Plants, green algae, and cyanobacteria synthesize storage polysaccharides by a similar ADPglucose-based pathway. Plant starch metabolism can be distinguished from that of bacterial glycogen by the presence of multiple forms of enzyme activities for each step of the pathway. This multiplicity does not coincide with any functional redundancy, as each form has seemingly acquired a distinctive and conserved role in starch metabolism. Comparisons of phenotypes generated by debranching enzyme-defective mutants in Escherichia coli and plants suggest that enzymes previously thought to be involved in polysaccharide degradation have been recruited during evolution to serve a particular purpose in starch biosynthesis. Speculations have been made that link this recruitment to the appearance of semicrystalline starch in photosynthetic eukaryotes. Besides the common core pathway, other enzymes of malto-oligosaccharide metabolism are required for normal starch metabolism. However, according to the genetic and physiological system under study, these enzymes may have acquired distinctive roles.

From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule.

Improving starch for food and industrial applications.

Starch synthesis in the cereal endosperm

Foods high in resistant starch have the potential to improve human health and lower the risk of serious noninfectious diseases. RNA interference was used to down-regulate the two different isoforms of starch-branching enzyme (SBE) II (SBEIIa and SBEIIb) in wheat endosperm to raise its amylose content. Suppression of SBEIIb expression alone had no effect on amylose content; however, suppression of both SBEIIa and SBEIIb expression resulted in starch containing >70% amylose. When the >70% amylose wheat grain was fed to rats in a diet as a wholemeal, several indices of large-bowel function, including short-chain fatty acids, were improved relative to standard wholemeal wheat. These results indicate that this high-amylose wheat has a significant potential to improve human health through its resistant starch content.

https://www.pnas.org/content/pnas/103/10/3546.full.pdf

High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.

/pdf/towards-a-better-understanding-of-the-metabolic-system-for-2myy9g26o2.pdf

Towards a Better Understanding of the Metabolic System for Amylopectin Biosynthesis in Plants: Rice Endosperm as a Model Tissue

Starch-branching enzymes (SBE) break the α-1,4 linkage of starch, re-attaching the chain to a glucan chain by an α-1,6 bond, altering starch structure. SBEs also facilitate starch accumulation by increasing the number of non-reducing ends on the growing chain. In maize ( Zea mays ), three isoforms of SBE have been identified. To examine the function of the SBEIIa isoform, a reverse genetics polymerase chain reaction-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe2a . To locate the insertion within the second exon of Sbe2a , the genomic sequence of Sbe2a containing the promoter and 5′ end was isolated and sequenced. Plants homozygous for sbe2a::Mu have undetectable levels of Sbe2a transcripts and SBEIIa in their leaves. Characterization of leaf starch from sbe2a::Mu mutants shows reduced branching similar to yet more extreme than that seen in kernels lacking SBEIIb activity. Characterization of endosperm starch from sbe2a::Mu mutants shows branching that is indistinguishable from wild-type controls. These mutant plants have a visible phenotype resembling accelerated senescence, which was correlated with the Mutator insertion within Sbe2a . This correlation suggests a specific role for SBEIIa in leaves, which may be necessary for normal plant development.

Identification of Mutator Insertional Mutants of Starch-Branching Enzyme 2a in Corn

Starch-branching enzymes (SBE) alter starch structure by breaking an α-1,4 linkage and attaching the reducing end of the new chain to a glucan chain by an α1,6 bond. In maize, three isoforms of SBE have been identified. In order to examine the function of the SBEI isoform, a reverse-genetics PCR-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe1. Compared to wild-type controls, Sbe1 transcripts accumulate at extremely low levels in leaves of the homozygous mutant. Antibodies failed to detect SBEI in leaf tissue of mutants or wild-type controls. In contrast, the level of SBEI in endosperm is undetectable in homozygous mutants while easily detected in wild-type controls. Starches extracted from mutant leaves and endosperm have structures indistinguishable from starches of wild-type controls as determined by size-exclusion chromatography (SEC) of intact starch and high-performance SEC of debranched starch. To investigate the possibility of compensation for the lack of SBEI by expression of the homologous sequence reported by Kim et al. (1998), a genomic fragment (Sbe1b) of this sequence was cloned. Northern hybridizations of mutant leaf, root, tassel, endosperm and embryo tissues with non-specific Sbe1b probes failed to reveal expression of the homologous sequence. These results suggest that the homologous sequence is not compensating for a lack of SBEI in sbe1::Mu mutants. Further study of this sbe1 mutation in the presence of other genetic mutations may help to understand the role of SBEI in determining starch structure in leaves and endosperm. Abbreviations: DAE, days after emergence; DAP, days after pollination; SBE, starch-branching enzyme

/pdf/identification-of-mutator-insertional-mutants-of-starch-51tyzs1fls.pdf

Susan L. Blauth

Papers

Identification of Mutator Insertional Mutants of Starch-Branching Enzyme 2a in Corn

Identification of Mutator insertional mutants of starch-branching enzyme 1 (sbe1) in Zea mays L.