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J C Shannon

Publications and source records attributed to J C Shannon.

At least 19 recordsLinked to original sources
PubMed2001

Identification of Mutator insertional mutants of starch-branching enzyme 2a in corn.

Starch-branching enzymes (SBE) break the alpha-1,4 linkage of starch, re-attaching the chain to a glucan chain by an alpha-1,6 bond, altering starch structure. SBEs also facilitate starch accumulation by increasing the number of non-reducing ends on the growing chain. In maize (Zea mays), three isoforms of SBE have been identified. To examine the function of the SBEIIa isoform, a reverse genetics polymerase chain reaction-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe2a. To locate the insertion within the second exon of Sbe2a, the genomic sequence of Sbe2a containing the promoter and 5' end was isolated and sequenced. Plants homozygous for sbe2a::Mu have undetectable levels of Sbe2a transcripts and SBEIIa in their leaves. Characterization of leaf starch from sbe2a::Mu mutants shows reduced branching similar to yet more extreme than that seen in kernels lacking SBEIIb activity. Characterization of endosperm starch from sbe2a::Mu mutants shows branching that is indistinguishable from wild-type controls. These mutant plants have a visible phenotype resembling accelerated senescence, which was correlated with the Mutator insertion within Sbe2a. This correlation suggests a specific role for SBEIIa in leaves, which may be necessary for normal plant development.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

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.

Adenosine Diphosphate Glucose↗
PubMed1997

Independent genetic control of maize starch-branching enzymes IIa and IIb. Isolation and characterization of a Sbe2a cDNA.

In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBEIIa, and SBEIIb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBEIIa, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify Sbe2-family cDNA from tissues lacking SBEIIb activity. The predicted amino acid sequence of Sbe2a cDNA matches the N-terminal sequence of SBEIIa protein purified from maize endosperm. The size of the mature protein deduced from the cDNA also matches that of SBEIIa. Features of the predicted protein are most similar to members of the SBEII family; however, it differs from maize SBEIIb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. Sbe2a mRNA levels are 10-fold higher in embryonic than in endosperm tissue, and are much lower than Sbe2b in both tissues. Unlike Sbe2b, Sbe2a-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBEIIa and SBEIIb activities.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

Evolutionary conservation and expression patterns of maize starch branching enzyme I and IIb genes suggests isoform specialization.

Expression of the maize (Zea mays L.) starch branching enzyme (SBE) genes Sbe1 and Sbe2 were characterized during kernel development and in vegetative tissues. The onset of Sbe1 and Sbe2 expression during endosperm development was similar to that of other genes involved in starch biosynthesis (Wx, Sh2 and Bt2). However, the expression of Sbe2 peaked earlier than that of Sbe1 in developing endosperm and embryos resulting in a shift in the ratio of Sbe1 to Sbe2 relative message levels during kernel and embryo development. Transcripts hybridizing to the Sbe2 probe were not detectable in leaves or roots which nonetheless have SBEII enzymatic activity, suggesting that there may be another divergent SBEII-like gene(s) in maize. A similar expression pattern is shared between the maize genes and related genes in pea, which together with their evolutionary conservation, suggests that the SBE isoforms may play unique roles in starch biosynthesis during plant development.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1992

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels.

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.

Journal Article↗
PubMed1992

Alterations in Carbohydrate Intermediates in the Endosperm of Starch-Deficient Maize (Zea mays L.) Genotypes.

Metabolite levels in kernels of selected starch-deficient mutants of maize (Zea mays L.) were investigated to gain insight into partitioning of carbohydrate metabolism during kernel development. Several free sugars, hexose phosphates, triose phosphates, fructose-2,6-bisphosphate, and pyrophosphate were measured in normal, shrunken, shrunken-2, amylose extender dull waxy, and brittle genotypes, which were in a near-isogenic W64A background. These mutants were selected to include at least one lesion in both the cytosolic (shrunken) and amyloplastic (shrunken-2) compartments. All the starch-deficient genotypes contained elevated levels of fructose-2,6-bisphosphate and triose phosphates but reduced levels of pyrophosphate, indicating an enhanced glycolytic utilization of carbohydrates in response to the reduced utilization of sugars for starch synthesis. The shrunken kernels (sucrose synthase deficient) contained reduced levels of glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, and this reduction paralleled the reduction in starch accumulation, but levels of triose phosphates were elevated. In shrunken-2 kernels, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate were increased, but fructose-1,6-bisphosphate was lower. These findings support the view that hexose phosphate transport across the amyloplast envelope is more important for starch biosynthesis than transport of triose phosphates. The amylose extender dull waxy mutation showed less dramatic effects on hexose phosphates, but the triose phosphates were greatly increased. The brittle mutation, which has an unknown lesion, showed distinctly similar changes in metabolite levels with shrunken-2, suggesting that the lesion may be associated with the amyloplast.

Journal Article↗
PubMed1990

Sugar uptake and starch biosynthesis by slices of developing maize endosperm.

(14)C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of (14)C among the soluble sugars extracted from endosperm slices incubated in (14)C-sugars. Competing hexoses reduced the incorporation of (14)C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.

Journal Article↗
PubMed1988

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.

Journal Article↗
PubMed1987

Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : I. Evidence for Regulation of Unloading by Cell Turgor.

Sugar and (14)C-assimilate release from the pedicel tissue of attached maize (Zea mays L.) kernels was studied following treatment with solute concentrations of up to 800 millimolal. Exposure and collection times ranged from 3 to 6 hours. Sugar and (14)C-assimilate unloading and collection in agar traps was reduced by 25 and 43%, respectively, following exposure to 800 millimolal mannitol. Inhibition of unloading was not specific to mannitol, since similar concentrations of glucose, fructose, or equimolar glucose plus fructose resulted in comparable inhibition. Ethylene glycol, a rapidly permeating solute which should not greatly influence cell turgor, did not inhibit (14)C-assimilate unloading. Based on these results, we suggest that inhibition of unloading by high concentrations of sugar or mannitol was due to reduced pedicel cell turgor. Changes in pedicel cell turgor may play a role in the regulation of assimilate transfer within the maize kernel.

Journal Article↗
PubMed1987

Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : II. Effects of Chemical Agents on Sugar, Amino Acid, and C-Assimilate Unloading.

Sugar, amino acid, and (14)C-assimilate release from attached maize (Zea mays L.) pedicels was studied following treatment with several chemical inhibitors. In the absence of these agents, sugar release was nearly linear over a 7-hour period. At least 13 amino acids were released with glutamine comprising over 30% of the total. Release was not affected by potassium concentration, 10-minute pretreatments with p-chloromercuribenzene sulfonic acid (PCMBS) or dithiothreitol, and low concentrations of CaCl(2). Three hours or more exposure to PCMBS, dinitrophenol, N-ethylmaleimide, or 2,4,6-trinitrobenzene sulfonic acid strongly inhibited (14)C-assimilate, sugar, and amino acid release from the pedicel. These treatments also reduced (14)C-assimilate movement into the kernel bases. It is, therefore, likely that reduced unloading, caused by these relatively long-term exposures to chemical inhibitors, was related to reduced translocation of assimilates into treated kernels. Whether this effect is due to disruption of kernel metabolism and sieve element function or reduced assimilate unloading and subsequent accumulation of unlabeled assimilates within the pedicel tissues cannot be determined at this time.

Journal Article↗
PubMed1985

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue.

Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of (14)C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl(2). Unloading was stimulated by 15 millimolar ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid which partially reversed the inhibitory effects of Ca(2+). Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses.

Journal Article↗
PubMed1981

A Nonaqueous Procedure for Isolating Starch Granules with Associated Metabolites from Maize (Zea mays L.) Endosperm.

A nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol was developed for the isolation from maize of starch granules with associated metabolites. In this procedure, immature endosperm tissue was quickly frozen at -156 C, freeze-dried, homogenized in cold glycerol, filtered through Miracloth, and centrifuged through a higher density medium of 3-chloro-1,2-propanediol. The procedure was used to isolate starch granules from the endosperm of normal and the mutant amylose-extender dull waxy. Starch and water-soluble polysaccharide recovery was high with low cytoplasmic (RNA) and nuclear (DNA) contamination.Electron microscopic examination of the isolated starch granules failed to demonstrate the presence of the amyloplast's membrane. However, based on an examination of fresh, freeze-dried, and rehydrated freeze-dried normal endosperm, it is suggested that the amyloplast membrane and enclosed stroma metabolites were dried onto the surface of the starch granules during the freeze-drying procedure. Chemical analysis of the glycerol-propanediol isolated granules showed the presence of alcohol-soluble sugars, inorganic phosphate, and phosphate-containing compounds. These soluble metabolites may represent amyloplast stroma metabolites which became bound to the starch granules during freeze-drying. Thus, this isolation procedure should be useful when metabolites closely associated with starch granules in situ are to be evaluated.

Journal Article↗
PubMed1981

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm.

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.

Journal Article↗
PubMed1980

Movement of C-labeled Assimilates into Kernels of Zea mays L: III. AN ANATOMICAL EXAMINATION AND MICROAUTORADIOGRAPHIC STUDY OF ASSIMILATE TRANSFER.

Basal tissue of developing maize kernels was examined by light micros-copy and by scanning and transmission electron microscopy. Plasmodesmata occur in pedicel and endosperm parenchyma but were not seen between the placento-chalazal cells and basal endosperm transfer cells. A layer of noncellular material separates the transfer cells from the placentochalazal cells. Microautoradiography of (14)C-labeled assimilates entering the kernels revealed that incoming sugars are not confined to the apoplast, but rather are present in the cytoplasm and vacuoles of pedicel and endosperm cells. No specific accumulation of radioactivity was seen in any particular tissue, although at later sampling times, a higher grain density in the pedicel than the endosperm indicated a general buildup of sugars in the pedicel. A possible model for sugar movement into developing kernels is discussed.

Journal Article↗
PubMed1972

Movement of C-Labeled Assimilates into Kernels of Zea mays L: I. Pattern and Rate of Sugar Movement.

Carbon-14, photosynthetically fixed in leaves of Zea mays L. and translocated to developing kernels, passed through specialized basal endosperm cells prior to movement into the starchy endosperm and embryo. Radioactivity migrated in the endosperm at a maximum rate of 2.7 millimeters per hour, and there was no difference in the rate of movement in kernels treated 14 to 30 days after pollination.Sucrose contained over three-fourths of the radioactivity in the kernal base (fruit stalk) 1 to 6 hours after (14)CO(2) treatment of the plant. Conversely, in the basal endosperm three-fourths of the radioactivity was in glucose and fructose. A high proportion of the radioactivity was retained in the monosaccharides of the starchy endosperm the first 3 hours after the (14)CO(2) treatment. With additional time after treatment there was a decline in the percentage of radioactivity in the monosaccharides and an increase in sucrose-(14)C. From these data we suggest that translocated sucrose is cleaved to glucose and fructose during entry into the endosperm and that the monosaccharides diffuse throughout the endosperm. Once the sugars arrive in the cells active in starch synthesis, they are rapidly converted to sucrose which in turn is utilized in the synthesis of starch.

Journal Article↗
PubMed1972

Movement of C-Labeled Assimilates into Kernels of Zea mays L: II. Invertase Activity of the Pedicel and Placento-Chalazal Tissues.

Invertases of the placento-chalazal and pedicel tissues are much more active than invertase from the pericarp of Zea mays L. kernels 12 to 40 days after pollination. Sucrose synthetase was not detected in the pedicel or placento-chalazal tissues. Sucrose content and percentage increased in the pedicel with advancing kernel age. Hexoses accounted for over half of the sugars extracted from the placento-chalazal tissues. These data are consistent with the hypothesis that sucrose translocated to the pedicel is hydrolyzed by acid invertase(s) prior to entry of sugar into the endosperm tissue. The placentochalazal tissue appears to be the primary site of sucrose inversion with the pedicel invertase contributing more or less to this process depending on kernel age.

Journal Article↗
PubMed1970

Starch Synthesis Studies in Zea mays: II. Molecular Distribution of Radioactivity in Starch.

The amylose and amylopectin fractions from kernel starch synthesized shortly after exposure of intact Zea mays L. plants to (14)CO(2) had similar specific radioactivities (counts per min per mg of carbohydrate). In both fractions the radioactivity was distributed throughout the molecules. These data are consistent with a model in which the polysaccharides are synthesized in the matrix of the amyloplast followed by crystallization of the completed molecules onto the starch granule.

Journal Article↗