Genetic Evidence That Chain Length and Branch Point Distributions Are Linked Determinants of Starch Granule Formation in Arabidopsis    

Abstract

The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. We varied both structural parameters by creating Arabidopsis (Arabidopsis thaliana) mutants lacking combinations of starch synthases (SSs) SS1, SS2, and SS3 (to vary chain lengths) and the debranching enzyme ISOAMYLASE1-ISOAMYLASE2 (ISA; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different SSs. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of SSs present affect the crystallinity and thus the solubility of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, glucan production was restored in the ss2ss3isa triple mutants, indicating that SS activity in ss2ss3 per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only SSs can cause phytoglycogen production. This is readily degraded by isoamylase and other enzymes so it does not accumulate and was previously unnoticed.Starch, the major storage carbohydrate in plants, is composed of two a-1,4-and a-1,6-linked glucan polymers: moderately branched amylopectin and predominantly linear amylose. Amylopectin, which constitutes approximately 80% of most starches, is synthesized by three enzyme activities. Starch synthases (SSs) transfer the glucosyl moiety of ADP-Glc to a glucan chain, forming a new a-1,4 glucosidic linkage, extending the linear chains. Branching enzymes (BEs) cleave some a-1,4 linkages and reattach chains of six Glc units or more via a-1,6 linkages, creating branch points. Debranching enzymes (DBEs) hydrolyze some of these branches, tailoring the structure of the polymer. However, the way in which the individual enzymes work together to create crystallizationcompetent amylopectin remains unclear.The coordinated actions of SSs, BEs, and DBEs are thought to produce a glucan with a tree-like architecture in which the branch points are nonrandomly positioned. According to models of amylopectin, clusters of unbranched chain segments are formed. Within these clusters, adjacent chains form double helices, which align in parallel giving rise to crystalline lamellae. These alternate with amorphous lamellae containing the branch points and chain segments that span the clusters . In the context of this amylopectin model, glucan cha...

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves

“…The chain length distribution of amylopectin differed slightly between esv1, lesv, ESV1, and LESV-overexpressing lines and wild-type starch ( Figure 6B), but these differences were small in comparison to those seen in Arabidopsis mutants deficient in enzymes of starch biosynthesis (Pfister et al, 2014). The chain length distributions of the b-limit dextrin of amylopectin from esv1 and lesv mutants were also similar to those of wild-type plants (Supplemental Figure 9F).…”

“…Second, abolition of amylose from the esv1 mutant (in the esv1 gbss mutant) does not alter its starch degradation phenotype. Third, the alterations in chain length distribution of amylopectin are much smaller than in many mutants that do not exhibit accelerated starch degradation (e.g., starch synthase mutants; Szydlowski et al, 2009;Pfister et al, 2014).…”

“…However, several potentially important features of leaf starch granules are known to be under tight control. These include the timing of granule initiation during leaf development, granule size, shape, and number per chloroplast (and therefore surface area), and the degree of crystallinity of the constituent glucan polymers (Roldán et al, 2007;Szydlowski et al, 2009;Crumpton-Taylor et al, 2012Pfister et al, 2014). These features may combine with more specific control mechanisms that modulate the activities of the enzymes of starch breakdown to confer the observed adjustment of the degradation rate to match the length of the night.…”

Molecular structural differences between maize leaf and endosperm starches

“…Many studies used high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the characterization of the chain-length distribution (CLD) of leaf starch but this can only give information up to ~DP 50 and also suffers from a mass bias, which is very laborious to correct (Pfister et al, 2014;Wong & Jane, 1997). Fluorophore-assisted carbohydrate electrophoresis (O'Shea, Samuel, Konik & Morell, 1998) (FACE) is regarded as the best technique for the characterization of starch chains (first level) below degree of polymerization (DP) of 180 (Wu, Li & Gilbert, 2014a) and thus is very useful in terms of revealing structural differences for longer amylopectin long chains as well as avoiding the mass bias of HPAEC.…”

“…Most work has focused on Arabidopsis, e.g. (Pfister, Lu, Eicke, Feil, Lunn, Streb & Zeeman, 2014), especially an elegant new study on crystallinity properties (Zhu, Bertoft, Wang, Emes, Tetlow & Seetharaman, 2015).…”