not increased. When Arabidopsis seedlings were grown on trehalose plus sucrose (Suc), root elongation was restored, but starch still accumulated to a much larger extent than during growth on Suc alone. The accumulation of starch in the shoots of trehalose-treated seedlings was accompanied by an increased activity of ADP-glucose pyrophosphorylase and an induction of the expression of the ADP-glucose pyrophosphorylase gene, ApL3. Even in the presence of 50 mM Suc, which itself also slightly induced ApL3, trehalose (5 mM) led to a further increase in ApL3 expression. These results suggest that trehalose interferes with carbon allocation to the sink tissues by inducing starch synthesis in the source tissues. Furthermore, trehalose induced the expression of the beta-amylase gene, AT-beta-Amy, in combination with Suc but not when trehalose was supplied alone, indicating that trehalose can modulate sugar-mediated gene expression.     

Investigation of subunit function in ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme. The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.     

Determining the role of Tie-dyed1 in starch metabolism: epistasis analysis with a maize ADP-glucose pyrophosphorylase mutant lacking leaf starch

In regions of their leaves, tdy1-R mutants hyperaccumulate starch. We propose 2 alternative hypotheses to account for the data, that Tdy1 functions in starch catabolism or that Tdy1 promotes sucrose export from leaves. To determine whether Tdy1 might function in starch breakdown, we exposed plants to extended darkness. We found that the tdy1-R mutant leaves retain large amounts of starch on prolonged dark treatment, consistent with a defect in starch catabolism. To further test this hypothesis, we identified a mutant allele of the leaf expressed small subunit of ADP-glucose pyrophosphorylase (agps-m1), an enzyme required for starch synthesis. We determined that the agps-m1 mutant allele is a molecular null and that plants homozygous for the mutation lack transitory leaf starch. Epistasis analysis of tdy1-R; agps-m1 double mutants demonstrates that Tdy1 function is independent of starch metabolism. These data suggest that Tdy1 may function in sucrose export from leaves.     

Crystal structure of potato tuber ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.     

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.     

Expression of a modified ADP-glucose pyrophosphorylase large subunit in wheat seeds stimulates photosynthesis and carbon metabolism

ADP-glucose pyrophosphorylase (AGP) is the rate-limiting step in seed starch biosynthesis. Expression of an altered maize AGP large subunit (Sh2r6hs) in wheat (Triticum aestivum L.) results in increased AGP activity in developing seed endosperm and seed yield. The yield phenotype involves increases in both seed number and total plant biomass. Here we describe stimulation of photosynthesis by the seed-specific Sh2r6hs transgene. Photosynthetic rates were increased in Sh2r6hs-expressing plants under high light but not low light growth conditions, peaking at roughly 7 days after flowering (DAF). In addition, there were significant increases in levels of fructose, glucose, and sucrose in flag leaves at both 7 and 14 DAF. In seeds, levels of carbon metabolites at 7 and 14 DAF were relatively unchanged but increases in glucose, ADP-glucose, and UDP-glucose were observed in seeds from Sh2r6hs positive plants at maturity. Increased photosynthetic rates relatively early in seed development appear to be key to the Sh2r6hs enhanced yield phenotype as no yield increase or photosynthetic rate changes were found when plants were grown in a suboptimal light environment. These findings demonstrate that stimulation of biochemical events in both source and sink tissues is associated with Sh2r6hs expression.     

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K _m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed. Received: 2 February 1999 / Accepted: 25 March 1999     

Induction of ApL3 expression by trehalose complements the starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the large subunit of ADP-glucose pyrophosphorylase

The disaccharide trehalose has strong effects on plant metabolism and development. In Arabidopsis seedlings, growth on trehalose-containing medium leads to an inhibition of root elongation, an accumulation of starch in the shoots, an increased activity of ADP-Glc pyrophosphorylase (AGPase), and an induction of the expression of the AGPase gene, ApL3 (A. Wingler, T. Fritzius, A. Wiemken, T. Boller, R.A. Aeschbacher [2000] Plant Physiol 124: 105-114). We used Arabidopsis mutants deficient in starch synthesis to examine whether the primary effect of trehalose was to affect carbohydrate allocation by the induction of AGPase in the photosynthetic tissue. In a mutant lacking the large AGPase subunit, ApL1, (adg2-1 mutant) growth on trehalose restored AGPase activity and led to a strong accumulation of starch in the shoots. In contrast, starch synthesis could not be induced in a mutant lacking the small AGPase subunit, ApS, (adg1-1 mutant) or in a mutant lacking plastidic phosphoglucomutase (pgm1-1 mutant). These results indicate that ApL3 can substitute for ApL1 in the AGPase complex. In addition, root elongation in the mutants, especially in the adg1-1 mutant, was partially resistant to trehalose, suggesting that the induction of ApL3 expression and the resulting accumulation of starch in the shoots were partially responsible for the effects of trehalose on the growth of wild-type plants.     

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.