Plants contain two types of ␣ (1 → 6) glucan hydrolase (starch-debranching enzyme [DBE]). Mutations that affect the pullulanase-type DBE have not been described, although defects in isoamylase-type DBE, known in many plant species, indicate a function in starch biosynthesis. We describe a null mutation of a pullulanase-type DBE gene, a Mutator insertion in maize Zpu1 . Plants homozygous for the zpu1-204 mutation are impaired in transient and storage starch degradation. Thus, hydrolytic activity of pullulanase-type DBE contributes to starch catabolism. Developing zpu1-204 endosperm accumulates branched maltooligosaccharides not found in the wild type and is deficient in linear maltooligosaccharides, indicating that the pullulanase-type DBE functions in glucan hydrolysis during kernel starch formation. Furthermore, in a background deficient in isoamylase-type DBE, zpu1-204 conditions a significant accumulation of phytoglycogen in the kernel that is not seen in the wild type. Therefore, pullulanase-type DBE partially compensates for the defect in isoamylase-type DBE, suggesting a function during starch synthesis as well as degradation.

Starch production in all plants examined is altered by mutations of isoamylase-type starch-debranching enzymes (DBE), although how these proteins affect glucan polymer assembly is not understood. Various allelic mutations in the maize (Zea mays) gene sugary1 (su1), which codes for an isoamylase-type DBE, condition distinct kernel phenotypes. This study characterized the recessive mutations su1-Ref, su1-R4582::Mu1, and su1-st, regarding their molecular basis, chemical phenotypes, and effects on starch metabolizing enzymes. The su1-Ref allele results in two specific amino acid substitutions without affecting the Su1 mRNA level. The su1-R4582::Mu1 mutation is a null allele that abolishes transcript accumulation. The su1-st mutation results from insertion of a novel transposon-like sequence, designated Toad, which causes alternative pre-mRNA splicing. Three su1-st mutant transcripts are produced, one that is nonfunctional and two that code for modified SU1 polypeptides. The su1-st mutation is dominant to the null allele su1-R4582::Mu1, but recessive to su1-Ref, suggestive of complex effects involving quaternary structure of the SU1 enzyme. All three su1-alleles severely reduce or eliminate isoamylase-type DBE activity, although su1-st kernels accumulate less phytoglycogen and Suc than su1-Ref or su1-R4582::Mu1 mutants. The chain length distribution of residual amylopectin is significantly altered by su1-Ref and su1-R4582::Mu1, whereas su1-st has modest effects. These results, together with su1 allele-specific effects on other starchmetabolizing enzymes detected in zymograms, suggest that total DBE catalytic activity is the not the sole determinant of Su1 function and that specific interactions between SU1 and other components of the starch biosynthetic system are required.

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.Starch biosynthesis is a central function in plant metabolism that is accomplished by a multiplicity of conserved enzymatic activities. Two known activities are starch synthase, which catalyzes the polymerization of glucosyl units into a(1/4)-linked "linear" chains, and starch-branching enzyme, which catalyzes the formation of a(1/6) glycoside bond branches that join linear chains. Acting together, the starch synthases and starch-branching enzymes assemble the relatively highly branched polymer amylopectin, with approximately 5% of the glucosyl residues participating in a (1/6) bonds, and the lightly branched molecule amylose. Amylopectin and amylose assemble into semicrystalline starch granules, which in land plants and green algae are located in plastids.A third activity necessary for normal starch biosynthesis is provided by starch-debranching enzyme (DBE), which hydrolyzes a(1/6) linkages. Two DBE classes have been conserved separately in plants (Beatty et al., 1999). These are referred to here as pullulanase-type DBE (PUL) and isoamylase-type DBE (ISA), based on similarity to prokaryotic enzymes with particular substrate specificity. ISA function in starch production is implied from genetic observations that mutations typically result in reduced ...

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A-and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.In the endosperm of all of the species of grasses so far investigated, there are both cytosolic and plastidial forms of the enzyme of ADP-Glc pyrophosphorylase (AGPase). However, there are indications that there may be differences between species in the relative amounts of the plastidial and cytosolic activities of AGPase in the endosperm and in the nature of the genes encoding their subunits. Studies of mutants of maize (Zea mays) show that the cytosolic form accounts for Ͼ95% of the total activity of AGPase in the endosperm (Denyer et al., 1996) and that this form is essential for normal rates of starch synthesis (Tsai and Nelson, 1966;Dickinson and Preiss, 1969). These studies also show that the plastidial and cytosolic forms of AGPase are encoded by different pairs of large and small subunit genes. For example, mutations in maize at the Bt2 locus specifically affect the cytosolic single small subunit (SSU) of AGPase and eliminate the activity of AGPase in the cytosol, but they do not affect the plastidial AGPase SSU or activity Hannah et al., 2001). For barley (Hordeum vulgare), the situation is less clear, but there is evidence to suggest that barley differs from maize in the following respects.First, the plastidial activity as a proportion of the total AGPase activity is considerably higher in barley endosperm (15%; Thorbjørnsen et al., 1996b) than in maize (Ͻ5%; Denyer et al., 1996). In maize endosperm, we calculate from published measurements of the rate of starch synthesis and total AGPase activity in maize (Singletary et al., 1997) that the plastidial AGPase activity alone is not sufficient to account for t...

SummaryConserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement.Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined.Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf.The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.

Abstract:The functions of the two known plant classes of a(1•¨6) glucan hydrolase were investigated by ge netic analysis in maize.

Introduction Results and Discussion Materials and Methods iv References CHAPTER 5. GENE EXPRESSION ANALYSIS OF MAIZE STARCH DEBRANCHING ENZYMES BY REAL-TIME QUANTITATIVE PCR Abstract Introduction Results and Discussion Conclusion Materials and Methods Acknowledgements References CHAPTER 6. SUMMARY AND GENERAL CONCLUSIONS General Discussion Recommendations for Future Research References APPENDIX. REVERSE GENETICS OF ZmIso2 and