Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage. Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGIc) as a substrate; ADPGIc pyrophosphorylase, the enzyme involved in ADPGIc formation, is regulated by allosteric effectors.
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In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield. Most if not all glucose for starch synthesis comes from the activated sugar, ADP-glucose. This sugar nucleotide is synthesized from glucose-I-phosphate and ATP by the enzyme ADP-glucose pyrophosphorylase (AGP). Recent observations from potato and maize point to AGP as a key limiting step in starch synthesis.
Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed.
- Precipitation with 18% PEG was carried out to concentrate and stabilise the activity.
- Transposon mutagenesis was used to create isogenic lines containing or lacking a functional structural gene for this enzyme.
- Also, the IbAGPaseS gene appears to be a member of multiple gene families and further, the differences in the copy numbers among the ten cultivars were considered to lead to differences in starch contents.
Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. During this same period UDP-glucose pyrophosphorylase increased approximately 2to 3-fold. This review outlines research performed in the last two decades on the structural, kinetic,regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzymefor starch biosynthesis.
Evolutionary, structural and expression analysis of core genes involved in starch synthesis
ADP-glucose pyrophosphorylase from walnut wood (J. regia L.) was purified 50 fold to a specific activity of 2.6 µmoles glucose-1-P produced per minute per milligram protein. Wood from young plants was ground in liquid N 2 after accurate removal of all the cortical parenchyma. The specific activity in the starting material was very low and the enzyme was quite unstable. Precipitation with 18% PEG was carried out to concentrate and stabilise the activity. Further purification was attained with DEAE Sepharose and MonoQ ion exchange chromatography.
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Transposon mutagenesis was used to create isogenic lines containing or lacking a functional structural gene for this enzyme. These lines were used to create otherwise genetically identical regulation of the amount of starch in plant tissues by adp glucose pyrophosphorylase seed differing only in amounts of AGP activity. Seed weight increased when the number of functional alleles increased from one to three doses although no difference was found between two and three doses. These data point to the physiological importance of the allosteric properties of AGP, although the data show that these properties cannot compensate for a 67 % reduction in total AGP activity.
Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues.
Regulation of starch biosynthesis in plant leaves: activation and inhibition of ADPglucose pyrophosphorylase
- 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.
- L’enzyme partiellement purifiée s’est montrée très sensible à l’activation allostérique par le 3-phosphoglycerate (3PGA) et à l’inhibition par Pi, même lorsqu’elle a été évaluée dans le sens pyrophosphorolitique.
- AGP constitutes the first committed and highly regulated step of starch synthesis in all plant tissues.
- The key regulatory step in starch biosynthesis is catalyzed by the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase).
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. Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C.
The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65° for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate.
Using heterologous probes from maize, two sets (B and S) of cDNA clones encoding potato AGPase were isolated from a tuberspecific cDNA library. 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.
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Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A0.5 (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 × 10−6 m while for the sorghum enzyme it was 3.7 × 10−4 m. The I0.5 (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 × 10−5 m (barley) to 1.9 × 10−4 m (sorghum). These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis. In all cases the enzymes showed stability to heat treatment at 65 degrees for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The A(0.5) (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 x 10(-6)m while for the sorghum enzyme it was 3.7 x 10(-4)m.
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.
The I(0.5) (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 x 10(-5)m (barley) to 1.9 x 10(-4)m (sorghum). In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of α-1,4-glucosidic chains. 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.
The partially purified enzyme was very sensitive to allosteric activation and inhibition by 3-phosphoglycerate (3PGA) and Pi respectively, even if assessed in the pyrophosphorolytic direction. ADP-glucose pyrophosphorylase from walnut wood, as those from many other higher plant tissues, may be strongly regulated by allosteric control, this could be one of the control sites of in vivo starch deposition in wood. Résumé De l’ADP-glucose pyrophosphorylase extrait de bois de noyer (J. regia L.) a été purifiée 50 fois jusqu’à une activité spécifique de 2,6 µmoles glucose-1-P produit par minute et par milligramme de protéine. Le bois des jeunes plants était plongé dans N 2 liquide après élimination totale du parenchyme cortical. Une précipitation avec 18 % de PEG fut réalisée pour concentrer et stabiliser cette activité.
Une purification ultérieure fut faite par chromographie d’échange d’ions (DEAE Sepharose et MonoQ). L’enzyme partiellement purifiée s’est montrée très sensible à l’activation allostérique par le 3-phosphoglycerate (3PGA) et à l’inhibition par Pi, même lorsqu’elle a été évaluée dans le sens pyrophosphorolitique. L’ADP-glucose pyrophosphorylase du bois de noyer comme celle de différents tissus de plantes supérieures peut être fortement régulée de manière allostérique. Cette régulation ts de contrôle de l’accumulation in vitro de l’amidon dans le bois. It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced.
Recent advances in studies on plant ADP-glucose pyrophosphorylase (AGP), the key enzyme of starch biosynthesis, are presented. AGP constitutes the first committed and highly regulated step of starch synthesis in all plant tissues. The open reading frame of IbAGPaseS gene is comprised of 1539-base pairs and encodes a polypeptide of 512 amino acid residues.
Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. 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-1-phosphate and ATP.
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