Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase

ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.     

Both subunits of ADP-glucose pyrophosphorylase are regulatory

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.     

ADP-glucose pyrophosphorylase from wheat endosperm. Purification and characterization of an enzyme with novel regulatory properties

ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) was purified and characterized from two wheat (Triticum aestivum L.) tissues: leaf and endosperm. The leaf enzyme, purified over 1,300-fold, was found to be a heterotetramer composed of subunits of 51 and 54 kDa and possessing regulatory properties typical of AGPases from photosynthetic tissues, being mainly regulated by 3-phosphoglycerate (activator; A0.5=0.01 mM) and orthophosphate (inhibitor; I0.5=0.2 mM). Conversely, the enzyme from wheat endosperm was insensitive to activation by 3-phosphoglycerate and other metabolites. It was, however, inhibited by orthophosphate (I0.5=0.7 mM), ADP (I0.5=3.2 mM) and fructose-1,6-bisphosphate (0.5 = 1.5 mM). All of these inhibitory actions were reversed by 3-phosphoglycerate and fructose-6-phosphate. The endosperm enzyme was found to be a heterotetramer composed of subunits of 52 and 53 kDa, which were recognized by antiserum raised to spinach leaf AGPase. The results suggest that wheat endosperm AGPase possesses distinctive regulatory properties that are relevant in vivo.     

ADP-glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis.

The accumulation of alpha-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the alpha-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation.     

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.     

Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase

In higher plants, ADP-glucose pyrophosphorylase (ADPGlc-PPase) is a heterotetrameric enzyme comprised of two small and two large subunits. Potato-Arabidopsis hybrid ADPGlc-PPases were generated and their regulatory properties analyzed. We show that ADPGlc-PPase subunits from two different species can interact, producing active enzymes with new regulatory properties. Depending on the subunit combinations, hybrid heterotetramers showed responses to allosteric effectors [3-phosphoglycerate (3-PGA) and Pi] in the micromolar or millimolar range. While hybrid potato small subunit (PSS) and the Arabidopsis large subunit APL1 showed an extremely sensitive response to 3-PGA and Pi, hybrid PSS/Arabidopsis APL2 was very insensitive to them. Intermediate responses were determined for other subunit combinations.     

Characterization of an autonomously activated plant ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1–198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1–198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1–198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1–198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1–198)/SH2.     

Isolation,Purification, and Characterization of Mitochondria from Chlamydomonas reinhardtii

Mitochondria were isolated from autotrophically grown Chlamydomonas reinhardtii cell-wall-less mutant CW 92. The cells were broken by vortexing with glass beads, and the mitochondria were collected by differential centrifugation and purified on a Percoll gradient. The isolated mitochondria oxidized malate, pyruvate, succinate, NADH, and [alpha]-ketoglutarate. Respiratory control was obtained with malate (2.0) and pyruvate (2.2) but not with the other substrates. From experiments with KCN and salicylhydroxamic acid, it was estimated that the capacity of the cytochrome pathway was at least 100 nmol O2 mg-1 protein min-1 and the capacity of the alternative oxidase was at least 50 nmol O2 mg-1 protein min-1. A low sensitivity to oligomycin indicates some difference in the properties of the mitochondrial ATPase from Chlamydomonas as compared to higher plants.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit.

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.     

Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize

Electrophoretic characterization of adenosine diphosphate glucose pyrophosphorylase from the developing endosperms of nine shrunken-2 and four brittle-2 mutants revealed that (1) all mutants had low but detectable levels of activity, (2) mutation at either locus decreased activity of pyrophosphorylases A and B, and (3) differences in mobility were not found. However, pyrophosphorylase B extracted from several shrunken-2 and brittle-2 mutants differed from normal in extent of urea denaturation, K _m (glucose-1-phosphate) or type of glucose-1-phosphate saturation kinetics. Pyrophosphorylase B from sh2-m (association of Dissociation with the sh2 locus) appears to differ from normal in K _m (glucose-1-phosphate).This research was supported by the College of Agricultural and Life Sciences and by National Institutes of Health Grant No. 15422.The investigations reported were included in the thesis submitted by L. C. Hannah to the Graduate School, University of Wisconsin, Madison, Wisconsin, in partial fulfillment of requirement for the Ph.D. degree. Laboratory of Genetics Paper No. 1922.     

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[¹⁴C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.     

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.     

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-113, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.     

Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.

We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii. The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms. The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6. The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM. The subunit Mr of Chlamydomonas ASL is approx. 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000. Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro. In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity. Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.     

Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources

Summary The primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.Offprint requests to: J. Preiss     

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn²⁺ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (K_i, 0.14 mm) and NADPH was a noncompetitive inhibitor (K_i, 0.42 mm) with respect to NAD⁺. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.IDH catalyzes the oxidative decarboxylation of isocitrate to produce 2-oxoglutarate. According to the specificity for the electron acceptor, two enzymes with IDH activity are known, NAD-IDH (EC 1.1.1.41) and NADP-IDH (EC 1.1.1.42) (Chen and Gadal, 1990a).In photosynthetic organisms NADP-IDH has been detected in the cytosol, chloroplasts, mitochondria, and peroxisomes. Cytosolic NADP-IDH has been purified from higher plants (Chen et al., 1988) and eukaryotic algae (Martínez-Rivas et al., 1996), and its cDNA has been cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et al., 1993), potato (Fieuw et al., 1995), and tobacco (Gálvez et al., 1996). This 80-kD isoenzyme is a dimer, and it is likely to be involved in the synthesis of NADPH for biosynthetic purposes in the cytosol (Chen et al., 1988), in the synthesis of 2-oxoglutarate for ammonium assimilation (Chen and Gadal, 1990b), and in the cycling, redistribution, and export of amino acids (Fieuw et al., 1995). Chloroplastic NADP-IDH has been studied in higher plants (Gálvez et al., 1994) and eukaryotic algae (Martínez-Rivas and Vega, 1994). It is a 154-kD dimer that has been proposed to be involved in the supply of NADPH for biosynthetic reactions in the chloroplast when photosynthetic NADPH production is low (Gálvez et al., 1994). The mitochondrial NADP-IDH of higher plants may have a physiological role in the production of NADPH, which can be converted to NADH by a transhydrogenase or used to reduce glutathione in the mitochondrial matrix (Rasmusson and Møller, 1990). NADP-IDH activity has also been detected in peroxisomes from spinach leaves (Yamazaki and Tolbert, 1970).NAD-IDH is localized exclusively in the mitochondria in association with the TCA cycle. This enzyme has been purified from several nonphotosynthetic eukaryotes such as fungi (Keys and McAlister-Henn, 1990; Alvarez-Villafañe et al., 1996) and animals (Giorgio et al., 1970), in which it appears to be a 300-kD octamer. Its key regulatory role in the TCA cycle is well documented. The NAD-IDH from yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963), whereas the animal enzyme is activated by ADP and citrate (Cohen and Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from yeast (Cupp and McAlister-Henn, 1991, 1992) and animals (Nichols et al., 1995; Zeng et al., 1995). In these organisms, the enzyme is composed of two (yeast) or more (animals) different subunits encoded by different genes.To our knowledge, no NAD-IDH from photosynthetic organisms has yet been purified to homogeneity, mainly because of the low stability of the enzyme (Oliver and McIntosh, 1995). However, partial purifications have been reported from pea (Cox and Davies, 1967; Cox, 1969; McIntosh and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al., 1996), and the eukaryotic microalga Chlamydomonas reinhardtii (Martínez-Rivas and Vega, 1994). Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967; McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et al., 1990), the regulatory importance of NAD-IDH in photosynthetic organisms is still under debate.To elucidate the regulatory significance of NAD-IDH in photosynthetic organisms and its apparent contribution to the 2-oxoglutarate supply for ammonium assimilation, we have purified and characterized the NAD-IDH from C. reinhardtii.     

Arabidopsis thaliana mutants lacking ADP-glucose pyrophosphorylase accumulate starch and wild-type ADP-glucose content: further evidence for the occurrence of important sources, other than ADP-glucose pyrophosphorylase, of ADP-glucose linked to leaf starch biosynthesis

It is widely considered that ADP-glucose pyrophosphorylase (AGP) is the sole source of ADP-glucose linked to bacterial glycogen and plant starch biosynthesis. Genetic evidence that bacterial glycogen biosynthesis occurs solely by the AGP pathway has been obtained with glgC? AGP mutants. However, recent studies have shown that (i) these mutants can accumulate high levels of ADP-glucose and glycogen, and (ii) there are sources other than GlgC, of ADP-glucose linked to glycogen biosynthesis. In Arabidopsis, evidence showing that starch biosynthesis occurs solely by the AGP pathway has been obtained with the starchless adg1-1 and aps1 AGP mutants. However, mounting evidence has been compiled previewing the occurrence of more than one important ADP-glucose source in plants. In attempting to solve this 20-year-old controversy, in this work we carried out a judicious characterization of both adg1-1 and aps1. Both mutants accumulated wild-type (WT) ADP-glucose and approximately 2% of WT starch, as further confirmed by confocal fluorescence microscopic observation of iodine-stained leaves and of leaves expressing granule-bound starch synthase fused with GFP. Introduction of the sex1 mutation affecting starch breakdown into adg1-1 and aps1 increased the starch content to 8-10% of the WT starch. Furthermore, aps1 leaves exposed to microbial volatiles for 10 h accumulated approximately 60% of the WT starch. aps1 plants expressing the bacterial ADP-glucose hydrolase EcASPP in the plastid accumulated normal ADP-glucose and reduced starch when compared with aps1 plants, whereas aps1 plants expressing EcASPP in the cytosol showed reduced ADP-glucose and starch. Moreover, aps1 plants expressing bacterial AGP in the plastid accumulated WT starch and ADP-glucose. The overall data show that (i) there occur important source(s), other than AGP, of ADP-glucose linked to starch biosynthesis, and (ii) AGP is a major determinant of starch accumulation but not of intracellular ADP-glucose content in Arabidopsis.