Amylase 2017; 1: 59–74 Review Article Cheng Li, Prudence O. Powell, Robert G. Gilbert* Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm DOI 10.1515/amylase-2017-0006 Abbreviations: ADPGlc, adenosine 5'-diphosphate Received July 31, 2017; accepted September 22, 2017 glucose; AGPase, ADP-glucose pyrophosphorylase; Abstract: Starch from cereal endosperm is a major CBM, carbohydrate-binding module; CLD, chain-length energy source for many mammals. The synthesis of this distribution; DAF, days after flowering; DBE, debranching starch involves a number of different enzymes whose enzyme; D-enzyme, disproportionating enzyme; mode of action is still not completely understood. ADP- DP, degree of polymerization; GBSS, granule bound glucose pyrophosphorylase is involved in the synthesis starch synthase; GH, glycoside hydrolase; GT, glycosyl of starch monomer (ADP-glucose), a process, which transferase; ISA, isoamylase; MOS, maltooligosaccharides; almost exclusively takes place in the cytosol. ADP- 3-PGA, 3-phosphoglyceric acid; Pi, inorganic phosphate; glucose is then transported into the amyloplast and PUL, pullulanase; SBE, starch-branching enzyme; SP, incorporated into starch granules by starch synthase, starch phosphorylase; SS, starch synthases; SuSy, sucrose starch-branching enzyme and debranching enzyme. synthase; UDPGlc, nucleoside diphosphate glucose. Additional enzymes, including starch phosphorylase and disproportionating enzyme, may be also involved in the formation of starch granules, although their exact 1 Introduction functions are still obscure. Interactions between these Starch is a highly branched d-glucose homopolymer enzymes in the form of functional complexes have been with a wide range of uses. It is accumulated in the cereal proposed and investigated, resulting more complicated endosperm as an energy reserve for seed germination, as starch biosynthetic pathways. An overall picture and well as serving as the primary carbohydrate component in recent advances in understanding of the functions of our diets, with the higher fraction for Asian diets. Besides, these enzymes is summarized in this review to provide it has numerous important industrial applications, with insights into how starch granules are synthesized in extensive use in paper-making, minerals processing, cereal endosperm. personal care, renewable and/or biodegradable packaging. There are two common components of starch: Keywords: starch biosynthesis; sucrose synthase; ADP- amylose, with moderate molecular weight and a small glucose pyrophosphorylase; starch synthase; starch- number of long branches, and amylopectin, with a much branching enzyme; debranching enzyme; enzyme higher molecular weight and a large number of short complex branches. Both have a wide distribution of molecular sizes and molecular weights. Each starch molecule contains a single reducing end and many non-reducing ends (at the *Corresponding author: Robert G. Gilbert, Joint International terminus of each branch). Research Laboratory of Agriculture and Agri-Product Safety, College Starch structure could be divided into six levels [1], of Agriculture, Yangzhou University, Yangzhou, Jiangsu 225009, with the lowest five levels schematically shown in Figure People’s Republic of China, E-mail: [email protected] 1. It starts with individual chains as the first level, which Cheng Li, Joint International Research Laboratory of Agriculture and Agri-Product Safety, College of Agriculture, Yangzhou University, are formed by ADP-glucose through (1→4)-α-glycosidic Yangzhou, Jiangsu 225009, People’s Republic of China linkages. Those chains in amylopectin have been further Prudence O. Powell Robert G. Gilbert, The University of Queens- denoted as C chains having the reducing end, A chains land, Centre for Nutrition & Food Sciences, Queensland Alliance for (degree of polymerization, DP, 6-12) carrying no branches, Agriculture & Food Innovations, Brisbane, QLD 4072, Australia B1 chains (DP 13-24) carrying A chains, B2 chains (DP Open Access. © 2017 Cheng Li et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 3.0 License. 60 C. Li, et al. # " " " ! ! Figure 1. The hierarchical structural levels of organization of cereal endosperm starch. Level 1 is individual chain and imbedded in the Level 1 box is the chain length of A and B chains of amylopectin molecules. Level 2 is the amylose and amylopectin molecules and the different colours in the Level 2 box show the A (red), B (purple) and C (blue) chains. Level 3 is the amylopectin double-helix cluster with the repeat distance of the crystalline (~6 nm) and amorphous (~3 nm) lamellae. The arrangement of the amylopectin double helices from the bottom view is shown on the right side of Level 3 box. The level 4 structure contains the semi-crystalline growth ring formed by the amylopectin molecules and amorphous growth ring, which finally forms the level 5 structure, starch granules. The reducing end is represented by the left-to-right crossed “O”. 25-36) carrying B1 chains, B3 chains (DP > 36) carrying B2 molecules and parts of longer amylopectin chains, to chains, and so on (for a recent review, see [2]). Although form the level 4 structure. The water-insoluble starch still occasionally one finds statements to the contrary, granules with varied sizes and morphologies are finally amylose is not solely linear, but contains a small but developed by these growth rings as level 5, and with significant number of long-chain branches [3]. This the other components, such as proteins, non-starch structural level is quantified as the number of weight of polysaccharides and lipids, form the whole grain (level 6 chains as a function of degree of DP, the chain-length structure). distribution (CLD). These chains are further connected The synthesis and determination of this architecturally together through (1→6)-α-glycosidic linkages to form complex polymer assembly is achieved through the amylose and amylopectin molecules as the second level coordinated interactions of a suite of starch biosynthetic of structure. The third level of structure is formed by enzymes. Although significant process in elucidating neighbouring amylopectin chains intertwined into double the mechanism of these enzymes, either individually or helices and further into clusters. These amylopectin co-operatively, has been made, many aspects regarding double helices can arrange as a monoclinic unit cell this complex process are still unclear. The various (A-type crystal polymorph) or hexagonal unit cell (B-type isoforms of the many starch metabolic enzymes can be crystal polymorph). A cluster has a regular repeat distance found in different plant or different tissues in the same of ~9 nm in granules (see [2]). These clusters lie alongside plant. In this review, we mainly focus on the starch to form the semi-crystalline growth rings, alternated with biosynthetic enzymes in the cereal endosperm, beginning amorphous growth rings, probably containing amylose with the formation of ADP-glucose monomer and ending Biosynthesis of starch granules 61 with the architecturally complex starch granule, to 3 ADP-glucose pyrophosphorylase summarize the progress in understanding the starch biosynthesis process and noting what is missing in the (AGPase) literature. The degradation of sucrose derived from The formation of starch monomer, ADPGlc, is by AGPase photosynthesis has also been included because of its (EC 2.7.7.27). AGPase is heterotetrameric in higher plants, significant contribution to the synthesis of ADP-glucose consisting of two large (AGP-L) subunits and two small and seed sink strength. For the potential application of (AGP-S) catalytic subunits encoded by distinct genes, these enzymes for future cereal starch bioengineering and respectively [6]. Genes expressing these subunits can some other complementary information of, e.g., granule be expressed differently in different parts of the same initiation and control of starch granule size, please refer plant and thus produce AGPase with varying degrees of to recent reviews like [4-6]. Throughout this review, sensitivity to allosteric effectors, which are suited to the affiliation of individual starch metabolizing enzymes to particular metabolic demands of a given tissue/organ various families of glycoside hydrolases (GH) and glycosyl [6]. The AGP-S subunits are generally responsible for transferases (GT) as well as their carbohydrate-binding enzymatic complex catalytic activity, whereas the AGP-L modules (CBM) reflects their classification within the subunits are thought to modulate the enzymatic regulatory CAZy database (http://www.cazy.org/). properties that increase the allosteric response of small subunit to 3-phosphoglyceric acid (3-PGA) and inorganic 2 Sucrose synthase (SuSy) phosphate (Pi) [23-25]. The enzyme is now known to be largely extra-plastidial (i.e. 85-95%) in cereal endosperm, SuSy (EC 2.4.1.13) catalyses the reversible conversion but plastidial in other cereal tissues and in all tissues of of sucrose and a nucleoside diphosphate into the non-cereal plants [16,26-30]. The starch-deficient kernel corresponding nucleoside diphosphate glucose (UDPGlc) phenotypes of maize shrunken2 and brittle2 is caused and fructose [7,8] and is of family GT4, possibly with by the loss of endosperm-specific cytosolic AGP-L and different families of CBM, e.g., CBM20. In cereal endosperm, AGP-S isoforms, respectively,