Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform

The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20-28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8-30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.     

Interaction of cytochrome b5 with surfactant vesicles.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.     

Enzymic properties of amyloplasts form suspension cultures of soybean

The aim of this work was to determine which enzymes of carbohydrate metabolism are present in amyloplasts. Protoplasts from 4- to 5-day-old suspension cultures of soybean, Glycine max, were lysed and fractionated on a sucrose gradient. This gave an amyloplast fraction that contained stromal enzymes and was not seriously contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. Studies of this fraction provide evidence that, in soybean cells, starch synthase and ADPglucose pyrophosphorylase are confined to amyloplasts; invertase, sucrose synthetase and UDPglucose pyrophosphorylase are absent from the amyloplast and probably confined to the cytosol; the following enzymes, though predominantly cytosolic, are present in the amyloplasts in activities high enough to mediate the rate of starch synthesis observed in vivo: glyceraldehyde-phosphate dehydrogenase (NAD), triosephosphate isomerase, fructose-1, 6-bisphosphate aldolase, fructose-bisphosphatase, glucosephosphate isomerase and phosphoglucomutase. The pathway from sucrose to starch in non-photosynthetic cells is discussed; particularly the possibility that sucrose is converted to triose phosphate for entry into the amyloplast.     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Is There an Alternative Pathway for Starch Synthesis?

In leaf tissue, carbon enters starch via the gluconeogenesis pathway where d-glycerate 3-phosphate formed from CO₂ fixation is converted into hexose monophosphates within the chloroplast stroma. In starch-containing sink organs, evidence has been obtained indicating that the flow of carbon into starch follows a different pathway whereby hexose monophosphates formed from sucrose are transported into the amyloplast, a plastid specialized in starch accumulation. In both chloroplasts and amyloplasts, the formation of ADPglucose, the substrate for starch synthase, is controlled by the activity of ADPglucose pyrophosphorylase, a key regulatory enzyme of starch synthesis localized in the plastid. Recently, an alternative pathway of starch synthesis has been proposed in which ADPglucose is synthesized from sucrose and transported directly into the plastid compartment, where it is used for starch synthesis. On the basis of the biochemical phenotypes exhibited by various plant mutants with defined genetic lesions, it is concluded that ADPglucose pyrophosphorylase is essential for starch synthesis, whereas the alternative pathway has only a minor role in this process.     

Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.     

Starch synthetase: Comparison of UDPG and ADPG as glucosyl donors in immature barley endosperm

Summary The activity of starch synthetase in developing barley endosperm was measured in amyloplasts and in the soluble endosperm fraction by incorporation of radioactively labelled glucose into starch. Both uridine diphosphate glucose (UDPG) and adenosine diphophate glucose (ADPG) were used as glucosyl donors. Enzyme activity was initially located in the soluble fraction, but increasing activity could be detected in the amyloplast fraction during endosperm maturation.     

A low-starch barley mutant,risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase,reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Ris? 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Ris? 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial.

Preparations enriched in plastids were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastid marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastids. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extraplastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit.     

Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.     

Btl, a structural gene for the major 39–44 kDa amyloplast membrane polypeptides

The aim of the present work was to investigate the relationship between the Btl gene (Btl) and the major 39–44 kDa amyloplast membrane polypeptides which were deficient in amyloplast membranes of brittlel (btl) kernels of maize (Zea mays L.). A rapid yet gentle procedure for the isolation of amyloplasts from immature kernels is described. These amyloplasts were relatively free of contamination by other cellular components, and immunological studies showed that they contained polypeptides which reacted with antibodies to maize starch branching enzyme and ADP-Gle pyrophosphorylase. Purified membranes isolated from the amyloplast contained a poly-peptide which reacted with antibodies to the P_i-translocator from spinach chloroplasts. However, a cluster of 39–44 kDa polypeptides accounted for about 40% of the total amyloplast membrane protein from W64A kernels. These polypeptides were specifically recognized by antibodies raised against a fusion protein consisting of 56 amino acids of the carboxyl terminus of the BTI protein and glutathione S-transferase. The BT1 antibodies also reacted with the abundant polypeptides in amyloplast membranes from hybrid kernels (Doebler 66XP and Pioneer 3780), and the shrunkenl and shrunken2 mutant genotypes, but no BTl reacting polypeptides were present in amyloplast membranes from btl mutant kernels. We were unable to detect BTl by the immunoblot procedure in microsomal membranes from embryo and pericarp tissues from the kernel, from seedling roots and shoots, or in membranes from mitochondria and chloroplasts. The same BTl immunoblot pattern was obtained for proteins extracted from microsomal membranes from developing endosperm and from purified amyloplast membranes. A linear relationship between the number of copies of Btl alleles and the levels of BTl in endosperm microsomal membranes was demonstrated in a gene dosage series. BTl was not extracted from amyloplast membranes by chloroform/methanol or by alkaline buffer at pH 11.5, but was partially extracted by 0.1 M NaOH. These lines of evidence support the conclusion that Btl is the structural gene for the major 39–44 kDa amyloplast membrane polypeptides and that these polypeptides are integral proteins specific to amyloplast membranes from the endosperm.