Functional Interactions between Starch Synthase III and Isoamylase-Type Starch-Debranching Enzyme in Maize Endosperm    

Abstract

This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1 -mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.Semicrystalline glucan polymers that form insoluble starch granules are found in the vast majority of organisms within the Archaeplastida lineage of primary photosynthetic eukaryotes. This group, which comprises glaucophytes, rhodophytes (red algae), and Chloroplastida (green algae and land plants), in general does not contain soluble glucan polymers of substantial size. Conversely, with a few exceptions, essentially all other eukaryotes and prokaryotes utilize the soluble polymer glycogen for the storage of Glc and lack any insoluble glucans. Starch granules and their constituent polymers are capable of storing many more Glc units than chemically similar but soluble glucans, and this is likely to have provided a selective advantage during the establishment and spread of the photosynthetic eukaryotes. Support for this suggestion comes from the facts that the primary photosynthetic eukaryotes are a monophyletic group (Rodríguez-Ezpeleta et al., 2005). In light of the important role of starch metabolism in these organisms, including the land plants, it is of interest to understand the molecular mechanisms that generate semicrystalline glucans and how these processes differ from those that generate glycogen.Starch granules are made up of two t...

FLOURY ENDOSPERM6 encodes a CBM48 domain‐containing protein involved in compound granule formation and starch synthesis in rice endosperm

“…Furthermore, SSI and SSIIa are associated with either BEIIa or BEIIb . In maize, ISA2 and SSIII work in a coordinated fashion, suggesting the possibility that they may exist in a complex (Lin et al, 2012). These data provide insight into the complex network of starch synthesis; however, few studies have reported such an association in rice.…”

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…In common with other enzymes of the starch biosynthetic pathway, the catalytic function of SSIII has largely been deduced from mutant studies. Cereals lacking SSIII show a marked reduction in longer, cluster-spanning B-chains (for nomenclature of glucan chains in amylopectin see review by Bertoft in this Special Issue), as well as alterations in the short (A and B) chains, suggesting that the primary role of this enzyme is amylopectin synthesis [235,236]. Maize mutants lacking SSIII produce an endosperm with a glassy, dull appearance (and are termed du1 mutants) [237], a phenotype that is only conspicuous in Waxy (amylose-free) backgrounds [180].…”

Functions of Multiple Genes Encoding ADP-Glucose Pyrophosphorylase Subunits in Maize Endosperm, Embryo, and Leaf

“…A third sample of the crude homogenate was dried at 80°C to determine the dry weight of the tissue analyzed. Starch in each duplicate sample was completely hydrolyzed to Glc by treatment with amyloglucosidase and then quantified by a colorimetic assay as described previously with minor but significant modifications (Lin et al, 2012). One difference was that the starch pellet was washed twice by suspension in cold 80% (v/v) ethanol.…”