STUDIES ON CARBOHYDRATE-METABOLIZING ENZYMES Part XXV. THE DEBRANCHING ENZYME SYSTEM IN GERMINATED BARLEY

Manners, D. J.; Rowe, Karen L.

doi:10.1002/j.2050-0416.1971.tb03385.x

Cited by 23 publications

(13 citation statements)

References 15 publications

(3 reference statements)

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“…There is little if any R-enzyme (debranching enzyme) activity in this fraction since the apparent debranching activity is 50% inhibited in the presence of 60 mm EDTA while the debranching activity eluted later from the column is not inhibited by EDTA. Thus, a third a-( 1, 6)-glucosidase in sweet corn which hydrolyzes pullulan as reported by Manners and Rowe (15) is likely a glucoamylase and not an isozyme of the major debranching enzyme. for the su enzymes as compared to the nonsugary enzymes.…”

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confidence: 61%

“…The partially debranched polysaccharide would constitute the amylopectin component ofstarch while the branches would be linked together to constitute the linear component (amylose). Subsequently, two laboratories (13,15) investigated debranching enzyme activity in su seeds, and both reported finding such activity. It is significant, ' however, in view of our findings that neither laboratory simultaneously investigated debranching enzyme activity in nonsugary endosperms.…”

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confidence: 99%

“…All data concerning enzyme activity in su endosperms came from preparations of the reference allele su-R except for the data in Table I presence of debranching enzymes in developing maize seeds has been reported for sugary genotypes (13,15). However, these studies did not test the possible difference in debranching enzyme activity between nonmutant and mutant genotypes.…”

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confidence: 99%

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A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize

Pan¹,

Nelson²

1984

Plant Physiol.

165

131

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Many of the sugary-i mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme la-(1, 6)-glucosidasel activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing a-1,6 branch points into the linear a-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one ofthese peaks ofactivity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (O to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.The sugary-l (sul) mutant of maize, Zea mays L., which is the usual sweet corn of commerce, has been known and utilized since preColumbian times (21), and specific efforts to improve particular varieties of sweet corn date back to the middle of the nineteenth century (18). In spite of this lengthy association with man, the specific biochemical lesion induced by mutations at the su locus has not been known. Sumner and Somers (20) reported that the principal polysaccharide storage product in su endosperms was not starch, but a highly branched, water-soluble polysaccharide of high mol wt which Sumner and Somers referred to as phytoglycogen. Erlander ( 11) MATERIALS AND METHODS Biological Materials. Self-pollinated seeds ofthe maize inbred W64A or the hybrid W64A x 182E were used as the nonsugary controls. The first group of sugary mutants examined contained the reference allele at the su locus, su-R, and an intermediate allele su-Bn2. These alleles had been placed in the W64A genotype by repeated backcrosses. A second group of sugary alleles contained su-8115, su-8116, su-8117, and su-8135. These were presumed to have been independently induced by ethylmethanesulfonate (EMS) treatment since they were isolated in the progeny of inbred W22 plants exposed to the mutagen. For simplicity, we refer to su endosperms throughout the report. It should be understood that this means homozygous sugary (sul su/su) endosperms.

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A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize

Pan¹,

Nelson²

1984

Plant Physiol.

165

131

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“…However, this observation by itself does not exclude the possibility that SU1 and isoamylase II are the same, because the size determination was made on relatively crude fractions and the possibility exists that SU1 is oligomeric in vivo. Another maize isoamylase was identified in extracts from mature su1-mutant sweet corn kernels (Manners and Rowe, 1969). The fact that SU1 is not present in su1-mutant kernels rules out identity with this sweet-corn isoamylase, which most likely participates in endosperm-starch degradation after seed germination.…”

Section: Discussionmentioning

confidence: 99%

“…The participation of a specific pullulanase or isoamylase in the biogenesis of kernel starch, however, has yet to be demonstrated directly. In addition to having potential biosynthetic functions, both types of DBE are believed to be involved in the degradation of endosperm starch after seed germination (Manners and Rowe, 1969;Toguri, 1991).…”

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Characterization of SU1 Isoamylase, a Determinant of Storage Starch Structure in Maize1

et al. 1998

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Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1-mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to ␣-(136) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes ␣-(136) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1-mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1-mutant kernels. These data support the hypothesis that DBEs are directly involved in starch biosynthesis.Starch is a reserve carbohydrate that accumulates in the storage organs of many higher plants. This storage compound consists of a mixture of two Glc homopolymers, amylopectin and amylose, in which linear chains are formed via ␣-(13 4) glucosyl linkages and branches are introduced by ␣-(13 6) glucosyl linkages. Starch synthesis in maize (Zea mays) occurs within the amyloplasts of endosperm cells during kernel development via the concerted actions of ADP-Glc pyrophosphorylase, starch synthases, and starch-branching enzymes (for reviews, see Preiss, 1991;Hannah et al., 1993;Martin and Smith, 1995;Nelson and Pan, 1995; Preiss and Sivak, 1996; Smith et al., 1996). In addition, selective removal of branch linkages by DBEs is proposed to play an essential role in the final determination of amylopectin structure (Ball et al., 1996).Physical and chemical analyses of granular starch have led to a widely accepted model for amylopectin structure called the "cluster model," in which amorphous and crystalline regions alternate with a defined periodicity (for reviews, see French, 1984;Manners, 1989;Jenkins et al., 1993). Within amylopectin the crystalline component is composed of parallel arrays of linear chains packed tightly in double helices. Branch linkages, which account for approximately 5% of the glucosyl linkages in amylopectin, are located at the root of each cluster in the amorphous region. This periodic clustering of branches allows for the alignment of the intervening linear chains and their dense packing into crystalline regions, thus providing an efficient mechanism for nutrient storage. Undoubtedly, the enzymatic processes requir...

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Starch Biosynthesis. I. The Size Distributions of Amylose and Amylopectin and Their Relationships to the Biosynthesis of Starch

Erlander¹

1998

Starch/Stärke

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STUDIES ON CARBOHYDRATE-METABOLIZING ENZYMES Part XXV. THE DEBRANCHING ENZYME SYSTEM IN GERMINATED BARLEY

Cited by 23 publications

References 15 publications

A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize

A Debranching Enzyme Deficiency in Endosperms of the Sugary-1 Mutants of Maize

Characterization of SU1 Isoamylase, a Determinant of Storage Starch Structure in Maize1

Starch Biosynthesis. I. The Size Distributions of Amylose and Amylopectin and Their Relationships to the Biosynthesis of Starch

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