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.

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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