J. Appl. Glycosci., 60, 3‒20 (2013) doi: 10.5458/jag.jag.JAG-2012_018 ©2013 The Japanese Society of Applied Glycoscience Special Issue: Starch Metabolism, Structure and Properties Review Starch Synthesizing Reactions and Paths: in vitro and in vivo Studies (Received November 5, 2012; Accepted November 19, 2012) (J-STAGE Advance Published Date: January 21, 2013) Henrike Brust,1 Slawomir Orzechowski,2 Joerg Fettke1,3 and Martin Steup1,＊ 1Department of Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam (Building 20, Karl-Liebknecht.Str. 24‒25, 14476 Potsdam-Golm, Germany) 2Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life Sciences―SGGW (159 Nowoursynowska, Warsaw 02‒776, Poland) 3Mass Spectrometry of Biopolymers, Institute of Biochemistry and Biology, University of Potsdam (Building 20, Karl-Liebknecht.Str. 24‒25, 14476 Potsdam-Golm, Germany)

Abstract: This review is written for plant biologists, biochemists and biotechnologists. It presents recent results and views on the metabolism of transitory and reserve starch. We discuss several topics related to starch biosynthesis and focus on the elongation of α-glucan chains as mediated by the various starch synthases and by the plastidial phosphorylase. We briefly discuss the two main glucosyl donors, i.e. ADPglucose or glucose 1-phosphate, that are used by the two types of glucosyl transferases. In the next section, we present a novel theoretical approach to analyse reiterating reactions mediated by a single enzyme. This type of reactions occurs frequently in carbohydrate metabolism of pro- and eukaryotic cells. We then give a schematic presentation of the structure of starch synthases from Arabidopsis thaliana and discuss the action of the different isoforms. The two types of phosphorylases that are common in plants are described and their in vivo actions are presented. We describe functional properties of heterogeneous protein complexes and explain why they are needed in starch metabolism. Finally, we discuss phenotypical analyses of starch-related mutants and explain some of the difficulties inherent to the attempt to deduce biochemical paths from phenotypical features.

Key words: starch synthesis, starch synthases, starch phosphorylases, starch excess phenotype, amylose, amylopectin

Essentially all living systems are capable of synthesizing As an extracellular product of plant cells, cellulose is large glycans, such as arabinogalactan, xylan, cellulose, composed of β-D-glucopyranosyl residues that are intercon- fructan, laminarin, glycogen or the two starch-related nected by β-1,4-bonds. By contrast, starch and glycogen are polyglucans, amylose and amylopectin. Each of these typical intracellular storage carbohydrate of plants and glycans exists as a large group of chemically closely related animals, respective. Both starch and glycogen are exclusive- but polydisperse biopolymers rather than as a distinct ly built of α-D-glucopyranosyl residues that are linked by macromolecule. The biological functions exerted by glycans two types of interglucose bonds, α-1,4- and α-1,6- linkages. are highly heterogeneous and range from shaping and/or Despite the chemical similarity, important physicochemical protecting cells or tissues to intra-cellular storage of reduced differences exist: Native starch is deposited as water-insolu- carbon including the realm of molecular interactions between ble particles designated as granules. Native starch particles plant cells or between a plant and a non-plant organism. are capable of an essentially unlimited growth. Glycogen is Glycans have an enormous impact on human diet.1,2) Further- a hydro-soluble polydispers molecule which appears to more, polysaccharides are gaining increasing importance for possess a strict upper size limit (for review see Ref. 9‒11)). several biotechnological applications and energy-converting In photosynthesis-competent eukaryots and their hetero- processes that all are based on photosynthesis-driven biosyn- trophic derivatives starch granules act as the almost ubiqui- thesis of carbohydrates.3‒8) tously occurring storage carbohydrate that allows central Naturally occurring polyglucans are usually composed of carbon metabolism and growth to continue when photosyn- either of two cyclic hemiacetals, designated as α- or thesis is not functional and, at the same time, to prevent β-glucopyranosyl residues. Both six-membered rings are carbon starvation. Starch may, however, share these interconnected by only one or two type(s) of precisely functions with other reduced carbon compounds, such as defined glucosidic linkages. The anomeric form of the sucrose, fructans or even soluble non-carbohydrates and pyranosyl residues and the type(s) of interglucose bonds both accumulation and utilization of the various compounds largely determine the structure of the entire polysaccharide. are remarkably flexible.12‒15) In green algae, mosses, ferns and higher plants, starch ＊ Corresponding author (Tel. +49‒331‒977‒2651, Fax. +49‒331‒977‒ granules are synthesized inside the plastidial compartment 2512, E-mail: [email protected]). but in many other eukaryotic algae the cytosol is the princi- 4 J. Appl. Glycosci., Vol. 60, No. 1 (2013) ple site of starch metabolism. In eukaryotic algae, cytosolic of α-glucan chains and the multifunctionality of glucosyl starch is assumed to represent an earlier evolutionary state as residues favour cyclisation but, to the best of our knowledge, compared to the starch granules in the plastid.16‒18) cyclodextrins have never been reported to occur in plants. Except in some mutants, native starch consists of two Based on the current knowledge, the entire process of types of polyglucan molecules, amylose and amylopectin. starch biosynthesis can be grouped into the following closely Amylopectin usually is quantitatively dominant and interconnected (but not strictly consecutive) steps: determines most structural features of native starch (for 1. formation of a suitable glucosyl acceptor (primer) details see Ref. 19, 20)). Likewise, in vivo amylose biosyn- 2. biosynthesis of suitable glucosyl donors thesis appears to require a pre-existing amylopectin-contain- 3. elongation of the primer by repetitive glucosyl transfer ing starch granule.21,22) The term ‘amylose’ covers a complex reactions and forming the dominant interglucose bond mixture of strictly unbranched and poorly branched 4. introduction of additional interglucose bonds that α-1,4-glucans having a wide range of degree of polymerisa- constitute branchings tion (DP). However, even large amylose molecules are 5. removal of excess branching points smaller in size as compared to amylopectin. In the latter (as 6. ordering of the polyglucose molecules yielding the final well as in glycogen) more than 90% of the total interglucose physical order linkages are α-1,4-bonds but the residual linkages are The formation of a suitable primer (step 1) is closely α-1,6-glucosidic bonds giving raise to branchings of glucan associated with the de novo starch biosynthesis of starch chains. Thereby, the number of non-reducing ends increases granules. This process is still incompletely understood but it but that of reducing ends remains unchanged. In amylopec- clearly deviates from the dominant mode of glycogen initia- tin, as opposed to glycogen, branchings are clustered rather tion.11,31‒34) In the eukaryotic marine picoalga, Ostreococcus than regularly distributed. This non-uniform organization tauri, the binary division of the plastidial starch granule leads to a more complex structure of the amylopectin precedes the binary cell division and, therefore, each molecule and, finally, to a hydro-insoluble starch particle. daughter cell contains a single starch granule as does the Clustering of branchings permits neighbouring chains to mother cell.35) In this case the number of algal cells is interact forming parallel oriented double helices which can massively increased by successive division without de novo be arranged in either of two physical orders, designated as biosynthesis of starch granules being essential. However, A- or B-type starch allomorph. If a single granule contains Ostreococcus tauri is likely to be exceptional in this respect both the A- and the B-type, starch is designated as C-type as in other eukaryotic algae a division of the plastidial starch allomorph. In any case, the highly ordered arrangements of granules preceding cell division has not been documented. the double helices defining the allomorph exist as layers Furthermore, algal cells typically contain several starch which in total represent a major proportion of but not the granules per chloroplast and often undergo multiple cell entire native starch particle. In addition, the starch granule divisions.36) In higher plants, the number of starch granules contains alternating layers of less ordered regions which are per organelle massively increases in growing tissues during enriched in branching points and, presumably, in these areas proplastid-chloroplast transition and, therefore, de novo amylose is located possessing an uneven distribution within biosynthesis of starch appears to be essential. By contrast, the granule. Unfortunately, many structural details of native formation of a suitable primer may be of limited relevance in starch have not yet been fully elaborated (for details see Ref. mature leaves at least from the Arabidopsis plant. Recently 19, 20)). it has been shown that in mesophyll cells the diel starch- Like ribosomal protein biosynthesis, elongation of starch- turnover largely proceeds by changes in granule size rather related α-glucan chains is strictly unidirectional as only the than by significantly altering the number of starch granules free hydroxyl group at C4 of non-reducing ends functions as per mesophyll cell. However, in leaves from wild type acceptor for a further transfer of glucosyl moieties from a Arabidopsis plants the number of starch granules per chloro- suitable donor. In pro- and eukaryots, some linear proteins plast varies.37,38) Likewise, in a synchronized photoautotro- (or peptides) undergo a complex posttranslational process phic Chlamydomonas the cellular starch contents (as well as leading to an extremely stable circular backbone.23‒25) the rates of starch biosynthesis and degradation) exhibit a Recently, an unusual path of extracellular starch degradation large cell-to-cell diversity.36) has been reported for several prokaryotes growing in a In this review, we largely focus on both in vitro and in vivo starch-containing medium. In the unusual path (which either aspects of the elongation of α-glucan chains as mediated by is the only functional route or co-exists with the convention- starch synthases (EC 2.4.1.21) and by α-glucan phosphory- al mode of starch degradation)26), cyclodextrins are formed lases (EC 2.4.1.1; step 3). Furthermore, we consider interac- from starch by the action of an extracellular cyclodextrin tions of both chain elongating enzymes with branching glucanotransferase. Following the import into the cell via an enzymes (EC 2.4.1.18; step 4). We include information on ABC-like transporter, cyclodextrins are linearized by an theoretical aspects of the elongation of α-glucans. We do, intracellular cyclodextrinase and hydrolysed to glucose and however, not specifically discuss the removal of excess maltose.27) Alternatively, the intracellular linear maltodex- branching points (step 5). This topic has been presented in a trins are largely converted to glucose 6-phosphate as mediat- recent review.39) Furthermore, the mechanisms leading to the ed by the sequential action of maltodextrin phosphorylase physical ordering of vicinal glucan chains (step 6) are largely and phosphoglucomutase.28,29) Similarly, some bacterial cells uncertain and, therefore, are not extensive considered here. are capable of converting linear α-glucans into various cyclodextrins which also can be used for several biotechno- logical applications.30) Presumably, both the helical structure Brust et al.: Starch Synthesizing Reactions and Paths 5

Adenosine diphosphoglucose and α-D-glucose 1-phosphate ed. In transgenic Arabidopsis plants expressing the mutated as starch-related glucosyl donors. AGPase, the total amount of the mutated protein is consider- In higher plants, adenosine diphosphoglucose (ADPglu- ably lower but transcript levels are elevated as compared to cose) and, to some extent, α-D-glucose 1-phosphate (G1P) the wild type and, therefore, the turnover rate of the mutated act as immediate glucosyl donors (step 2) for the elongation AGPase seems to be higher. Because of the lowered steady- of α-glucan chains. G1P is an intermediate of the central state level of the mutated protein, the total AGPase activity carbon metabolism and is formed by utilizing other hexose (Vmax) is only 10 to 20% of that of the wild type. Neverthe- monophosphates, as mediated by phosphoglucomutase and less, leaves of the transgenic Arabidopsis plants possess phosphohexoisomerase. G1P exists as several intracellular elevated ADPglucose levels and accumulate more starch pools and can be imported into the cell as well as into the than the wild type. Furthermore, the transgenic Arabidopsis plastid.40‒42) Depending on the subcellular location, the lines possess a higher rate of starch degradation and elevated anomeric glucose phosphate is involved in several other maltose levels.61) Taken together, these data clearly show paths, such as the biosynthesis of uridine diphosphoglucose that, at least under some conditions, the in vivo rates of (UDPglucose) and ADPglucose, and, thereby, indirectly starch biosynthesis and the measured Vmax values of the leads to the accumulation of various end products, such as AGPase activity do not correlate. Currently, the complex sucrose, cellulose and starch. For the latter, the action of control of starch metabolism as exerted by the AGPase is not ADPglucose pyrophosphorylase (AGPase; EC 2.7.7.27) is fully understood. Complexity of balancing carbon and essential which converts glucose 1-phosphate plus ATP to nitrogen metabolism may be envisaged by recent results ADPglucose and pyrophosphate. Thus, within the complex obtained with Saccharomyces cerevisiae.62,63) process of starch biosynthesis G1P exerts a dual function as The starch-related phenotype of AGPase-reduced it serves both as an immediate glucosyl donor for starch seedlings of Vicia narbonensis L. is by far less severe. Due biosynthesis (step 3; see below) and as a substrate for the to an antisense construct that is derived from the small formation of another immediate glucosyl donor, ADPglu- subunit of AGPase and placed under the control of a cose (step 2). seed-specific promotor, both the activity of the target enzyme Traditionally, the synthesis of ADPglucose is considered and the level of ADPglucose are reduced to approximately to be the first committed step in the starch synthesizing path. 5% of that of the wild type. By contrast, the starch content of In higher plants, the AGPase holoenzyme is frequently a the cotyledons is only moderately affected and accounts for, heterotetrameric complex (designated as α2β2 or S2L2) at least, 60% of the wild type control. Cotyledons of these consisting of two slightly smaller (α or S monomers) and transgenic lines possess, however, starch granules whose two slightly larger (β or L) subunits.43‒45) The α- and the morphology is altered. Furthermore, the parenchyma cells of β-subunit encoding genes are derived from the same the cotyledons are vacuolated and the seed-filling period is ancestral gene. Deletion of either subunit type affects starch prolonged.64,65) These results strongly suggest that starch metabolism. biosynthesis is, to a large extend, qualitatively rather than Arabidopsis mutants lacking the functional AGPase are quantitatively altered and point to an unexpectedly complex largely (but not completely) incapable of accumulating leaf cellular phenotype. Similar results have been obtained using starch.46‒48) Likewise, reserve starch biosynthesis is impaired embryos from transgenic pea lines in which, due to a RNAi in AGPase-related mutants from Zea mays L.49) and Pisum construct, expression of the AGPase is strongly decreased. sativum L.50) As revealed by flux control analyses, AGPase is RNAi inhibition of the expression of the target gene is reported to significantly contribute to the control of starch associated with many changes in carbon-nitrogen metabo- biosynthesis in leaves of Arabidopsis thaliana.51‒53) However, lism but the fresh-weight based starch content of the seeds these results are largely based on measurements of the accounts for approximately 50% of the wild type control.66) maximal catalytic activity, Vmax, of AGPase which is unlike- Likewise, a low flux control coefficient of the AGPase was ly to reflect the in vivo situation. In higher plants, the AGPase observed in developing seeds of Vicia narbonensis L. and activity is controlled by several posttranscriptional Pisum sativum L.65,67) Based on these data, a metabolic route mechanisms, including allosteric activation/inhibition, is likely to exist that contributes to starch accumulation but redox-regulation, oligomerization/dissociation and proteo- does not depend on the formation of ADPglucose. In a strict lysis.54‒56) In addition, light and sugars exert a transcriptional sense, the phenotype even of the AGPase-deficient Arabidop- control of the AGPase57,58) whereas both nitrate and sis mutants mentioned above proves the requirement of the orthophosphate inhibit gene expression59,60) and, thereby, ADPglucose-dependent path of starch biosynthesis rather counteract. Presumably, all these transcriptional and than its exclusiveness (see below). post-translational control mechanisms are part of a more In cereal endosperms, amyloplastic starch biosynthesis complex regulatory network that balances intra- and intercel- relies on both cytosolic and the plastidial AGPase isozymes. lular carbon fluxes under a wide range of external conditions. The former represents the dominant AGPase form whose Recently, redox-regulation of the AGPase in Arabidopsis product, ADPglucose, is imported into the plastid by the leaves has been studied in more detail. In the small subunit, Brittle1 transport protein mediating an ADPglucose/ADP APS1, the cysteine residue that is essential for the redox antiport.1,68,69) In transgenic rice lines that constitutively control and both necessary and sufficient for the dimeriza- express an unregulated AGPase from E. coli in the cytosol, a tion of two small subunits has been identified. Oxidation of 3- to 6-fold higher AGPase activity but only a modest this residue results in a linkage of two subunits by a disulfide increase in starch accumulation was observed.70) The lack of bridge.61) If the cysteine residue is exchanged by a serine, the a clear correlation between the measured AGPase activity mutated AGPase is constitutively monomerized and activat- and the accumulation of starch strongly suggests more 6 J. Appl. Glycosci., Vol. 60, No. 1 (2013) complex mechanisms being functional that control reserve of the α-glucan. This limitation is due to the fact that the starch synthesis in the cereal endosperm. action of a transferase on a given α-glucan molecule usually Finally, it should be mentioned that in mesophyll cells of cannot be distinguished (or physically separated) from C3 plants a cytosolic synthesis of ADPglucose by sucrose consecutive reactions that utilize the elongated carbohydrate synthase has been claimed to follow the equation as a substrate. Second, the increasingly complex mixture of sucrose + NDP ↔ NDPglucose + fructose (1) reactants requires a large (and, possibly, unlimited) number where N stands for uridine, adenosine, guanosine, cytidine, of differential equations to comprehensively describe the thymidine or inosine. Therefore, cleavage of sucrose is rates of reiterating glucosyl transfer reactions. Third, the expected to result in the formation of both UDPglucose and force that actually drives the series of reiterating reactions ADPglucose. Because of the subcellular distribution of the needs to be identified. Finally, it remains to be clarified what sucrose synthase isozymes, the proposed mode of ADPglu- actually defines the equilibrium distribution of the α-glucans. cose formation implies a cytosolic origin and a subsequent Recently, statistical thermodynamics has been used as a import into plastids. If so, the proposed alternative path of conceptual framework for the analysis of complex transfer starch biosynthesis would be similar to that in the cereal reactions.78) As a first approach, reactions have been consid- endosperm but would deviate in the assumed reaction that ered that are catalyzed by disproportionating enzymes, such actually forms ADPglucose. In fact, dicotyledonous plant as the plastidial DPE1 (EC 2.4.1.25; GH77) or the cytosolic species, such as Arabidopsis thaliana and Solanum DPE2 (also designated as transglucosidase; EC 2.4.1.25; tumberosum L., possess a putative Brittle1 homologue GH77). During degradation of transitory starch, DPE1 acts located in the inner chloroplast envelope. However, in terms on the plastidial pool of intermediate maltodextrins that are of functionality this protein clearly deviates from the derived from starch granules by direct debranching and monocotyledonous ADPglucose transporter as it mediates β-amylase activity. DPE1 mediates a repetitive oligosaccha- an import of ATP, ADP and AMP but not that of ADPglu- ryl transfer between linear oligoglucans. Each transfer cose. Furthermore, Arabidopsis mutants strongly reduced in reaction results in the release of glucose that, in vivo, is the expression of the Brittle1 homologue possess similar leaf exported into the cytosol by the recently identified glucose starch levels as the wild type.71) The assumed cytosolic transporter.79‒81) DPE2 is essential for the cytosolic metabo- generation of ADPglucose in leaves (reaction 1) has initiated lism of maltose that is derived from starch by the action of a lasting debate that will not be continued here (for details plastidial β-amylases and exported into the cytosol by the see Ref. 72, 73) and references therein). maltose transporter.82,83) In an easily reversible reaction, It is, however, known for decades that isolated intact DPE2 transfers a glucosyl residue from maltose to a chloroplasts perform photosynthesis-driven transitory starch non-reducing end of an oligo- or polyglycan and releases the synthesis. The rate of 14C incorporation into starch is inverse- other glucosyl moiety of the disaccharide (the one carrying ly related to the efflux of labelled carbon into the medium the reducing end) as free glucose. Under in vitro conditions, (which varies depending on the external orthophosphate DPE2 utilizes glycogen as glucosyl acceptor.82) In planta, concentration) and remains essentially constant for up to 30 DPE2 acts on the non-reducing ends of cytosolic heterogly- min. Therefore, the isolated organells are capable of provid- cans that have been described for several higher plant ing ADPglucose for an extended period of time under a wide species.84‒87) To some extent, DPE2 is functionally similar to range of experimental conditions.74‒77) the prokaryotic MalQ that is essential for the bacterial maltose utilization.88) Theoretical aspects of α-glucan chain elongation by repeti- Both DPE1 and DPE2 catalyze a series of easily revers- tive glucosyl transfer reactions. ible reactions which transfer glucosyl or oligosaccharyl Starch biosynthesis proceeds, to a large extent, by a repeti- residues from an oligoglucan donor to a carbohydrate tive glucosyl transfer to the non-reducing end(s) of an acceptor that consists of glucosyl moieties or represents an existing α-glucan. Each glucosyl transfer includes loosening α-glycan. When exclusively acting on carbohydrates of a phosphoester bond in the glucosyl donor and formation consisting of glucose or glucosyl residues only, all reactions of an additional α-1,4-interglucose linkage at the acceptor follow the equation site. The elongated α-glucan chain can immediately be used Gn + Gm ↔ Gn－q + Gm＋q (2) by the same enzyme as a substrate for the next catalytic where Gn and Gm means an α-glucan consisting of n and m process but the concentration of the glucosyl acceptor sites glucosyl residues, respectively. The number of glucosyl remains unchanged. Thus, structural features of α-glucan moieties transferred by a single catalytic cycle is indicated chains that act as glucosyl acceptors are not precisely defined by q = 1,2,3. Each cycle results in a ‘disproportionation’ of and the transferases preferentially recognize distinct the two carbohydrate substrates as the size of one carbohy- submolecular features of the carbohydrates rather than the drate is increased and that of the other one is decreased. entire molecule. Because of the size of the polysaccharides, Thereby, the total number of both the glucosyl residues and the latter is largely inconceivable. A series of consecutive the interglucose bonds remains constant. With respect to net glucosyl transfer reactions results in a massive but strictly enthalpy, all interglucose linkages are essentially equal and, unidirectional growth of the α-glucan chain. therefore, all transfer reactions mediated by DPE1 or DPE2 Several theoretical implications are obvious: First, based do not noticeably change net enthalpy.89) Nevertheless, a on the classical Michaelis-Menten theory a comprehensive ‘disproportionating’ enzyme is capable of massively increas- theoretical analysis of the action of these transferases is ing the degree of polymerization of α-glucans and can hardly possible as, under most conditions, neither the km nor convert maltose or maltodextrins to polysaccharides very the Vmax value can precisely be determined for a distinct size similar to native glycogen provided a branching enzyme is Brust et al.: Starch Synthesizing Reactions and Paths 7 present.90) chain within the glycogen molecule. Therefore, the concept When applying statistical thermodynamics, all oligoglu- of the statistical thermodynamics for the DPE reactions can species are considered to represent different energy described above cannot be directly transferred to the starch- levels each of which is defined by the number of interglu- synthesizing reactions that are mediated by starch synthases cose bonds and of the glucosyl moieties within the respec- or phospho rylases (as well as to glycogen biosynthesis). tive α-glucan. The complex mixture of all carbohydrate Nevertheless, the glucosyl transfer reactions mediated by reactants are described as a statistical ensemble each constit- starch synthases and phosphorylases share remarkable uent of which is attributed to a discrete energy level. Transi- similarities to those of the disproportionating isozymes, tions between the energy levels, as mediated by the dispro- DPE1 and DPE2. This is verified by an example shown in portionating enzymes, are entropy-driven and the equilibrium Fig. 1. A distinct recombinant starch synthase isozyme, SS1 distribution of the glucan mixture can be calculated by from Arabidopsis thaliana (for details see below) was determining the maximum entropy. Model-based predic- incubated with commercially available maltoheptaose tions have been confirmed by time-resolved analyses of the (which contains a small amount of maltohexaose) plus either pattern of the α-glucans by capillary electrophoresis.78) ADPglucose (Fig. 1(A)) or ADP (Fig. 1(B)) and were Evidence has been presented that in vivo entropy-driven incubated for 22 h. At intervals, aliquots of the reaction reactions result into a significant buffering capacity and mixtures were withdrawn and the maltodextrin patterns were thereby stabilize the central carbon metabolism that largely determined by High-Performance Anion Exchange Chroma- consists of variable and multiple intracellular fluxes.78) tography coupled to Pulsed Amperometric Detection Starch biosynthesis largely proceeds by two other glucosyl (HPAEC-PAD). When glucosyl transfer reactions were transferases, starch synthases and phosphorylases. Both initiated by the addition of either ADPglucose or ADP, transferase types differ from the disproportionating (iso－) complexity of the glucan patterns increased with time and in enzymes DPE1 and DPE2 as they do not utilize oligoglucans both reaction mixtures α-glucans were formed whose DPs as carbohydrate donor but rather strictly rely on ADPglucose are below and above that of maltoheptaose serving as starting and glucose 1-phosphate (G1P), respectively. The reactions maltodextrin. When the recombinant starch synthase was mediated by starch synthases release one ADP molecule for heat-inactivated and incubated for 22 h under otherwise each glucosyl residue transferred. Similarly, phosphorylases unchanged conditions, the initial maltodextrin pattern convert G1P to orthophosphate when transferring a glucosyl remained unchanged. moiety to an α-glucan. When using either of the two donors, Thus, active starch synthase mediates a complex series of the glucosyl transfer includes the cleavage of a phosphoryl transfer reactions. When starting the reaction with ADPglu- ester bond that is energetically similar but not equal to the cose (Fig. 1 (A)), ADP is formed with time and, due to the interglucose bond to be formed. Likewise, glycogen biosyn- reversibility of the glucosyl transfer reactions, leads to the thesis by the UDPglucose-dependent glycogen synthase (EC formation of maltodextrins with a DP of less than 7. Similar- 2.4.1.11) cleaves a phosphoryl ester bond when transferring ly, initiation of the incubation by adding ADP plus maltohep- a glucosyl residue to a non-reducing end of an α-glucan taose (in the absence of any exogenous ADPglucose) result-

Fig. 1. Repetitive and reversible glucosyl transfers mediated by recombinant soluble starch synthase (SSI) from Arabidopsis thaliana. In a final volume of 50 µL, 10 µg of purified recombinant SSI was incubated with 0.4 mM (Fig. 1 (A)) and 0.3 mM (Fig. 1 (B)) maltoheptaose (DP 7, containing a small amount of maltohexaose; DP 6), 10 mM Tricin-NaOH (pH 8.0), 5 mM potassium acetate, 0.4 mM EDTA, 100 mM so- dium citrate (pH 8.0), 0.025% (w/v) bovine serum albumin and either 2 mM ADPglucose (Fig. 1 (A)) or 15 mM ADP (Fig. 1 (B)) at 30°C. At in- tervals (as indicated), reaction was terminated by heating for 5 min at 95°C. Subsequently, samples were passed through a 30 kDa filter and the filtrate was lyophilized. The lyophilisate was dissolved in water and was then subjected to an anion exchange chromatography. Finally, maltodex- trins were analyzed by HPAEC-PAD. As a control, the recombinant SSI was heat-inactivated prior to incubation for 22 h. DP 3, 4, 7 and 11 mark the position of maltodextrins consisting of 3, 4, 7 and 11 glucosyl moieties. x, position of an unknown compound that is present in the incubation mixtures but does not change with time. 8 J. Appl. Glycosci., Vol. 60, No. 1 (2013) ed in the formation of α-glucans having both lower and also a more advanced evolutionary state.96) higher degrees of polymerization (DPs). When replacing The five starch synthase classes comprise the granule- functional SSI by the heat-inactivated recombinant enzyme, bound starch synthase (GBSS; one class) and the so-called the maltodextrin pattern remained unchanged over the entire soluble starch synthases that form four distinct classes (SSI incubation period (Figs. 1 (A) and 1 (B), control). In a further to SSIV). GBSS is essentially restricted to the granule bound control experiment, ADPglucose (Fig. 1 (A)) or ADP (Fig. 1 state and, therefore, enzymes of this class cannot be (B)) was omitted under otherwise unchanged conditions. solubilised without disintegration of native starch granules. Incubation for 22 h did not lead to any alteration of the Soluble starch synthases are partitioned between the insolu- maltodextrin pattern indicating that reactions other than the ble (i.e., granule-bound) and the soluble state but factors SSI mediated glucosyl transfers (such as hydrolytic degrada- and/or mechanisms determining partitioning are not yet tions) are undetectable (data not shown). Presumably, the known. When applying conventional protein extraction reversibility of the reactions mediated by starch synthases procedures to plant tissues, only those SS enzymes (and can also be demonstrated when the linear oligoglucans are activities) are covered that reside in the stromal space of the replaced by branched α-glucans. It is reasonable to assume plastids whereas synthases tightly bound to starch granules that the only limitation imposed on the reverse (i.e., ADPglu- are usually lost. cose forming) reactions is due to kinetic properties of the In Fig. 2, the domain structure of five starch synthases respective starch synthase. For the action of the various from Arabidopsis thaliana is given. They share in common synthases, a minimum distance between the non-reducing a core sequence of approximately 60 kDa which forms the ends and the branching point(s) of the α-glucans is needed. C-terminus whereas the N-terminal sequences are largely In the case of maltodextrins, a similar limitation is given by different both in sequence and length. The core sequence the minimum chain length that is required for the catalytic which also exists in prokaryotic and eukaryotic glycogen action of the various starch synthases (see below). synthases (data not shown) is indispensible for the catalytic When taken together, two conclusions are reached from activity and consists of two conserved regions that in the the results shown in Fig. 1: First, the reactions mediated by linear presentation are separated by a more variable linker. starch synthases are easily reversible and, therefore, cause The two conserved regions are often designated as GT5 and even a decreased DP of the maltodextrins. Second, the GT1 domain. The former is frequently found in glucosyl ADP-dependent formation of ADPglucose utilizes initially transferases which follow the retaining mechanism.97) In maltoheptaose as glucosyl donor and thereby starts a series both glycogen and starch synthases, the N-terminal regions of transfer reactions that elongate maltodextrins at the of the GT5 domain carry a conserved pentapeptide, KXGGL. expense of ADPglucose. The increasingly complex pattern The other conserved region, i.e. the GT1 domain, is located of the oligoglucans indicates that the bidirectional glucosyl close to the C-terminus of the entire enzyme and contains a transfer reactions (i.e., both the decrease and the increase in discontinuous series of short motifs that are found in a DPs) largely occur simultaneously but, depending on the functionally heterogeneous group of glycosyl transferases actual composition of the incubation mixture, the rates of the mediating an inverting mode of glycosyl transfer.98) Both polymerizing and depolymerizing reactions vary. GT5 and GT1 appear to be involved in the binding of the glucosyl donor, ADPglucose or UDPglucose. Gene-based multiplicity of starch synthases. The N-terminal regions of the five starch synthase classes Starch synthases (ADP-Glc: α-1,4 glucan α-4-glucosyl are divers and, to some extent, differ even in higher plants. transferases; EC 2.4.1.21) catalyze similar reactions as do In this review, we focus on the sequences of the Arabidopsis prokaryotic and eukaryotic glycogen synthases. Prokaryotic starch synthases. AtGBSS (At1g32900) is the smallest starch glycogen synthases usually rely on ADPglucose (ADP-Glc: synthase mainly consisting of the core sequence and carrying α-1,4 glucan α-4-glucosyl transferases; EC 2.4.1.21) only a short N-terminal extension. All soluble starch synthas- whereas the respective eukaryotic synthases typically utilize es (AtSSI to AtSSIV) possess N-terminal extensions which, UDPglucose as glucosyl donor (UDP-Glc: α-1,4 glucan presumably, affect the catalytic activity of the entire enzyme α-4-glucosyl transferases; EC 2.4.1.11). Evolutionary by intra- and/or intermolecular interactions. The length of implications of the use of ADPglucose and UDPglucose as the extensions varies depending on both the starch synthase glucosyl donors are discussed elsewhere.18) class and the plant species. Among the so-called soluble Starch synthases are classified as members of the glucosyl starch synthases, AtSSI (At5g24300) contains the shortest transfer family 5 (GT5). Based on sequence comparison, at N-terminal extension. An unique feature of AtSSII least five classes of starch synthases exist in green algae and (At3g01180) is a serine-rich region whose function has, higher plants. In some plant species, such as Arabidopsis however, not yet fully been elucidated. Both AtSSIII thaliana, each class is represented by a single gene91) but in (At1g11720) and AtSIV (At4g18240) possess large some unicellular algae as well as in many higher plant N-terminal extensions that are similar in size and exceed that species, a given class may comprise two or even more genes. of the conserved C-terminal core region but largely differ in Multiplicity is caused by early gene or genome duplications, the amino acid sequence. In maize the N-terminal extensions diversification and retainment of the diversified genes.1,18,92‒95) of both classes are significantly smaller as compared to Heterotrophic cells performing a massive starch accumula- Arabidopsis.1) Interestingly, the extension of AtSSIII tion often express genes encoding starch synthase isozymes contains three repeats of a distinct carbohydrate binding that differ from those of photoautotrophic cells. However, module (CBM) that is similar to CBM2599,100) but the AtSSIV duplicated and diversified genes might be lost and, therefore, sequence does not possess any of these repeats (Fig. 2). in principle a lower number of genes per class may reflect The biochemical function of the various starch synthases Brust et al.: Starch Synthesizing Reactions and Paths 9

Fig. 2. Sequence-based scheme of the five starch synthase classes from Arabidopsis thaliana. Granule-bound starch synthase (AtGBSS) and four so-called soluble starch synthase isozymes (AtSSI to AtSSIV) are shown. N-terminus is at the left, the C-terminus at the right side. The conserved sequences of the glycosyl transferase family 5 (GT5, grey) and the glycosyl transferase family 1 (GT1, dark grey) are separated by the linker region (white). At the N-ter- minal region of GT5, the position of the highly conserved motif KXGGL is marked by an arrow. The N-terminal extension of AtSSII contains a serine-rich (Ser-rich) region. In the extension of AtSSIII, three copies of the carbohydrate binding modul of family 25 (CBM25) exist. The N-terminal transit peptides, given in light grey, considerably vary in length. From all starch synthases, the topogenic sequence of the precursor of AtSSIII is the shortest, that of the precursor of AtGBSS the largest. aa, amino acid residues. has been largely deduced from starch-related phenotypes of on α-glucan chains that are located within the clusters of naturally occurring or generated mutants that lack distinct amylopectin, SIII enzymes are believed to elongate the long isozymes (see below). Biosynthesis of amylose is largely chains that connect several clusters of the amylopectin attributed to GBSSI which, however, also mediates the molecule.110,115‒117) formation of extra long chains (ELCs) of amylopectin.101) In Classes SSI to SSIII are evolutionary conserved and terms of evolution of starch metabolism, the latter action appear to generally exist in starch-storing cells but in might be original. In cereals as well as in several dicotyle- glycogen-metabolizing organisms multiplicity of glycogen donous species, gene duplication has lead to two GBSS synthase is not essential. In starch-storing cells, rhodophy- encoding genes possessing a similar intron/exon structure ceae are exceptional as the cytosolic amylopectin biosynthe- but differing in expression patterns.102) Functional redundan- sis appears to proceed by using only a single starch synthase cy or diversity of the GBSS isoforms is uncertain.1,103) In enzyme which utilizes UDPglucose as donor. It implies that vivo, the major GBSS isoform (or the only existing GBSS in red algae the total number of enzymes involved in starch protein) transfers many glucosyl residues from ADPglucose metabolism is lower than in other eukaryotic cells.16,18) to the non-reducing end(s) of a single amylose-like acceptor.1) Currently, the biochemical implications of this exception are Mutants deficient in (the major) GBSS (isoform) synthesize difficult to understand. starch that consists essentially of amylopectin and lacks detectable amounts of amylose (so-called waxy starch). Functional multiplicity of starch synthases. Polymorphism in the GBSS 1 isoform encoding gene The primer dependence of the Arabidopsis starch syntha- (Gbss1) from barley is associated with varying amylose ses was tested by using recombinant enzymes. This approach contents of the starch granules.104) In a granule-free state, was used as not all of the starch synthases can be easily GBSS is, however, capable of acting on maltodextrins and isolated from plant tissue because of low abundancy of the soluble amylopectin but appears to be not able to elongate respective isozymes. glycogen.21,105,106) Each of the four (SSI-IV) recombinant soluble starch SS classes have been preferentially but not exclusively synthases from Arabidopsis was incubated with ADPglu- studied in heterotrophic tissues, such as the endosperm from cose for an extended period of time either in the presence or cereals. SSI, SSII and SSIII all are involved in the elongation the absence of a primer. It should be noted that we did not of α-glucan chains of amylopectin and, therefore, mediate intend to measure the initial rates of the α-glucan elonga- distinct although, to some extent, overlapping functions tion. In principle, all starch synthases require an α-glucan as within the biosynthesis of amylopectin. In rice, SSI elongates glucosyl acceptor but do not differ in the minimum size of short side chains until a degree of polymerization of 8 to 12 the primer used. The latter was determined by adding is reached.107,108) Similarly, in Arabidopsis leaves AtSSI equimolar levels of glucose or maltose. Following a appears to act on short chains from amylopectin.109,110) For prolonged incubation, carbohydrates were analyzed by many plant species, evidence has been presented that SSII High-Performance Anion Exchange Chromatography catalyses the synthesis of intermediate chains lengths (see coupled to Pulsed Amperometric Detection (HPAEC-PAD). Ref. 111) and references therein). Importantly, the chain As revealed by these in vitro assays, all soluble starch lengths formed by SSII enzymes strongly affect physico- synthases are capable of using maltose as glucosyl acceptor chemical features of starch, such as crystalline structure and but the disaccharide cannot be replaced by glucose (Fig. 3). the gelatinization temperature (see Ref. 112‒114) and These results do not exclude that, depending on the DP of references therein). Whilst both the SSI and SSII classes act the primer used, the various SS isozymes differ in the initial 10 J. Appl. Glycosci., Vol. 60, No. 1 (2013) rates of elongation. For several reasons, we did not include synthase isoform, the oligoglucan patterns vary (Fig. 3). branched polyglucans (such as amylopectin). These When maltodextrin primers having a DP of 5 or 7 are applied, α-glucans are polydispers macromolecules and the actual all soluble starch synthases form larger products as compared number of non-reducing ends that can be used by the to maltotriose or maltose acting as initial primers but differ- recombinant starch synthases is difficult to determine. ences between the four classes are obvious: As compared to Furthermore, due to the complex size distribution a precise the other three soluble starch synthases, recombinant AtSSI analysis of the the elongated polyglucan primers is difficult tends to form shorter maltodextrins when DP 5 or 7 is added to achieve. For AtSSIII, amylopectin is reported to be a by as primer. AtSSIV forms products having the highest DPs. It far superior primer as compared to maltodextrins but the should be noted that in HPAEC-PAD the sensitivity of uncertainties inherent to branched polyglucans limit the detection decreases with the size of the target and, therefore, comparability of the various assay mixtures.33) The data only relative differences in the maltodextrin patterns can be shown in Fig. 3 do, however, imply that none of the plant stated. Nevertheless, the differences shown in Fig. 3 are starch synthases appears to be capable of an autogluco- likely to reflect different kinetic properties, such as glucan sylating reaction. Autoglucosylation (and, subsequently, de selectivities of the various starch synthases and/or rates of novo biosynthesis of oligoglucans) has been reported for the glucosyl transfer reactions differing with the size of the glycogen synthase from Agrobacterium tumefaciens.118) glucosyl acceptor. In summary, the data clearly demonstrate Following an extended incubation, all recombinant starch that all soluble synthases mediate reiterating glucosyl synthases from Arabidopsis thaliana converted maltose into transfer reactions, provided a primer having a mimimum DP a complex mixture of oligoglucans but, depending on the of 2 is added, and catalyse a series of related reactions.

Fig. 3. The enzymatic action of the four recombinant soluble starch synthases from Arabidopsis thaliana as revealed by High Performance Anion Exchange Chromatography coupled to Pulsed Amperometric Detection (HPAEC-PAD). The reaction mixtures (final volume of 100 µL each) contained 10 mM Tricin-NaOH pH 8.0, 5 mM potassium acetate, 0.4 mM EDTA, 1 mM dithioerythritol, 3.5 mM ADPglucose, 1.2 mM maltodextrins having a DP ranging from 1 to 7, and 5 µg recombinant purified starch synthase each. Following incubation for approximately 22 h at 30°C, samples were heated for 5 min at 95°C. The heat-treated samples were passed through a 30 kDa cellulose filter and the filtrates were dried by lyophilisation. They were then dissolved in water and analysed by HPAEC-PAD (oligosaccharide mode). SSI to SSIV, soluble starch synthases class I to class IV; DP, degree of polymerization of a linear α-glucan; x, unknown compound that is present in the reaction mixture but does not participate in the glucosyl transfer reactions. The early eluting peaks (1 to 8 min elution time) are due to buffer compounds as well as ADPglucose and glucose (DP 1). Brust et al.: Starch Synthesizing Reactions and Paths 11

Confusing results have been reported for the effect of tion is achieved in either of two gels: When using a separa- various buffer compounds on the availability of ADPglu- tion gel lacking any immobilized carbohydrate, mobility of cose.119) Tris-type buffer (such as Tris, Bicine or Tricine) the proteins (or of protein complexes) is determined by their have been reported to completely prevent starch synthase physicochemical features (such as net charge, size and activity by complexing ADPglucose and, thereby, decreas- shape). However, glycogen-containing gels are also ing the usability of one substrate of the starch synthases, frequently used to resolve soluble starch synthases. During ADPglucose. The addition of glycogen or maltotetraose has affinity electrophoresis,120) interaction between a given been claimed to partially reverse complex formation, thus protein (or protein complex) and the immobilized polyglu- (though incompletely) restoring the availability of ADPglu- can also affects the migration velocity and may even be cose. Following this line, a putatively unprimed activity has dominant. Selectivity of the interaction between the been described for a potato starch synthase but any primer immobile ligand and the target protein can be easily ensured dependence has been attributed to the unavailability of by using a separation gel in which glycogen is replaced by a ADPglucose and considered to be artificial119) (and references non-interacting (and non-stainable) large-size polysaccha- therein). In our experiments, we did not observe such buffer ride, such as dextrans. In the glycogen-containing gel, starch effects. ADPglucose was consumed by recombinant starch synthases have direct access to glucosyl acceptors and, synthases using various buffer (including Tris-type buffers) therefore, the glucan-elongating activity can be monitored and a wide range of experimental conditions (data not when ADPglucose, as the only substrate, is added to the shown). Both time-dependent consumption of ADPglucose incubation mixture. Under otherwise unchanged conditions, and formation of α-glucans as well as the strict primer the starch synthase patterns obtained by electrophoresis or requirement (see Fig. 3) were consistently observed when affinity electrophoresis may be largely different as the applying various analytical procedures (data not shown). presence of the immobilized polyglucan can both increase or Functional analyses of the plant-derived starch synthases decrease resolution (Fig. 4). are often restricted to the soluble synthases (SSI to SSIV). The color of the iodine-stained glucan products that are Synthase patterns are frequently determined by using native formed by the starch synthases in a glycogen-containing or polyacrylamide gel electrophoresis. Electrophoretic separa- glycogen-free separation gel differs: It is more brownish in the presence of glycogen and bluish if soluble starch is added as primer following electrophoresis. The color is, to some extent, indicative for chemical features of the α-glucans formed. Iodine noncovalently interacts with single helices of α-glucan chains but the length of the helix affects the color of the iodine-glucan complex. Long α-glucan chains are stained bluish, shorter chains have a more brownish color. It implies that the mode of action of the starch synthases differs in the two types of polyacrylamide gels. In leaves from Arabidopsis thaliana, all four soluble starch synthases are expressed but the zymograms usually are dominated by AtSSI and AtSSIII whereas the two other starch synthases (AtSSII and AtSSIV) are not detected (Fig. 4). This result was confirmed when all bands of starch synthase activity were identified by comparing the zymograms of the wild type with those of all single knock- out lines that are deficient in one soluble starch synthase (data not shown). It concurs with recently published data on Fig. 4. Native PAGE of Arabidopsis leaf extracts followed the total activity of soluble starch synthases measured in leaf by starch synthase activity staining. extracts. When quantifying the total enzyme activity, the The leaf extracts were prepared from wild type (Ws, left Arabidopsis mutant deficient in AtSSII did contain still lanes) and a single knock-out line lacking functional AtSSI 100% of the starch synthase activity indicating that AtSSII (ssI, right lanes). For native PAGE, a discontinuous electro- contributes little to the total activity measured.110) The phoresis system was used that separates at a moderately alka- reasons for these results are not yet fully understood. When line pH value (for details see Ref. 81)). Migration is from top to bottom. A glycogen-containing (0.2% (w/v) from bovine recombinant starch synthase isozymes were subjected to liver (Sigma-Aldrich, Taufkirchen, Germany); two lanes on the native PAGE, all four isozymes from Arabidopsis formed left) and a glycogen-free (two lines on the right) separation gel iodine-stainable bands. Therefore, during electrophoresis were loaded with the same leaf crude extract (60 µg protein they retained, at least partially, their glucan elongating per line). Following electrophoresis, the slab gels were incu- bated over night at room temperature in mixture containing 50 activity (data not shown). mM Tricine-KOH, pH 8.0, 0.025% (w/v) bovine serum albu- The starch synthase zymograms of Arabidopsis leaf mine, 5 mM dithiothreitol, 2 mM EDTA, 25 mM potassium extracts that are obtained in the presence or absence of acetate and 1 mM ADPG (left). The glycogen-free separation glycogen differed largely (Fig. 4). When glycogen was gel (right) was incubated in the same mixture supplemented included in the separation gel, AtSSI was recovered as a with 0.1% (w/v) soluble starch from potato (Sigma-Aldrich). Following incubation, gels were washed with water and were relatively broad band of activity which was undetectable in then stained with iodine. the AtSSI-deficient mutant. Using the same electrophoretic conditions, AtSSIII was strongly retarded and remained 12 J. Appl. Glycosci., Vol. 60, No. 1 (2013) close to the top of the separation gel. Mobility of AtSSI was bound cofactor whose aldehyde group forms a Schiff-base high in a glycogen-free separation gel but AtSSIII formed linkage to a distinct lysine residue. The 5’-phosphate group three distinct bands of activity located in the middle of the is thought to serve as a general acid-base catalyst and to separation gel. All these bands were absent in the AtSSIII- precisely position the orthophosphate or the anomeric deficient mutant (data not shown). The reasons for the phosphate ester during catalysis.133,134) multiplicity of the AtSSIII protein that are observed in the In the mammalian system, phosphorylase is the essential glycogen-free gel are not yet clear. In principle, it can be due degrading enzyme for the cytosolic glycogen breakdown to covalent modification of the synthase protein, to whose activity is regulated by both covalent modification non-covalent interactions with carbohydrates and/or other and allosteric effects.135,136) By contrast, hydrolysis of proteins. In any case, during affinity electrophoresis these glycogen appears to be restricted to the lysosomal or effects are largely dominated by the interaction of the vacuolar compartment.137,138) Phosphorolysis converts most AtSSIII states with the immobilized glycogen. In the of the glucosyl residues stored as glycogen to G1P and, as mammalian system, glycogen synthase is regulated by compared to hydrolysis, is generally believed to offer two multiple phosphorylation that is mediated by a hormone- advantages: An ATP consuming conversion of the liberated triggered cAMP cascade.11,121) However, less is known about hexose moiety to glucose monophosphate is not required phosphorylation and functional implications of starch and, unlike glucose, G1P is incapable of being rapidly synthases. It should be noted that the zymograms (Fig. 4) are exported from the subcellular site where it is formed. In based on a selective enzyme activity staining but do not mammals distinct isozymes of the glycogen phosphorylase specify the entity performing the field-driven movement and exist that differ in regulatory properties. They are differently containing the starch synthase activity. expressed in various tissues (such as muscle and liver) and In many plant tissues, functional interactions between their expression patterns contribute to tissue-specific distinct isozymes of starch synthases and other starch-related metabolic functions (for review see Ref. 135, 136)). enzymes exist leading to the reversible formation of high For plant phosphorylases, the direction of the net reaction molecular weight heteromeric protein complexes that are has been frequently deduced from the ratio of G1P to relatively stable in cell-free systems and are retained during orthophosphate using a simple thermodynamic calculation several purification procedures.122,123) As an example, and this procedure has been applied even to the in vivo complexes consisting of starch synthase and branching situation. Following this line, the molar concentration of the isoforms124‒126) or of starch synthase and isoamylase glucosyl acceptor remains unchanged and the thermody- isoforms127) have been described. More complex protein namical equilibrium is defined exclusively by the orthophos- associations comprise more than one isozyme of starch phate to G1P ratio. If this value deviates from that of the synthase and branching enzyme plus phosphorylase.122,128) equilibrium, the phosphorylase-mediated net reaction is Formation of these complexes is often restricted to distinct either elongation or phosphorolytic degradation of developmental stages and is likely to offer the advantage that α-glucans. It can, however, easily be demonstrated that net several starch-related biochemical reactions are locally phosphorolysis includes a transient elongation of α-glucans. concentrated and closely coordinated. In many cases, Likewise, net elongation of α-glucans (which occurs when complexes of several starch-related enzymes appear to be the glucose 1-phosphate concentration largely exceeds that based on reversible protein-protein interactions including of orthophosphate) is associated with transient glucan covalent protein modifications122,123) but in some cases degradation. Furthermore, a detailed study on kinetic carbohydrates are likely to play an essential role in the features of the dominant phosphorylase from rice, especially interaction of different proteins.129) For further discussion of of the inhibitory effect that orthophosphate exerts on the the starch-related protein complexes see below. glucan elongating activity, strongly suggest a higher complexity of the in vivo function of the enzyme. Orthophos- Gene-based multiplicity of phosphorylases. phate is reported to inhibit the elongating activity of In the functional state, phosphorylase is typically a phosphorylase when amylopectin is used as primer but the dimeric protein. The enzyme is common in prokaryotic and inhibitory effect is much less effective provided maltodex- eukaryotic cells and was first identified as the activity that trins act as primers. Likewise, ADPglucose appears to massively elongates α-glucan chains and, therefore, was strongly inhibit the phosphorylase and, thereby, might considered to be an α-polyglucan synthesizing enzyme.130,131) restrict the action of the enzyme to distinct metabolic In an easily reversible reaction, phosphorylase repeatedly situations.139) Because of these kinetic properties, at least transfers glucosyl residues from G1P to the non-reducing plastidial phosphorylases might be capable of elongating ends of α-glucan chain chains. short α-glucans even at high orthophosphate and low glucose The catalytic mechanism appears to be conserved in pro- 1-phosphate levels. Furthermore, theoretical approaches to and eukaryotic phosphorylases but carbohydrate specifici- analyse the complete catalytic action of the phosphorylases ties, regulatory mechanisms and in vivo functions are divers are still lacking (see above). despite the fact that all the glycan phosphorylases possess In any case, phosphorolysis of glucans is incomplete as the same E.C. number, 2.4.1.1. Catalysis is complex and the enzyme cannot bypass branching points of glucans or includes formation or cleavage of an α-1,4-interglucose glucan chains140,141) and is incapable of degrading minimum linkage but retaining the α-configuration at C1 of the size maltodextrins (usually maltotetraose142)). glucosyl residue transferred.132) Selectivity of the reaction is For several reasons, the cellular functions of plant ensured by excluding water molecules from the catalytic phosphorylases are even more complex as compared to site. Pyridoxal phosphate is an indispensible and covalently glycogen metabolizing cells: Brust et al.: Starch Synthesizing Reactions and Paths 13

First, two types of phosphorylases are common in plants monas reinhardtii is reported to form G1P from native that occur as plastid and cytosol specific isozymes within the starch.146) The reason for this difference is not yet clear. Even same cell.143) Each type may exist as more than a single in higher plants, the plastidial phosphorylase is capable to protein and, if so, the phosphorylase isoforms residing in the act on native starch granules when incubated at high G1P same compartment appear to possess non-identical kinetic levels which favour net elongation of α-glucan chains. When properties.144‒146) The plastidial and the cytosolic phosphory- recombinant Pho1 from rice was incubated with reserve lase types are designated as Pho1 (or, in Arabidopsis starch granules isolated from potato tubers and [U-14C] G1P, thaliana, PHS1) and Pho2 (in Arabidopsis PHS2), respec- labelling of the native starch was observed.41) Following tively. Based on the affinities towards glycogen, the plasti- solubilisation and enzymatic debranching of the starch, the dial and the cytosolic phosphorylases are occasionally incorporation of 14C into α-glucan chains was determined. named L-type (low affinity) and H-type (high affinity), Interestingly, during short incubation periods (5 min) most respectively. Following translation in the cytosol, Pho1 of the 14C-label was recovered in short chains having a DP of (PHS1) exists as a precursor having a topogenic signal 3 to 4 but with time, 14C-labelling was shifted to higher DPs. (transit peptide) at the N-terminus whereas Pho2 (PHS2) is Labelling of starch was considerably lower as compared to translated as mature monomer which is converted into the in situ experiments using potato tuber discs incubated with functional state by covalently binding pyridoxal phosphate [U-14C]G1P. Therefore, it appears that initially Pho1 transfers and dimerization.147,148) The precursor of Pho1 (PHS1) is glucosyl moieties to very short side chains but this transfer posttranslationally imported into the plastid, processed to reaction occurs rarely. With time, more suitable glucan the mature protein and assembled to a dimer containing two chains are generated and preferred for further glucosyl pyridoxal phosphate groups. The topogenic sequence of the transfer reactions.81) precursor is rapidly degraded by a plastidial peptidasome.149) The cytosolic counterpart, Pho2 (PHS2), possesses an Second, in higher plants the Pho1 (PHS1) type isozymes extremely high affinity towards highly branched polyglu- possess a large, approximately 80 residues comprising cans, such as glycogen. This effect also explains the strong insertion that is located between the C- and the N-terminal retardation in glycogen-containing polyacrylamide gels (see domain and might originate from an intron.150‒152) Using the below). As compared to Pho1 (PHS1), the cytosolic muscle glycogen phosphorylase from rabbit as reference phosphorylase isozymes exhibit an approximately one order sequence, the insertion is located within the glycogen storage of magnitude lower affinity to maltodextrins having a degree site. The amino acid sequence of the insertion is less of polymerization (DP) of 5‒10.142) Based on these features, conserved as compared to the C- and N-terminal domains the cytosolic phosphorylase isozymes resemble the glycogen but consistently possesses an unusually high content of phosphorylase isozymes from mammals. Plant Pho2/ charged residues and the isoelectric point of the insertions is PHS2-type phosphorylases do, however, clearly deviate low ranging from 3.7 to 4.153) For steric reasons, the insertion from glycogen phosphorylases as they are capable of using has been attributed to the distinct glucan specificity of Pho1/ the cytosolic heteroglycans from plant cells as both glucosyl PHS1 isozymes (i.e., their preference of small α-glucans and donor and acceptor. By contrast, the mammalian muscle poor binding to large polyglucan molecules142,154)). However, phosphorylases do not noticeably interact with these polysac- the precise structure of the large insertion has not yet been charides.85) Thus, in a strict sense the cytosolic phosphory- elaborated and their functional implications are still lase isozymes (Pho2/PHS2) are considered to be glycan incompletely understood. The large insertion is absent in all phosphorylases. other phosphorylase sequences, including Pho2/PHS2 and the prokaryotic maltodextrin phosphorylase. Functional multiplicity of phosphorylases. Third, due to compartmentation, Pho2 (PHS2), as opposed At the protein level, the plastidial and the cytosolic to Pho1 (PHS1), does not have direct access to starch and phosphorylases are usually separated by affinity chromatog- starch-derived plastidial α-glucans. Thus, the cytosolic raphy,157) by ion-exchange chromatography158) or by electro- phosphorylases are not immediately involved in the plasti- phoretic techniques.157) Chromatographic methods are dial starch metabolism. frequently applied for preparative separation of phosphory- Fourth, the plastidial and the cytosolic phosphorylase lase isozyme types taking advantage of their large differ- isozymes differ largely in their carbohydrate specificity and, ences in glucan specificities and/or net charges. For the therefore, are unlikely to mediate closely related reactions. following reasons, electrophoretic separations in polyacryl- In their kinetic features, plastidial phosphorylase isozymes amide gels are preferred as analytical tools: resemble the prokaryotic maltodextrin phosphorylase which, First, electrophoretic separation results in a high resolu- however, lacks the large insertion mentioned above.155) They tion when performed under appropriate conditions. Second, have a high affinity towards maltodextrins but interact very functionality of the target isozymes can be easily demonstrat- poorly with glycogen. Maltotetraose is an efficient competi- ed by activity staining which is based on the α-glucan tive inhibitor of the phosphorolytic degradation of maltodex- elongating (i.e., glucose 1-phosphate dependent) or degrad- trins by Pho1 (PHS1) but the cytosolic isozyme, Pho2 ing (orthophosphate dependent) action of phosphorylases.159) (PHS2), exhibits a much lower sensitivity to the tetraose.142) In both cases, separation gels are stained with iodine follow- Fifth, under in vitro conditions purified plastidial ing the enzymatic actions and the positions of active phosphorylase from higher plants appears to be incapable of phosphorylase isoforms are visible as dark zone on an performing phosphorolysis when incubated with native essentially unstained background (glucan elongation) or, transitory starch granules and high orthophosphate levels.156) alternatively, as essentially unstained areas in an stained gel By contrast, the plastidial phosphorylase from Chlamydo- (phosphorolytic degradation). Using the latter approach, 14 J. Appl. Glycosci., Vol. 60, No. 1 (2013) phosphorylases can be distinguished from hydrolases by synthases and all phosphorylases transfer glucosyl residues their strict dependence of orthophosphate. When detected as to the non-reducing end of a given α-glucan (or glucan an α-glucan elongating activity, high molecular weight chain) and, therefore, mediate a strictly unidirectional products are formed which diffuse slowly inside the gel and growth of the chain. During elongation, the number of into the medium permitting an extended incubation and an glucosyl acceptor sites remains unchanged unless the prolonged accumulation of the product of the enzymatic elongated α-glucan or glucan chain is branched. Recently, a reaction. Therefore, detection of the phosphorylase activity close functional interaction and a mutual enhancement of the is highly sensitive. activities of plastidial (Pho1-type) phosphorylase and Third, plastidial (Pho1/PHS1) phosphorylases can be branching isozymes has been reported that has been analyzed easily distinguished from the cytosolic (Pho2/PHS2) under in vitro conditions.129) The interaction between the two isozymes by affinity electrophoresis using a glycogen- enzymes was not affected by the addition of isoamylase containing separation gel. suggesting that the dextrins involved in the functional As a cautionary remark, a disadvantage of some electro- interaction are tightly bound to the phosphorylase and/or the phoresis systems should be mentioned. During anionic branching enzyme. Together with the complex kinetic electrophoresis, phosphorylase isozymes exposed to strong- properties of the phosphorylase139) mentioned above, these ly alkaline conditions tend to be labile. Therefore, electro- data suggest that Pho1-type phosphorylase could be involved phoretic separation performed at pH values of approximate- in one path of the initiation of starch granule formation. This ly 9 leads to a decreased sensitivity of detection and some view is consistent with the expression of plastidial phosphor- phosphorylase isozymes may even remain undetected.157) ylase isozymes during starch accumulation and its induction In the simplest case (as in Arabidopsis thaliana in which by exogenous sugars which favour starch biosynthe- At3g29320 and At3g46970 encodes PHS1 and PHS2, sis.145,153,164,165) However, several mechanisms of the starch respectively), a plant genome encodes for only a single granule initiation are likely to exist as Arabidopsis mutants plastidial and cytoplasmic phosphorylase isozyme. However, constitutively lacking functional PHS1 are capable of the phosphorylase activity pattern of extracts from Arabidop- accumulating starch.166) sis leaves consists of up to four bands as each of the two As opposed to glycogen synthesis, the formation of isozymes is recovered as two distinct zones of activity.160) parallel oriented double helices is essential for the biosyn- The unexpected complexity appears to reflect posttransla- thesis of amylopectin as well as for growth of the entire tional modifications that occur in vivo but neither reasons starch granule. Currently, it is uncertain whether double nor implications are yet fully clear. In other plant species, helices are spontaneously formed and do not require proteins more than a single compartment-specific phosphorylase to be involved. Likewise, the subsequent ordering of double isozyme exists.144,145,153,159,161) helices to form the three-dimensional elementary unit of the More detailed studies have been performed with phospho- A-type or B-type starch allomorph19) may be the result of a rylases from heterotrophic tissues. In roots of sweet potato spontaneous macromolecular self-organization or it may be (Ipomoea batatas L.) the dominant plastidial phosphorylase mediated by proteins. If protein-mediated, the roles of these isozyme, Pho1 has been reported to undergo both covalent proteins would be the rearrangement of non-covalent interac- modification by phosphorylation and partial proteolytic tions of α-glucan chains similar to (but not identical with) degradation.162,163) A distinct serine residue located in the 78 protein functions described by Purich167) more than a decade residues comprising insertion has been identified as main ago. Similar problems are inherent to the biosynthesis of phosphorylation site. In a phosphorylated state, the Pho1 highly ordered polysaccharides, such as cellulose, that are phosphorylase appears to be more susceptible to proteolytic constituents of the cell wall. Cellulose biosynthesis includes degradation.163) Proteolysis is reported to proceed by con- the formation of microfibrils possessing a defined supramo- verting the Pho1 protein into a high molecular weight complex lecular organization.168) containing the 20S proteasome. Within this complex, Pho1 In any case, formation of the amylopectin-related double is partially degraded to a 50 kDa fragment. Under in vitro helical α-glucan chains and their subsequent three- conditions, the partially degraded protein still retains dimensional arrangements require that vicinal chains have a phosphorylase activity although the affinity towards G1P is similar length. Vicinity is achieved by the clustering of slightly altered. When subjected to native polyacrylamide branching points. Mechanistically it includes that single gel electrophoresis, it forms a distinct band of activity.162) It chain branching and elongating reactions are coordinated. is, however, uncertain whether in vivo the partial degrada- Recently, it has been shown that branching isozymes differ tion of Pho1 is process relevant for the normal starch in kinetic properties, especially the length of the glucan metabolism or rather represents a distinct cellular response chains transferred and, therefore, within a single amylopec- to heat stress conditions.162) tin molecule the functions of the various branching isozymes are divers.169) Presumably, correcting reactions are required Protein complexes operating during starch biosynthesis. that remove unwanted branchings which are likely to disturb At least two processes within the biosynthesis of starch double helix formation and their ordering (‘preamylopectin are likely to be based on a close and coordinated interaction processing’). Within a double helix, a similar length of the between several enzyme activities: the initiation (i.e., the de two single chains is difficult to achieve by using a single novo biosynthesis) of starch granules and the formation of glucosyl transferase that acts on a single carbohydrate chain. double helices during the biosynthesis of amylopectin. De Rather, the elongation process taking place at two chains novo biosynthesis of granules includes the elongation of an that form (or will interact to yield) a double helix is likely to α-glucan whose minimum size is maltose (Fig. 3). All starch be somehow coordinated. A series of uncoordinated reactions Brust et al.: Starch Synthesizing Reactions and Paths 15 mediated by individual enzymes that act on single carbohy- Despite the indisputable progress, phenotypical analyses drate chains is clearly insufficient to lead to the precisely of starch-related mutants are complex and often cannot be defined structure of a native starch granule. Therefore, it is directly used for biochemical analyses. In some cases, reasonable to assume that the various coordinating functions phenotypical data may even lead to wrong conclusions. are exerted by heterogeneous protein complexes mentioned The constitutive loss of function within the entire starch above. Formation of these complexes is both reversible and biosynthesis may cause a largely diminished starch level flexible. Reversible protein phosphorylation appears to be an which can be easily detected by a simple iodone staining of important step associated with the assembly of the complex- leaves. As an example, this is the case in Arabidopsis mutants es.122‒124,126,128,170) Flexibility of complex formation is indicat- deficient in the plastidial AGPase,46) the plastidial phospho- ed by mutants that constitutively lack a single protein glucomutase174) or hexose phosphate isomerase.175) This is, constituent of the wild type complex. For these mutants, however, a relatively rare phenotype and, even then, the protein complexes different from those of the wild type were biochemical implications are not always clear (see above). observed.126,128,171) Furthermore, these results clearly support Furthermore, a mutation of a single starch-related gene is the functional need of the heterogeneous protein complexes often associated with an altered expression of many other in starch biosynthesis. In some cases, the close functional genes175,176) and, therefore, it is often uncertain whether the interaction appears to include a physical interaction with phenotypical features observed reflect the mutated target α-glucans. It is likely that a detailed analysis of heteroge- gene or altered gene expression. Higher plant mutants related neous complexes acting as functional unit will strongly to starch biosynthesis possess, however, often a less dramatic improve the current understanding of starch biosynthesis. phenotype as structural properties of the starch granules (such as size and shape of the starch particle or the length of In situ analyses of starch biosynthesis. side chains) are affected rather than the total amount of Carbon fluxes towards starch are often analysed by using starch. In these cases, a detailed analysis of the starch granule radioactive isotopes. Using this approach, the flux of carbon is required.33,110,114,177‒182) For practical reasons, this approach from the operating Calvin cycle to transitory starch has been is not applicable if a large number of mutants need to be demonstrated (see above). For biochemical analyses, screened. labelling in situ studies often are limited by the fact that the Another complication should be mentioned which has fluxes to starch can be demonstrated but the unlying both theoretical consequences and practical implications. biochemical reactions are often difficult to assess. As an Recently, it has been demonstrated that in situ starch synthe- example, recently the import of glucose 1-phosphate into sizing reactions (and, therefore, phenotypical features) are starch accumulating parenchyma cells from potato tubers largely affected by the temperature of the biological system was demonstrated when tubers discs were incubated with under study. Rice mutants deficient in the plastidial labelled G1P.41) Likewise, mesophyll protoplasts from phosphorylase isoform (Pho1) form seeds that are Arabidopsis leaves import G1P and convert the labelled macroscopically indiscernible from the wild type control carbon into assimilatory starch.42) The addition of various when the rice plants were grown at elevated temperatures unlabelled carbohydrates did not interfere with the flux of (18°C or higher). At lower temperatures, however, seed labelled carbon towards starch and, therefore, it was development of the mutants largely deviates from the concluded that G1P is directly imported into the cells and a control.183) As supported by detailed phenotypical analyses, selective transporter of the anomeric glucose monophos- the Pho1-mediated starch synthesizing path is blocked in the phate was postulated. The intracellular processes leading to mutants but can be compensated by other routes at higher the incorporation of labelled carbon into starch can, however, temperatures. As the compensating routes differ in their not directly studied. Permeabilisation of cells is not an temperature dependence from that of the Pho1-mediated approach commonly used in plant biochemistry. Instead, the path, compensation is restricted to a relatively narrow range biological system is frequently simplified by using isolated of temperatures. Experiments recently performed with intact chloroplasts rather than cells or tissues.42) Neverthe- potato tuber discs are fully consistent with this view. Potato less, the actual intraplastidial paths usually cannot be tuber discs were placed a mixture containing labelled G1P analysed. If the assumed path is relatively short and mutants plus unlabelled sucrose or vice versa and incubation was are available that are deficient in a single step within this performed at different temperatures. By using this approach, route, labelling experiments with these mutants are very two carbon fluxes to reserve starch (as mediated by either helpful although only indirect evidence is obtained. Pho1 or AGPase/starch synthases) occur simultaneously but During the last decades, the biochemistry of starch has can be measured separately. At lower temperatures, the strongly benefite from the availability of large sets of G1P-dependent path is functional reaching maximal activity insertion mutants that are deficient in a single starch-related at approximately 20°C. By contrast, the rate of the alterna- gene product. The phenotypical characterization of these tive route strongly increases above 20°C. This effect was mutants as well as the identification of the respective loci in confirmed by in vitro studies using the respective glucosyl the genome lead to the discovery of novel starch-related transferases in a wide range of temperatures.81) proteins that reside inside the plastid, in the cytosol or even Thus it appears that plants have developed a complex in the nucleus. Furthermore, using this approach novel system permitting starch biosynthesis to be functional in a protein functions, such as the maltose transport through the relatively wide range of temperatures. The system appears to plastidial envelope membranes, were identified. These be composed of at least two biosynthetic routes that differ in results have largely altered the current view on starch their temperature dependence and, therefore, redundancy is metabolism (for details see Ref. 106, 172, 173)). given only in a certain range of temperatures. 16 J. Appl. Glycosci., Vol. 60, No. 1 (2013)

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