Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions

Abstract

amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

“…2; Supplemental Fig. S1) through a decrease in the number of available nonreducing termini of glucan chains available for SSs (Liu et al, 2012a). Further dynamic expression analysis of the three SBEs indicated that the SBEs were inhibited gradually along with endosperm development in TRS, while in TQ, it showed an increased protein level until 10 DAF but then a decreasing trend ( Fig.…”

A review of starch‐branching enzymes and their role in amylopectin biosynthesis

“…The precise mechanism underlying the association of specific enzymes of starch synthesis with the starch granule are not known, although it has been suggested that alternative splicing of SBEII in bean (Phaseolus vulgaris L.) leads to partitioning within the starch granule (27). Recent evidence suggests that granule-associated proteins (including the SBEII class) become entrapped in the granule through association in heteromeric protein complexes (8,53). Distinct roles for SBEI and SBEII classes in amylopectin synthesis were suggested by the analysis of glucans produced when different combinations of maize SSs and SBEs were heterologously expressed in E. coli (54).…”

“…It is notable that both the Ser 649 and Ser 286 motifs are completely absent from SBEI, again suggesting a distinct class of functions for these sites. There is evidence of competition for the different phosphorylation sites by the plastidial CDPKs, as an allelic mutant of amylose extender maize expressing a truncated form of SBEIIb lacking Ser 286 and Ser 297 becomes hyperphosphorylated at the remaining Ser 649 residue (53). Phosphorylation of SBEs plays a critical role in the formation of heteromeric protein complexes with SSs and other SBE isoforms (see below).…”

“…In the soluble trimeric protein complex, found in cereal endosperm amyloplasts, SBEIIb is phosphorylated as is granule-associated SBEIIb, consistent with the observation that enzymes found in the trimeric protein complex become entrapped in the starch granule. Uncomplexed SBEIIb is also found phosphorylated in the plastid stroma (8,53). Regulation of the starch pathway by protein phosphorylation and protein-protein interactions is axiomatic of a highly regulated metabolic process, ultimately impacting the plant's carbon resource.…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…The developing endosperms of maize su2 (ΔSSIIa), amylose extender (ae, ΔBEIIb), sbe2a (ΔBEIIa), and dull1 (ΔSSIII) mutants have demonstrated the absence of the high molecular weight (>600 kDa) starch biosynthetic enzyme complexes, suggesting that SSIIa, SBEIIb, SBEIIa and SSIII are essential components for this complex [135]. SBEIIb-deficient maize mutant recruited SBEI, SBEIIa and Pho1 to form the SSI, SSIIa, SBEI, SBEIIa and Pho1 complex instead of SSI, SSIIa and SBEIIb [135], while the maize mutant containing an inactive SBEIIb formed a SSI, SSIIa, SBEI and SBEIIa complex [136]. This difference might result in the difference in starch structure observed from these studies.…”