Abstract Starch arose in chloroplast-containing species from a combination of prokaryotic and eukaryotic genes involved in the metabolism of soluble branched α-polyglucan, i.e., glycogen. Non-mutant plants entirely lack such soluble polymers and instead contain amylopectin in insoluble starch granules. The transition between soluble and insoluble branched α-polyglucans during plant evolution is not well understood. This study generated maize (Zea mays L.) lines exhibiting a gradually varying distribution between soluble α-polyglucan and starch in the endosperm. These chemotypes were determined by complexes of conserved α-(1→6)-glucosidases of the isoamylase class (ISA). Four independent spontaneous missense substitutions in the ISA1 subunit of these complexes each cause a distinct soluble/insoluble α-polyglucan ratio, even though all four ISA1 variants lack detectable catalytic activity. These substitutions are located near each other in a domain distant from the active site. A separate region of ISA1 binds its non-catalytic paralog ISA2. Removal of ISA2 from the ISA1 mutant lines conditions further variability in the proportions of soluble α-polyglucan and starch. Thus, the extent of precursor α-polyglucan crystallization is determined by aspects of the ISA complexes beyond enzymatic activity. Various arrangements of multiple glucan-binding sites in different forms of the ISA1/ISA2 assemblies are proposed to determine how those complexes interact with precursor polymers. In turn, structural organization of the polymers is proposed to influence their crystallization, independent of α-1,6-glucosidase activity. Gradual change from soluble α-polyglucan metabolism to starch metabolism is proposed as a selective advantage leading to ISA2 conservation despite its lack of a functional catalytic site.
Hennen‐Bierwagen et al. (2025) studied this question.