Recent Progress Toward Understanding the Role of Starch Biosynthetic Enzymes in the Cereal Endosperm

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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

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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, suggesting that plastidial the biosynthesis of starch granules starts with importing AGPase by itself is not sufficient to support the normal the sucrose derived from photosynthesis. Thus SuSy is a processes of starch biosynthesis in cereal endosperm [31- major determinant of sink strength that highly controls 34]. In non-graminaceous plant tissues and algae, storage the channelling of incoming sucrose into starch [9,10]. The starch biosynthesis appears to be solely dependent on a UDPGlc is converted to adenosine 5'-diphosphate glucose plastidial AGPase (for more details, see review [35]). The (ADPGlc) in the cytosol by the stepwise reactions of UDPGlc cytosolic localization of AGPase in cereal endosperm pyrophosphorylase and ADPGlc pyrophosphorylase may have functional significance for partitioning large (AGPase), while the conversion of fructose to ADPGlc amounts of carbon into starch when sucrose is plentiful, involves hexokinase, phosphoglucoisomerase and as in plants that have exclusively plastidial AGPase, the phosphoglucomutase [11]. Although UDP is the preferred sucrose-to-starch pathway involves plastid import of nucleoside diphosphate substrate for SuSy, ADP could hexose phosphates that can also be used in pathways also be an acceptor molecule of this sucrolytic enzyme to other than starch synthesis [26]. The reaction scheme produce ADPGlc [12-14]. Cytosolic ADPGlc is then thought for AGPase is shown in Figure 3 [36]. In in vivo systems, to be transported into the amyloplast by means of Brittle- the plastidial reaction is shifted in favour of ADPGlc 1, a membrane protein located in the envelope membranes synthesis by converting inorganic pyrophosphate (PPi) of amyloplasts [15], whose absence results in reduced into Pi by alkaline inorganic pyrophosphatase (PPase) starch content [16,17]. For a summary of the transporters [37]. The Pi can then be transported across the plastid found from the plastid envelope membrane, see [18]. SuSy- envelope membrane via a variety of different routes [6]. overexpressed cotton plants have an increase of biomass AGPase is required to control the flux of carbon from the and fibre yield [19,20], while maize sh1 mutants possessing Calvin-Benson cycle into starch synthesis. Much research approximately 10% of the wild-type SuSy activity show a into this enzyme has focused on its redox properties substantial (~50-70%) reduction in starch levels in the and have determined that both light levels and sugars seed endosperm [16,21]. The SuSy activity is known to be influence this state in vivo [38]. In addition, AGPase regulated by reversible phosphorylation, possibly by a activity is tightly regulated, with the catalytic activity seed-development specific protein kinase (SPK) [22]. These of the enzyme increased by the presence of 3-PGA and processes are summarized in Figure 2. inhibited by the presence of Pi. Therefore, the ratio of 62 C. Li, et al.

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Figure 2. Pathways of starch biosynthesis in cereal endosperm. ADPGlc is almost exclusively produced in the cytosol by different enzymes (see the text for the actual functions of each enzymes). A minor amount of AGPase (5-15% of total AGPase amount) found in the amyloplast could also produce the ADPGlc using the Glc 6-P imported from the cytosol via the Glc 6-P/Pi transporter. See [18] for a list of different solute transporters of the plastid envelope membrane. The activity of plastidial AGPase is regulated by its redox status and positively by 3-PGA and negatively by Pi concentration, while the activity of cytosolic AGPase is relatively insensitive to these regulations. ADPGlc is then incorporated into starch granules by a coordinated function of SS, SBE, DBE, SP and D-enzyme. SSI, SSIIa and SSIIIa are responsible for the biosynthesis of short (A chains, DP 6-12), intermediate (B1 chains, DP 13-24) and long chains (B2 chains, DP 25-36 and B3 chains, DP > 36), respectively. The loss of SSI’s activity can be partially compensated by SSIIa and SSIIIa, although SSIIIa has an inhibitory effect on the SSI’s activity when both exist. SSIIa and SSIIIa can partially compensate each other in their respective deficient mutant. GBSSI may have a role in the biosynthesis of long amylopectin chains and partially compensate for the loss of SSIIIa’s activity, while SSIIIa has an inhibitory effect on GBSSI’s activity. Finally, PUL could partially compensate ISA’s activity in ISA’s deficient mutant. SBEI and SBEIIb are responsive for the production of inner and outer branches. GBSSI produces amylose molecules from both MOS or short amylopectin chains and SBE may be involved in creating amylose branches. ISA1 trims the improperly placed branches, which could be further used by DPE1 and Pho1. Pho1 is also able to add glucosyl unit to amylopectin chains on the granule surface directly from Glc 1-P. Some enzymes have been found within the starch granules (SSIIa, SSI, SSIVa, SBEIIb) except GBSSI, while the mechanism involved is still unclear. This figure includes general features of the starch biosynthetic pathways in cereal endosperm. However, it may differ from plant to plant and tissue to tissue. Box 1 shows the responsible enzymes for respective chains length range, Box 2 the transporters and Box 3 the full names of each enzyme. Biosynthesis of starch granules 63

NH2 N N O O O N N O O - O P O P O P O CH2 O -O P O P O- - - - O O O O- O- ATP OH OH Pyrophosphate + + NH2 CH2OH CH2OH N H O H H O H N H H O O O N OH H OH H N O P O- O P OH OH O P O CH2 O H OH O- H OH O- O- -Glucose-1-phosphate ADP-glucose OH OH

Figure 3. Mechanisms of the actions of some main starch biosynthetic enzymes. AGPase: ADP-glucose pyrophosphorylase; SS: starch synthase; SBE: starch-branching enzyme; DBE: debranching enzyme; D-enzyme: disproportionating enzyme; empty hexagon: anhydroglucose unit; filled hexagon: ADP-glucose; dashed line: starch chains; X0 and Xmin are the minimum chain-length constraints on SBE. these two allosteric effectors plays a key role in starch might also be involved with amylose synthesis. Detailed biosynthesis [6]. biochemical studies have indicated the importance of Modulating the activity of AGPase has been used glutamate and aspartate residues for the catalytic activity in order to increase the starch yield. Expression of an and substrate binding [43], and the involvement of lysine allosterically insensitive Escherichia coli mutant AGPase in the K-X-G-G-L domain in determining glucan primer in rice endosperm results the increase of its sink strength preference [44]. and starch production [39]. In addition, the overexpression Granule bound starch synthase (GBSS) consists of a heat stable variant of maize AGP-L with reduced Pi of two isoforms, GBSSI and GBSSII. GBSSI is encoded sensitivity also results an increase of starch yield by by the Waxy (wx) locus in cereals and is contained 38% and 23% in wheat [40] and rice [41] endosperm, completely within the starch granule [45,46]. It can use respectively. both maltooligosaccharides (MOS) and short amylopectin chains as primers to synthesize amylose molecules [47]. Many mutations in the wx locus lead to a complete loss 4 Starch synthases (SSs) of amylose. It is worth noting that PROTEIN TARGETING TO STARCH (PTST) is needed for the targeting of GBSS to SSs (EC 2.4.1.21) in higher plants catalyse the transfer of starch granule in Arabidopsis [48]. In addition, GBSSI is a glucose unit from the soluble precursor ADPGlc, to the thought to be responsible for the extension of long chains non-reducing end of a pre-existing (1→4)-α-glucan primer, of amylopectin (species dependent), remaining associated which will eventually form amylose or amylopectin [42] with the chain after the addition of each glucose unit to and is of family GT5 with CBM53. There are multiple add further units [49,50]. GBSSII is encoded by a separate isoforms of SSs in plants, of which the major classes gene to GBSSI and is thought to be responsible for the can be divided into two groups: the first involved in biosynthesis of amylose in leaves and other non-storage amylose biosynthesis and the second concerned with tissues, which accumulate transient starch [6,51]. amylopectin synthesis, although it is possible that the Soluble SSs are thought to be exclusively involved in mainly-amylopectin SSs and other amylopectin enzymes amylopectin biosynthesis (but see the remark above) and 64 C. Li, et al. the individual isoforms (SSI, SSII, SSIII and SSIV) each released reducing end to a C6 hydroxyl group, which is playing unique roles; for example, there is evidence that either a part of the original glucan chain (known as intra- each isoform predominantly (but probably not exclusively) chain transfer) or an adjacent chain (known as inter-chain synthesises different chain lengths [49]. Although these transfer), to create a new α-(1→6)-α-glucosidic linkage. It enzymes may have different activities depending on the is of family GH13 with CBM48. There are two minimum plant species and tissues, they are all present in starch- chain-length constraints on the transferred and the synthesising cells. SSI appears to be primarily responsible residual segments [65-67], which have been termed as Xmin for the synthesis of short glucan chains, as evidenced from and X0, respectively [68]. Biotechnological modification of SSI mutants in Arabidopsis [52]. Further investigation of these constraints on SBE has the potential of developing the chain-length specificities of maize endosperm SSI has nutritionally desirable plants [4,68,69]. In addition, suggested that SSI prefers the shortest amylopectin chains various conserved amino-acid sites have been proved as substrates [53]. The isoforms of SS and other enzymes to be important for SBE catalytic activity, site-directed often exhibit a DP range where they dominate over other mutagenesis of which have resulted in various degrees of isoforms, but it is important to be aware that “dominate” reduction of SBE’s activity [69,70]. does not mean that other isoforms have no effects in that SBE activity in cereals is a function of multiple range. The amylopectin synthesized in the SSI-deficient isoforms (SBEI and SBEII), which may be tissue- or rice endosperm showed a reduced amount of chains with developmentally-specific in their expression. While dicots DP 8-12 and an increased amount of chains with DP 6-7, typically have a single SBEII enzyme, in monocots, SBEII suggesting that SSI generates chains of DP 8-12 from short can be further be split into SBEIIa and SBEIIb isoforms; chains of DP 6-7 [54]. SSII has been determined to play a role SBEIIa is present in nearly every tissue and SBEIIb is in the synthesis of intermediate-length branch chains of usually confined to the endosperm [6]. The catalytic amylopectin [55]. There are two classes of SSII genes found activity of SBEII is regulated by protein phosphorylation, in monocots: SSIIa and SSIIb. The role of SSIIb in starch as inferred from wheat endosperm studies [71]. In the biosynthesis is unknown, as no mutants have been identified developing wheat endosperm, the expression of SBEIIb is as yet. SSIIa is mainly present in cereal endosperms, while at a much lower level than SBEIIa [72,73], in contrast to SSIIb is mainly found in photosynthetic tissues [6]. Both the maize endosperm [74]. In rice, SBEIIa is at the earliest SSI and SSII are localised within the starch granules in expressed 3 days after flowering (DAF) and maximally many cereals [56-58]. SSIII has two genes responsible for its at 5-7 DAF, while SBEI and SBEIIb are most abundant at expression in the endosperms and leaves of rice. Mutants of 7-10 DAF [75,76]. SBEII isoforms are partitioned between SSIII in maize and rice display altered granule morphology the plastid stroma and the starch granules [57], although and crystallinity, and a reduction in long-branch chains the factors or mechanisms involved remain incompletely extending between clusters [59-60]. Loss of SSIII has also determined. been associated with pleiotropic effects affecting other These SBE isoforms also differ in terms of the length of SSs, where SSs have an increase in activity, while SBEIIa glucan chain transferred and their substrate specificities. activity is decreased. This has led to the suggestion that An in vitro study showed that SBEI transfers longer chains SSIII acts as a regulator for the starch biosynthetic process. (DP ≥ 16) and has a higher affinity for amylose, while SBEII SSIV is the most recently discovered form of SS [61] and transfers shorter chains (DP ≤ 12) and appears to have a as such, little is known about its contributions to starch higher affinity for amylopectin [45]. However, in maize biosynthesis. Two isoforms of SSIV can be found in plants, endosperm the amylopectin CLD is not affected by a SSIVa and SSIVb, which are expressed in endosperm and deficiency in SBEI activity, although a further loss of SBEIIb leaf tissues, respectively. Studies conducted in Arabidopsis causes increased branching, suggestive of a regulatory have indicated that SSIV may have a role in controlling the role for SBEI in influencing other SBEs [77,78]. This is number of starch granules within a plastid, although it may supported by the observation of the physical interaction also be involved in making short chains [62-64]. between SBEI and SBEIIb in the wheat endosperm amyloplast [71]. Interestingly, amylopectin from rice SBEI- deficient endosperm showed a significant reduction of 5 Starch-branching enzymes (SBEs) chains with DP 12-21 and DP ≥ 37, together with an increase of chains with DP ≤ 10 and DP 24-34, indicating that rice SBEs (EC 2.4.1.18) generate new branches on starch SBEI synthesizes B1 chains and cluster-connecting B molecules, mainly amylopectin, by cleaving internal chains of amylopectin [79]. Although the SBEIIa activity (1→4)-α bonds of a branch chain and transferring the was detected at a very early stage in the developing rice Biosynthesis of starch granules 65 endosperm [80], the SBEIIa mutation in maize and rice maize and barley sugary-1 (ISA1) mutants have shown only caused a slight reduction in the short-chain content heterogeneous distributions of starch and phytoglycogen of leaf starch and did not show any significant changes in their endosperms, while Arabidopsis mutants of ISA1 in the endosperm amylopectin CLDs [81,82]. SBEIIa might and ISA2 display accumulation of phytoglycogen in the thus play a role in leaf starch biosynthesis and be involved leach mesophyll and slightly altered amylopectin in in the initiation of starch biosynthesis. SBEIIb mutant the epidermal cells and vascular bundle-sheath cells was originally found to have a high amylose content [83], [97.98]. In Arabidopsis and potato, accumulation of which was later shown in maize and rice to be because the phytoglycogen is due to the loss of either ISA1 or ISA2. In higher amount of amylose is actually from the A (internal) addition to a reduction in starch content, a loss of ISA1 amylopectin chains [80,84,85]. Moreover, a rice SBEIIb has been linked to a reduction in granule size and an mutant had a 50% reduction of SSI activity, suggesting increase in granule number [93,99], indicating ISA1 may that SBEIIb and SSI may interact in vivo [80]. However, be involved in granule initiation. In maize, a loss of ISA1 high amylose starch was only developed by suppressing had a pleiotropic effects on other enzymes, particularly both SBEIIa and SBEIIb in wheat [73]. The amylopectin SBEIIa [91], while the presence of a catalytically inactive from these mutants appeared to have greatly elongated ISA1 eliminates some of these effects, suggesting that the chains and fewer short chains of DP ≤ 17, especially ISA1 has both enzymatic and non-enzymatic functions in chains of DP 8-12, suggesting that SBEIIb plays an and may form enzymatic complex with other enzymes. important role in the formation of amylopectin A chains. In rice sugary1 mutant, the introduction of wheat ISA1 Indeed, an in vitro study has shown that recombinant results in essentially the replacement of phytoglycogen rice SBEIIb preferentially produces short chains of DP 6-7, by amylopectin [100]. In these plants, there appears to while recombinant rice SBEIIa forms short chains with a be a consistent relationship between ISA1 expression and relatively broader range of DP 6-11 [86]. The loss of short the percentage of amylopectin chains of DP ≤ 12. Starches chains from SBEIIb mutant has also resulted in the shift from ISA1 mutants also show different physiochemical of A-type X-ray diffraction pattern of starch granules from properties in the grain, such as lower viscosity and wild-type rice to a B-type pattern [87]. gelatinization temperature, reduced and less stable crystalline structure and altered granule structure [51,92]. ISA2 has no activity itself, and the absence of ISA2 in 6 Starch debranching enzymes maize and rice results in near normal kernels, in contrast (DBEs) to ISA1 mutants. However, this enzyme is believed to be associated with starch biosynthesis in leaves and DBEs (pullulanase – EC 3.2.1.41 and isoamylase – EC potato tuber and is required, along with ISA1, for the 3.2.1.68) hydrolyse (or debranch) the (1→6)-α-glycosidic activity of the ISA heteromeric enzyme [98,99,101,102]. linkages of starch (Fig. 3) and are of the GH13 family This hetero-oligomer is less important compared to the possibly with different CBMs, e.g., CBM 48. Two groups homo-oligomers of ISA1 in rice and maize endosperm of DBEs exist in plants: the isoamylase-type (ISA), of for the trimming of improper branches [103-105]. Indeed, which there are at least three forms that have been the overexpression of ISA2 in rice endosperm results in recognised (ISA1, ISA2 and ISA3) and the pullulanase more soluble polysaccharide, indicating that the increase type (PUL, also known as limit-dextrinases, LDA) [88,89]. of ISA2 leads to the increase of non-functional hetero- ISA has the ability to debranch both phytoglycogen and oligomers of ISA1 and ISA2, and the decrease of the amylopectin, while PUL is able to debranch both pullulan functional homo-oligomers of ISA1. and amylopectin, but not phytoglycogen [82]. Mutations affecting PUL or ISA3 do not appear to The loss of ISA1 activity in cereal endosperms result in the accumulation of phytoglycogen on their own. has been shown to result in the loss of the highly ISA3 is mainly expressed in rice leaves and to a lesser ordered amylopectin structure and the accumulation extent in endosperm [100], and appears to be essential of phytoglycogen and/or modified amylopectin [82,90]. for starch degradation in Arabidopsis leaves [99]. The Different levels of ISA activity, such as in maize (su1), function of PUL is less understood than that of ISA. PUL barley (isa-1), and rice (sug-1), can result in a range of is believed to be associated with the degradation of starch phenotypes associated with the endosperm, ranging from during kernel germination; however, its activity has a mild phenotype of sugary amylopectin with more short been detected in developing rice and maize endosperms chains, to a severe phenotype resulting in the replacement [106-108], suggesting PUL may also be involved in of amylopectin with phytoglycogen [91-96]. Rice, starch biosynthesis. Loss of just PUL in maize mutants 66 C. Li, et al. results in no significant alterations in starch structure is sketched in Figure 3. A mutant of Chlamydomonas or composition in maize endosperm, while loss of both reinhardtii specifically lacking D-enzyme (in the STA11 ISA1 and PUL results in a significant accumulation of locus) exhibited no detectable effects on any other phytoglycogen in maize endosperm [109]. Thus it has been known starch biosynthetic enzymes, but showed a suggested that PUL can partially compensate for the loss severe decrease in starch content, and the amylopectin of ISA activity in starch biosynthesis in cereal endosperms molecules showed a significant increase in the frequency [45,89,109]. It has also been suggested that PUL is subject of short A chains, clearly indicating a role for the enzyme to activation by changes in redox status, and inhibition in in starch synthesis [116-118]. Linear MOS also accumulate the presence of high sugar concentration [110]. abnormally in the soluble fraction from the mutants. Different models have been put forth to explain the Analysis of D-enzyme levels in the developing endosperm accumulation of phytoglycogen in the absence of DBEs. of wheat is consistent with a role in starch biosynthesis for Currently, the most prevalent view is termed the ‘glucan this enzyme [119]. However, the involvement of D-enzyme trimming model’, which postulates that DBEs modify the in amylopectin biosynthesis may vary depending on structure of amylopectin during biosynthesis [42,111]. physiological conditions or tissue, as the effects of sta11 This model indicates that the actions of SS and SBE were only observed in conditions of nutrient starvation, alone are unable to produce a glucan able to crystallize. not in exponential-phase cells. Furthermore, no obvious This may be due to the greater number of branches, the changes in starch biosynthesis occurred in potato tubers transferred chains being too short or the branch points bearing an antisense transgene targeting D-enzyme [120]. incorrectly positioned. DBEs are theorised to selectively Two possibilities have been proposed for the role of remove errantly-placed branch points, thereby facilitating D-enzyme in starch synthesis [111]. It is likely that DBEs crystallization; however, there is no evidence as yet to function prior to the involvement of D-enzyme, providing indicate, which branch points are removed, how these are MOS as the D-enzyme substrate, consistent with the role of selected or how this contributes to amylopectin synthesis DBEs as proposed in either the WSP-clearing model or the [42,11,112]. The finding of numerous short chains on glucan-trimming model. The D-enzyme may convert MOS the surface of immature starch granules supports this to glucose and other longer MOS chains [121]. The latter model [65]. The other model proposes that DBEs have a would be suitable substrates for phosphorylase (discussed ‘clearing’ role in starch biosynthesis rather than being as below) and would thus be converted to Glc-1-P, and directly involved in amylopectin biosynthesis. Rather, further converted to ADPGlc and re-enter the biosynthetic DBEs prevent the formation of phytoglycogen by removing process via SS. Although this model could account for soluble glucans from the stroma, which otherwise would the decrease in total starch content that correlates with serve as the primer for phytoglycogen biosynthesis and D-enzyme deficiency, it does not explain the altered further inhibit the starch synthesis rate. This proposes chain lengths observed in amylopectin from C. reinhardtii that phytoglycogen is a separate soluble product rather sta11 mutant. Thus, a second model was proposed: that than an intermediate for amylopectin synthesis [6,97]. D-enzyme directly transfers glucan chains from MOS into This model is consistent with that in DBE mutants, the the A chains of amylopectin, which could then explain accumulation of phytoglycogen is always at the expense both the decrease in amylopectin biosynthesis and the of amylopectin [89,99,113]. altered chain length. SP catalyses the reversible transfer of glucosyl units from Glc-1-P to the non-reducing end of (1→4)-α-linked 7 Additional enzymes glucan chains and may be driven in either a synthetic or (disproportionating enzyme and a degradative direction by the relative concentrations of the soluble substrates. Plants possess two types of SP, Pho1 starch phosphorylase) and Pho2, localized to the plastid and cytosol, respectively. A number of studies have found that Pho1 gene expression Other enzymes may also be involved in starch biosynthesis, and activity correlate with other starch biosynthetic such as disproportionating enzyme (D-enzyme; EC 2.4.1.25) enzymes, like SS and SBE, suggesting a role in contributing and starch phosphorylase (SP; EC 2.4.1.1), although their to starch biosynthesis [122,123]. Rice pho1 mutant shows exact role is still unclear. an accumulation of smaller starch granules and abnormal D-enzyme transfers two of the glucosyl units from endosperm phenotype [124], and the amylopectin showed maltotriose on to a longer glucan chain and is present a higher proportion of short chains of DP < 11 and a lower in many different plants [114,115]. Its working mode proportion of intermediate chains of DP 13-21. Biosynthesis of starch granules 67

Different models explaining the role of SP in the activity tests showed that the activities of these enzymes starch biosynthesis has been proposed, including the were maintained. In developing barley endosperm, a suggestion that D-enzymes work in conjunction with SP. phosphorylation-dependent SSI, SSIIa, SBEIIa and SBEIIb In this model, short-chain MOS liberated in the trimming complex was identified [132]. In vitro study has found an reaction by DBEs are converted to longer-chain glucans by interaction between Pho1 with SBE [133], and Pho1 with D-enzyme, which in turn are available for phosphorolysis D-enzyme [134], which may play a role in starch granule by SP, liberating Glc-1-P used to synthesize ADPGlc by initiation [130]. plastidial AGPase [117,120]. Analysis of mutants lacking one or more starch biosynthetic enzymes has also shed new light on the mechanism of enzyme complex formation. The 8 Enzyme complexes developing endosperms of maize su2 (ΔSSIIa), amylose extender (ae, ΔBEIIb), sbe2a (ΔBEIIa), and dull1 (ΔSSIII) There is evidence that several starch biosynthetic enzymes mutants have demonstrated the absence of the high depend on other biosynthetic components for their molecular weight (>600 kDa) starch biosynthetic enzyme activity; for example, a mutation affecting SSIII reduces complexes, suggesting that SSIIa, SBEIIb, SBEIIa and the activity of SBEIIa whilst also increasing the activity of SSIII are essential components for this complex [135]. SSI in cell extracts [125], indicating that starch biosynthesis SBEIIb-deficient maize mutant recruited SBEI, SBEIIa is most likely to be the result of several enzymes acting and Pho1 to form the SSI, SSIIa, SBEI, SBEIIa and Pho1 in a coordinated manner. The physical and biochemical complex instead of SSI, SSIIa and SBEIIb [135], while the interactions between starch-synthesising enzymes must maize mutant containing an inactive SBEIIb formed a thus be taken into account in order to fully understand the SSI, SSIIa, SBEI and SBEIIa complex [136]. This difference process of starch biosynthesis. might result in the difference in starch structure observed It has been found that some enzymes involved in starch from these studies. Hence, starch biosynthesis probably synthesis form complexes with one another: for example, involves the recruitment of other starch biosynthetic the homo-oligomers of ISA1 and hetero-oligomers of ISA1 isozymes, and alternative protein complexes form when and ISA2 as outlined above. In wheat, a protein complex specific starch biosynthetic enzymes lost. has been found involving SBEI, SBEIIb and SP, the Starch biosynthetic enzyme complexes are formation of which has been proposed to be dependent summarized in Table 1. on protein phosphorylation [71]. Further study showed a 260 kDa enzyme complex of SSI, SBEIIa and SBEIIb was formed within mid-late-developing wheat seeds, and this 9 Conclusions and future work enzyme complex was not present in seeds during the early stage of development [126]. SBEIIb in the same complex Our understanding of starch synthesis in cereal found in maize was proven to be phosphorylated [127]. endosperms has advanced substantially from both in vivo Three phosphorylation sites (Ser286, Ser297 and Ser649) and in vitro studies. The cytosolic form of AGPase that is were then experimentally identified in maize SBEIIb, with unique to cereals may serve to commit excess carbon to Ser286 and Ser297 conserved among cereal SBEIIs [128]. starch production. Roles for SS and SBE isoforms in chain- Maize SSIIa was found at the centre of a trimeric enzyme length determination and branch placement have been complex of SSI and SBEIIb both within the stroma and proposed, as well as the possible working mechanisms for granule, and it conferred starch binding properties to the DBEs. The coordinated combinations of multiple isoforms complex [129]. According to the specificities of individual of SS, SBE and DBE lead to a uniform number of chains isozymes, this complex was proposed to contribute to per amylopectin cluster. However, to precisely understand the synthesis of short and intermediate amylopectin starch biosynthesis in cereal endosperm, detailed branches to form the crystalline lamellae [127,130]. In characterisation of the properties of additional enzymes addition, maize SSI, SSIIa, SSIII, SBEI, SBEIIa and SBEIIb is needed, e.g., the functional roles of SSIV, D-enzyme (SSIIa, SSIII, SBEIIa, SBEIIb in ~600 kDa complex; SSIIa, and SP. An improved understanding of the starch granule SBEIIa, SBEIIb in ~300kDa complex) were found within initiation is also required. More work is required to discover protein-protein complexes [125]. A larger protein complex new interactions between starch synthetic enzymes and to including SSIIa, SSIIIa, SSIVb, SBEI, SBEIIb and PUL elucidate the mechanisms and signalling cues that govern (>700 kDa) was eluted by gel filtration chromatography this aspect of metabolic regulation, as well as to identify from soluble rice endosperm extracts [131]. Zymogram the components controlling protein complex formation. 68 C. Li, et al. Note - biosynthe starch for important is It endosperm; other than in leaves sis a play may and inactive is ISA2 higher affinity has it role; regulatory hom - than phytoglycogen towards ooligomer The amount of ISA2 to that of ISA1 ISA1 of that to ISA2 of The amount the amount of the ratio determines hetero-oligomer of that homo to of Phosphorylation could activated the activated could Phosphorylation SBEIIa stromal Most well studied; formed at Mid Mid at formed studied; well Most the development; seed of stage of capacity binding granule starch of the affinity determine may SSIIa for complexes protein trimeric this at located is SSIIa granules; starch the complex of the center The interaction of these presumed presumed these of The interaction with enzymes cytosol-localized questions raise enzymes plastidial - comple whether these concerning vivo in present actually are xes Reference [89, 98, 99, 102-105, 131, 137- 139] [103-105, 131, 139] [131, 140] [71] [126, 127, 129, 131] [125, 135] [125, 135] [127] [126] - Possible function Possible tin CLD; accelerate the crystallization the crystallization accelerate tin CLD; and molecules amylopectin nascent of of the interference prevent thereby - in the bio amylases as such enzymes, process synthetic Control granule initiation and number; number; and initiation granule Control biosynthesis, amylopectin to contribute the amylopec change will which of loss Contribute to starch biosynthesis by by biosynthesis starch to Contribute rate the crystallization accelerating Starch initiation Starch na glucan glucan in vitro for the affinity Increase - the amylo synthesize and substrates and the short including pectin cluster chains intermedium May account for the ordered construction construction the ordered for account May - crys to destined polymer the glucan of granules starch form and tallize Plastidal PPDK may direct the AGPase the AGPase direct may PPDK Plastidal degradation ADPGlc towards activity direction Complementation for the loss of SBEIIb SBEIIb of the loss for Complementation cluster the amylopectin synthesize and intermedium and the short including chains glucan glucan in vitro for the affinity Increase substrates Regulation na na na Post-translational phosphorylation Post-translational phosphorylation Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation SP; on SBEI and effect hydrophobic Post-translational phosphorylation; effect hydrophobic Location Within stroma and and stroma Within granule starch Within stroma Within na Within stroma Within Within stroma and and stroma Within granule starch Within stroma Within Within stroma Within Within stroma and and stroma Within granule starch Within stroma Within Complex size size Complex (kDa) 450-500 (potato); 510-550 400 (rice); (maize) 420-480 300 (rice); 190 (maize); - ( Chlamydo monas ) ~243 ~286 260 (wheat, 260 (wheat, 300 rice), (maize) ~300 670 ~440 180 Plant origin Plant - Arabi tuber, Potato rice leaves, dopsis leaf, and endosperm endosperm, maize Chlamydomonas Rice endosperm, endosperm, Rice endosperm, maize Chlamydomonas Rice Pho1 expressed Pho1 expressed Rice in E.coli Wheat endosperm Wheat Wheat endosperm, endosperm, Wheat endosperm, maize endosperm rice Maize endosperm Maize Maize endosperm Maize mutant ae mutant Maize endosperm Wheat endosperm Wheat a endosperm. in cereal found complexes enzyme Different Table1. complex Enzymatic Heteromer of ISA1 ISA1 of Heteromer ISA2 and Homomer of ISA1 Homomer of Pho1, Pho1 SBEI, SBEIIb, Pho1 SBEIIb, SBEI, SSI, SSIIa, SBEIIb SSIIa, SSI, SSIIa, SBEIIa, SBEIIb SBEIIa, SSIIa, SSIIa, SSIII, SBEIIa, SBEIIa, SSIII, SSIIa, PPDK, SBEIIb, SuSy AGPase, SSI, SSIIa, SBEIIa, SBEIIa, SSIIa, SSI, SP SBEI, SBEIIa, SBEIIa SBEIIa, Biosynthesis of starch granules 69 Note Formed in mutant Formed SSIIa is inactive in Japonica rice in Japonica inactive is SSIIa SSIIa is inactive in Japonica rice in Japonica inactive is SSIIa Reference [126] [126] [126] [126] [126] [126] [132] [132] [134] [131] [131] Possible function Possible glucan glucan in vitro for the affinity Increase substrates glucan glucan in vitro for the affinity Increase substrates glucan glucan in vitro for the affinity Increase substrates glucan glucan in vitro for the affinity Increase substrates glucan glucan in vitro for the affinity Increase substrates glucan glucan in vitro for the affinity Increase substrates Biosynthesis of both A and B type barley barley B type both A and of Biosynthesis granules Biosynthesis of A type barley granules in granules barley A type of Biosynthesis endosperm the mutant The complex utilizes a broader range of of range a broader utilizes The complex substrates for enhanced synthesis of each than maltooligosaccharides larger significantly and enzyme individual Pho1; of affinities the substrate elevate is the Pho1 in complex moreover, - transglyco of products utilize to able G3, G1 and involving reactions sylation directly catalyze cannot it that sugars na na Regulation Post-translational phosphorylation; effect hydrophobic Post-translational lation; phosphory effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic Post-translational phosphorylation; effect hydrophobic na na na Location Within stroma Within Within stroma Within Within stroma Within Within stroma Within Within stroma Within Within stroma Within Within stroma and and stroma Within granule starch Within stroma and and stroma Within granule starch Within stroma Within Within stroma Within Within stroma Within Complex size size Complex (kDa) 180 260 260 260 260 260 338 376 168 >700 200-400 Plant origin Plant Wheat endosperm Wheat Wheat endosperm Wheat Wheat endosperm Wheat Wheat endosperm Wheat Wheat endosperm Wheat Wheat endosperm Wheat Barley endosperm Barley Barley (ΔSBEIIa/ Barley mutant) ΔSBEIIb endosperm Rice endosperm Rice Rice endosperm Rice Rice endosperm Rice Enzymatic complex Enzymatic SBEIIb, SBEIIb SBEIIb, SSI, SBEIIa, SBEIIa SBEIIa, SSI, SSIIa, SBEIIa, SBEIIa SBEIIa, SSIIa, SSI, SBEIIb, SBEIIb SBEIIb, SSI, SSIIa, SBEIIb, SBEIIb SBEIIb, SSIIa, SSI, SSIIa, SBEIIa SSIIa, SSI, SSI, SSIIa, SBEIIa, SBEIIa, SSIIa, SSI, SBEIIb SBEIIa, SBEIIb, SBEI, SBEI, SBEIIb, SBEIIa, SP DPE1, Pho1 SSIIa, SSIIIa, SSIVb, SSIVb, SSIIIa, SSIIa, PUL SBEIIb, SBEI, SSI, SSIIa, SBEIIb, SBEIIb, SSIIa, SSI, PUL, Pho1 ISA1, Note: this is only a list of main enzymatic complexes found to date. For more enzymatic complexes, see [125, 131, 136]. complexes, enzymatic more For date. to found complexes enzymatic main of a list only is this a Note: 70 C. Li, et al.

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