Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf.

Conserved 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.

Functions of heteromeric and homomeric isoamylase-type starch-debranching enzymes in developing maize endosperm.

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.

Mutational analysis of the pullulanase-type debranching enzyme of maize indicates multiple functions in starch metabolism.

Plants contain two types of alpha(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.

A low-starch barley mutant, risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit.

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.