scholarly journals ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope

1997 ◽  
Vol 324 (2) ◽  
pp. 503-509 ◽  
Author(s):  
Torsten MÖHLMANN ◽  
Joachim TJADEN ◽  
Gundrun HENRICHS ◽  
Paul W. QUICK ◽  
Rainer HÄUSLER ◽  
...  
Keyword(s):  
Starch Synthesis ◽  
Dihydroxyacetone Phosphate ◽  
Maize Endosperm ◽  
Endosperm Tissue ◽  
Plastid Envelope ◽  
Phosphorylated Compounds ◽  
Saturation Kinetics ◽  
Uptake Data ◽  
Hexose Phosphates

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395–401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2 mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.

scholarly journals Functional Characterization of fst1 in Fusarium verticillioides During Colonization of Maize Kernels

2011 ◽  
Vol 24 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Hun Kim ◽  
Charles P. Woloshuk
Keyword(s):  
Fusarium Verticillioides ◽  
Functional Characterization ◽  
Hexose Transporter ◽  
Maize Endosperm ◽  
Endosperm Tissue ◽  
Genes Expression ◽  
Fluorescent Tag ◽  
Maize Kernels ◽  
Delayed Growth

The putative hexose transporter gene fst1 in Fusarium verticillioides was identified previously by microarray analysis as a gene that was more highly expressed during colonization of autoclaved maize endosperm than germ. In contrast to a previous study, in which disruption of fst1 did not affect growth of the pathogen on autoclaved maize kernels, in the current study, we demonstrated that disruption of fst1 delayed growth and symptom development on wounded maize ears. Characterization of the fst1 promoter revealed that regulation of fst1 expression was similar to that of fumonisin biosynthetic (fum) genes; expression was highest during growth on endosperm tissue and repressed by elevated concentrations of ammonium in the growth medium. With a fluorescent tag attached to FST1, the protein localized transiently to the periphery of the cells near the plasma membrane and in vacuole-like structures, suggesting that membrane-localized FST1 was internalized and degraded in vacuoles. Expression of fst1 in a yeast strain lacking hexose transporter genes did not complement the yeast mutation, suggesting that FST1 does not transport glucose, fructose, or mannose. The results indicate a functional role for FST1 in pathogenesis during the colonization of living kernels.

scholarly journals Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis

1993 ◽  
Vol 296 (2) ◽  
pp. 395-401 ◽  
Author(s):  
H E Neuhaus ◽  
O Batz ◽  
E Thom ◽  
R Scheibe
Keyword(s):  
Starch Synthesis ◽  
Dihydroxyacetone Phosphate ◽  
Low Degree ◽  
Fructose 1,6 Bisphosphatase ◽  
Percoll Gradients ◽  
Phosphoglyceric Acid ◽  
Barley Leaves ◽  
Centrifugation Step ◽  
Cellular Components ◽  
Hexose Phosphates

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.

Regioselective Functionalization of Starch:  Synthesis and1H NMR Characterization of 6-O-Silyl Ethers‖

2001 ◽  
Vol 2 (3) ◽  
pp. 965-969 ◽  
Author(s):  
Katrin Petzold ◽  
Lars Einfeldt ◽  
Wolfgang Günther ◽  
Armin Stein ◽  
Dieter Klemm
Keyword(s):  
Starch Synthesis ◽  
Silyl Ethers ◽  
Nmr Characterization ◽  
Regioselective Functionalization

scholarly journals Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.

1997 ◽  
Vol 11 (1) ◽  
pp. 73-82 ◽  
Author(s):  
H.E. Neuhaus ◽  
E. Thom ◽  
T. Mohlmann ◽  
M. Steup ◽  
K. Kampfenkel
Keyword(s):  
Arabidopsis Thaliana ◽  
Plastid Envelope

scholarly journals Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

1996 ◽  
Vol 319 (3) ◽  
pp. 717-723 ◽  
Author(s):  
Ian J TETLOW ◽  
Caroline G BOWSHER ◽  
Michael J EMES
Keyword(s):  
Inorganic Phosphate ◽  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Carrier Protein ◽  
Sugar Phosphate ◽  
Wheat Endosperm ◽  
Single Carrier ◽  
Phosphate Translocator ◽  
Envelope Membranes ◽  
Hexose Phosphates

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent Km for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.

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