Charles D. Boyer

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Abstract Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A 1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d -glucose into the growing α- d -glucan and the synthesis of high molecular-weight polymers. However, the α- d -glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than II a.

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Abstract Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, sugary and amylose-extender , showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from sugary kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by sugary branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of amylose-extender kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of amylose-extender maize, but this activity was regenerated by the addition of any branching enzyme.

Citrus Leaf Chlorosis Induced by Sink Removal: Starch, Nitrogen, and Chloroplast Ultrastructure

Summary Chlorosis was observed in the mature source leaves of Citrus reticulata Blanco when branches were girdled and defruited, thereby in effect removing all major sinks. Starch levels slightly increased when either the fruit sink (defruiting) or the root sink (girdling) was removed. However, only when both sinks were removed did starch levels increase dramatically. Total nitrogen decreased and proline, arginine, and serine increased only when both sinks were removed. The non-protein amino acid, gamma-aminobutyric acid, decreased in response to total sink removal and was unaffected by removal of only one sink. In a time course study it was observed that the increase in starch levels preceeded the decrease in chlorophyll levels. A developmental study of chloroplast ultrastructure showed that starch granules increased in size and number and appeared to play a role in the deformation of the chloroplast lamellar organization. Concomitant with thylakoid breakdown was an increase in plastoglobules which were extruded from the plastid. Finally, plastids of the chlorotic leaves resembled amyloplasts or chloro-amyloplasts. The results are discussed in terms of source-sink relationships and chloroplast integrity.

Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen sythase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.

Allelic Analysis of the Maize amylose-extender Locus Suggests That Independent Genes Encode Starch-Branching Enzymes IIa and IIb.

Starch branching enzymes (SBE) catalyze the formation of [alpha]-1,6-glucan linkages in the biosynthesis of starch. Three distinct SBE isoforms have been identified in maize (Zea mays L.) endosperm, SBEI, IIa, and IIb. Independent genes have been identified that encode maize SBEI and IIb; however, it has remained controversial as to whether SBEIIa and IIb result from posttranscriptional processes acting on the product of a single gene or whether they are encoded by separate genes. To investigate this question, we analyzed 16 isogenic lines carrying independent alleles of the maize amylose-extender (ae) locus, the structural gene for SBEIIb. We show that 22 d after pollination ae-B1 endosperm expressed little Sbe2b (ae)-hybridizing transcript, and as expected, ae-B1 endosperm also lacked detectable SBEIIb enzymatic activity. Significantly, we show that ae-B1 endosperm contained SBEIIa enzymatic activity, strongly supporting the hypothesis that endosperm SBEIIa and IIb are encoded by separate genes. Furthermore, we show that in addition to encoding the predominant Sbe2b-hybridizing message expressed in endosperm, the ae gene also encodes the major Sbe2b-like transcript expressed in developing embryos and tassels.

Maize leaf and kernel starch synthases and starch branching enzymes

Abstract Soluble starch synthases and branching enzymes were partially purified from developing leaves and kernels of maize using DEAE-cellulose chromatography. One form of starch synthase and two forms of branching enzyme were detected in leaves as compared to two forms of starch synthase and three forms of branching enzyme isolated from the kernels. The starch synthase fraction from the leaves and the first starch synthase fraction from the kernels showed greater activity in reactions containing various glycogens as primers than in those containing amylopectin. In addition, both were capable of synthesizing a polyglucan in the absence of an added primer but in the presence of sodium citrate and bovine serum albumin (citrate-stimulated starch synthesis). The second starch synthase fraction from kernels showed greater activity with amylopectin as primer and had no citrate-stimulated activity. We suggest that the leaf enzyme and endosperm starch synthase I are the same enzyme and that it is ‘constitutively’ expressed. Branching enzymes from leaves and kernels differed not only in their elution profiles but also their stimulation of phosphorylase a (assay A) and amylose branching (assay B) activities. A minor branching enzyme fraction from leaves (leaf branching enzyme I) eluted from the DEAE-cellulose column after the addition of a salt gradient, whereas branching enzyme I from kernels eluted in the buffer wash prior to the application of the gradient. However, the ratios of assay A to assay B suggested that branching enzyme I from leaves was catalytically similar to branching enzyme I from the kernels. The major leaf branching enzyme (branching enzyme II) eluted at the same position from the DEAE-cellulose column as endosperm branching enzyme IIa. These enzymes had similar ratios of activity (Assay A/Assay B). The cross reaction of leaf branching enzymes with antisera prepared against maize endosperm branching enzymes in immunodiffusion experiments and enzyme activity neutralization experiments further demonstrated the relationship of the leaf and endosperm branching enzymes.

Biotechnological modification of carbohydrates for sweet corn and maize improvement

Abstract The many mutants of maize affecting starch biosynthesis have been used in the production of specialized corn, including sweet corn. The advent of molecular biology techniques will expand the types of corn used in commerce as the new, in vitro-produced mutations have not been found in nature. Essential to this work is a firm understanding of the path by which sucrose is converted to starch in the maize endosperm. Summarized below is our current understanding of the function of many of the starch synthetic genes based on genetic, molecular, biochemical, and physiological techniques. Emphasized are the unpredicted effects of various sugars and glucose polymers.

Two closely related cDNAs encoding starch branching enzyme from Arabidopsis thaliana

Two starch branching enzyme (SBE) cDNAs were identified in an Arabidopsis seedling hypocotyl library using maize Sbe1 and Sbe2 cDNAs as probes. The two cDNAs have diverged 5′ and 3′ ends, but encode proteins which share 90% identity over an extensive region with 70% identity to maize SBE IIb [12]. Genomic Southern blots suggest that the two cDNAs are the products of single, independent genes, and that additional, more distantly related SBE genes may exist in the Arabidopsis genome. The two cDNAs hybridize to transcripts which show similar expression patterns in Arabidopsis vegetative and reproductive tissues, including seedlings, inflorescence rachis, mature leaves, and flowers. This is the first report of the identification of cDNAs encoding two closely related starch branching enzymes from the same species.

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Abstract Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.

Starch and water-soluble polysaccharides from sugary endosperm of sorghum☆

Abstract The starch water-soluble polysaccharides from sugary ( su ) endosperm of sorghum were isolated and characterized. Starch granule structure and co