Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes a-amylase, ,6-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sexl at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.
ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803.The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (Ao.s = 0.81 millimolar; nH = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (Ao.5 = 0.12 millimolar, n,H = 1.0), having a maximal stimulation of 17-fold. 10.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (Ao.5 = 10 micromolar; n,H = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia co/i) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate-and two-dimensional polyacrylamide gel electrophoresis and probed with Westem blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.
Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbibition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose--phosphate dehydrogenase) or and that, with removal of the testa from seeds, the "leakage phenomenon" is enhanced (14,32). The amount of leakage during imbibition has been shown to correlate negatively with viability in studies with seeds of soybean (6, 39), pea (14,24), bean (17), and peanut (1) and has suggested to some that the leaked substances may, in some way, decrease viability. Another study has shown that removal of the testa of pea seeds results in death of the outer layers of cotyledonary cells during imbibition (25). The question arises: does the testa protect against leakage or is the leakage only a symptom of a fundamental dysfunction which can occur in imbibition?Two hypotheses have been promoted to explain the mechanism(s) ofleakage of solutes during the imbibition ofseeds. Larson (14) has suggested that cell membranes are ruptured during the initial phases of imbibition. Simon (31, 33) has proposed that the membranes of dry seeds are formed into hexagonal plates with pores formed in the areas of the phospholipid heads through which low-molecular weight solutes can leak from cells by passive diffusion during initial stages of membrane hydration (e.g. before phospholipids form typical bilayer membranes). Recently Powell and Matthews (25) have suggested that, in peas, cellular rupture and leakage through membranes may both occur when the testae are removed from seeds. To date, there has been a paucity of data that any macromolecules could move through the cell membrane during imbibition. Here, we have examined the leakage of imbibing seeds with and without testae for the presence of cytoplasmic, organelle, and organelle marker enzymes which would not pass through small membrane pores but which could only pass through very large membrane discontinuities or which would be the result of membrane rupture. In this way, we have tested both of the aforementioned hypotheses in a more definitive manner than has been hitherto reported.In the development of the legume seed, the testa appears to function in interconverting amino acids and sugars supplied by the phloem to the developing embryo (19,35,36) and in preventing injury by differentiating into a sclerified integument as the embryo matures (27). It has also been proposed that the testa protects seeds against "leakage" of intracellular substances during imbibition (32). This function has been suggested to be of great importance in the initial stages of germination of legume seeds in that many substances which leak from seeds may offer a substrate for potential pathogens (32). Past studies have demonstrated that electrolytes, sugars, amino acids, organic acids, and proteins are released from seeds during imbibition (1,6, 14,18,23,29,33,34) Mammoth Virgi...
Regulation of the ADPGlc pathwayThere is evidence in vitro suggesting that ADPGlc synthesis is regulated by activation of the plant ADPGlc synthetase by 3-phosphoglycerate (3PGA) and inhibition by inorganic phosphate (Pi). In vivo or in situ evidence showing a correlation between the concentrations of 3PGA and starch and inverse correlations between Pi and starch levels have been obtained and were reviewed [5, 61. Recently, Pettersson & Ryde-Pettersson [ 81 have applied modern control theory as developed by Kacser & Burns [9, 101 to develop a kinetic model to determine the extent that stromal metabolites, known to affect leaf ADPGlc synthetase activity in vitro, controlled the rate of photosynthetic starch production under conditions of light and CO, saturation. The model consists of the 13 enzyme-catalysed steps of 539 - Properties of ADPGlc synthetase Summary of regulatory and structural propertiesRegulation ly 3-P-glycerate and ly Pi. The properties
Pea (Pisum sativum L.) chloroplast D-enzyme (4-a-D-glucanotransferase, EC 2.4.1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M, of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's Km for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of a-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by Denzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed.Pea chloroplasts contain 4-a-glucanotransferase (transglycosylase) activity equal to that of the total hydrolytic starch degrading activity, and eightfold greater than starch phosphorylase activity ( 12). 4-a-Glucanotransferase alters the degrees of polymerization in a population of maltodextrins by the simultaneous hydrolysis and condensation of a-1,4-linkages between maltodextrins. For example, potato 4-a-glucanotransferase can utilize two maltotrioses (G33) to produce glucose (GI) and maltopentaose (G5) or it can utilize G5 and GI to produce two G3s (1 1
Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.
Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max IL.I Menf.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25°C) of 3.0 and 15.8 kilocalones per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intraceDlular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.ferences one would see in the kinetics of leakage of various seed components. If the assays used for seed leakage do not correlate with one another under most experimental conditions they should not be used interchangeably, as they have in the past, to study the leakage processes.In this study, we utilized imbibing soybean seeds to examine commonly used seed leakage assays to gain insight on the contribution of passive diffusion through membranes and cellular rupture (preexisting or induced) to each measurement. Seeds with various degrees of testa integrity were used because testa integrity is a primary factor in controlling legume seed leakage and cellular damage during germination (7,11,12,21,24). Effects of temperature were studied to gain insight into the energetics of the leakage of various seed components during imbibition and effects of 4,2 were studied because of previous studies which indicate a 4iw effect on soybean seed leakage and seedling vigor (28, 29). Our findings indicate that passive diffusion of low mol wt solutes and membrane rupture-dependent diffusion of macromolecules during seed imbibition are quite different kinetically and that the contribution of each process is variable among the methods used for measuring seed leaching.
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