Kram, Anita Margarita

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Dissertations of the University of Groningen

   University of GroningenStructure and biosynthesis of starch. The localization of starch synthesizing enzymes with theuse of immunoelectron microscopy.Kram, Anita MargaritaIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1995Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Kram, A. M. (1995). Structure and biosynthesis of starch. The localization of starch synthesizing enzymeswith the use of immunoelectron microscopy. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022f the granule does not: granule. Since assayss-Scheperkeuter et al.,semi-crystal l ine starchthe granule. However,granule. Thus, unless,ar the surface of thegrowing starch granuleting that the amyloset starch indicates thatcwie and Greenwood,ylopectin, the crystal l i -ignif icantly co-crystal -trthermore, if amylose:rhelical structure, theparently this is not the) .at the surface of thee in the semi-crystal-:ration of branchingn purif ied SSS-I andrted.SUMMARYStarch comprises two carbohydrate polymers: amylose and amylopectin. Amyloseconsists of a largely linear chain of a-1,4-linked glucose units, whereas amylopectin ishighly branched. Amylopectin is built of short linear chains which are interconnected bycr-1,6-bonds, forming branch points. The branch points are clustered along the molecule.Since the branch points are concentrated in one half of the cluster, the other half compris-es the linear parts of the chains. The molecules are radially oriented in the granule withtheir growing ends pointirrg to the surface. Linear chains of adjacent amylopectinmolecules are crystallized into a superhelical structure.Starch, as found in potato tubers and cereals, is synthesized in amyloplasts, an organellebounded by two membranes. Inside the amyloplasts the starch granules are formed,surrounded by a thin layer of stroma.The final steps in the biosynthesis of starch are catalyzed by ADPglucose pyrophospho-rylase (AGPase), starch synthase and branching enzyme. AGPase catalyzes the formationof ADPglucose. Starch synthase elongates linear chains with the use of glucose residuesfrom ADPglucose. Two forms of starch synthase are distinguished in higher plants: solublestarch synthase (SSS) and granule bound starch synthase (GBSS). Although in essenceboth enzymes catalyze the same reaction, they play distinct roles in starch synthesis. Mostplants contain two soluble starch synthases (SSS-I and SSS-II) and one GBSS. Finally,branching enzyme catalyzes the formation of branch points by breaking one of the 6;.-1,4-bonds in a chain and subsequently linking the severed fragment to a similar chain via ancr-1.6-bond.The formation of amylose is dependent on the presence of GBSS only. Starch mutantslacking GBSS do not form amylose, although the formation of amylopectin is nothampered. Amylopectin is formed by the concerted action of soluble starch synthases andbranching enzymes. Amylose and amylopectin are therefore formed via separate routes.Although amylose is not branched in vivo, branching enzyme is capable of branchingamylose in vitro. This raises the question, therefore, why amylose is not branched ín vivoas well. A possible explanation would be that the synthesis of amylose is spatiallyseparated from the synthesis of amylopectin.Evidently, there are still questions concerning the synthesis of starch in vivo, whichcannot be answered by studying the activity of isolated enzymes in vitro. One of thequestions we tried to solve is where the starch synthesizing enzymes are active, bydetermining their subcellular location. First, the localization of AGPase, branching enzymeand GBSS was studied in potato tubers. In addition the localization of AGPase in barleyendosperm was studied.The enzymes were localized with the use of immunoelectron microscopy (IEM). Withthis technique proteins in tissue sections are labeled with the use of specific antibodies105coupled to colloidal gold as a probe. The resolution of this technique is 25-40 nm, whichenables us to determine the localization of proteins inside the amyloplasts. For IEM notonly specific antibodies are needed, but also ultrathin tissue sections with preservation ofultrastructure as well as antigenicity. A suitable method for the preparation of starchstorage tissue was not yet available and had to be developed. Specific antibodies werekindly provided by others.In chapter 2 the suitability of different methods for the preparation of ultrathin sectionsof starch storage tissue is discussed. Ultrathin sections for electron microscopy (-100 nmin thickness) must withstand the electron beam and the vacuum in the microscope. Thisusually means that the ultrastructure of fresh tissue is stabilized (fixation), dehydrated withan organic solvent (dehydration), and finally embedded in a resin. From the resultant hardblock, containing the tissue, ultrathin sections are then cut. For every tissue and concomi-tant problem under study, a suitable preparation method has to be developed.Starch granules consist largely of carbohydrates. However, for starch no chemicalfixatives are known which are also compatible with IEM. Starch granules swell andshrink, dependent on the amount of water incorporated. During a conventional preparationmethod, as described above, the granules shrink upon dehydration and become embeddedin this state. Upon sectioning, however, the starch granules come into contact with waterand swell again. The swollen granules will then fold and cover other parts of the section.The result can be very rnessy and unsuitable for further research. In developing apreparation method for starch storage tissue we have tried to avoid or minimize shrinkingand swelling of the starch granules.The problems associated with starch are directly related to the size of the starchgranules. Mature potato tubers contain starch granules up to 100-200 trrm. Using smalldeveloping potatoes, containing smaller granules, would therefore be an advantage. Inaddition, we used potato microtuber. Microtubers are in vitro grown tuber of approximate-ly I cm in diameter, containing smaller cells and also smaller starch granules (10-20 pm).These microtubers were very suitable for electron microcopy. Small developing tuber, witha fresh weight of approximately 20 g, could also be used. Tissue of large tubers wasdifficult to embed. Further studies were, therefore, done with small developing tubers andpotato microtubers.For ultrastructural research, fresh tissue was fixed with glutaraldehyde and osmium-tetroxide, dehydrated in a graded series of ethanol and embedded in Epon. The embeddedmaterial had to be trimmed down carefully to avoid pulling starch granules from theblock. Cutting ultrathin sections of material embedded in Epon was relatively easy. Theultrastructure of the cells was well preserved. Subsequently a suitable embedding methodfor IEM was developed. The use of freeze-substitution and cryosectioning was comparedto more conventional embeddins methods. Material was embedded in Lowicryl K4M orLR White.The first slin the t issueding in the remethod is attissue usuallyIt is essentialwater turns idone by ul lRecrystallizatfreezing potaprinciple, t issthen substitudamaged, durfreezing or tlwatery cells rFor cryostliquid nitrogultrathin seclabeled, andin theory, iCof potato tuare probablstay intact aTissue etTissue wasthe temperultrastructustarch gratlargely ovretrieving4"C was :preservedeasy to sebound prcChapteembeddetenzyme {spinach Ithe stron106h\,-que is 25-40 nm, whichryloplasts. For IEM notrns with preservation ofe preparation of starchipecific antibodies wereion of ultrathin sectionsr microscopy (-100 nmin the microscope. Thisration), dehydrated withFrom the resultant hardery t issue and concomi-leveloped.br starch no chemical:ch granules swell and:onventional preparationand become embeddedinto contact with water1er parts of the section.:arch. In developing aI or minimize shrinkinsthe size of the starch)-200 pm. Using smalle be an advantage. Inn tuber of approximate-:h granules (10-20 prm).developing tuber, withre of large tubers wasI developing tubers andaldehyde and osmium-n Epon. The embeddedrch granules from theas relatively easy. Theble embedding methodctioning was comparedI in Lowicryl K4M orThe first step in freeze-substitution is a physical fixation by means of freezing. The icein the tissue is then substituted by an organic solvent (dehydration), followed by embed-ding in the resin of choice. Additional chemical fixation is optional. The advantage of thismethod is an excellent preservation of ultrastructure. Membranes in freeze-substitutedtissue usually appear smooth and turgid. Freezing the samples is, however, a critical step.It is essential that upon freezing the water in the cells is vitrified. Vitrification means thatwater turns into amorphous ice and that no ice crystals are formed. Freezing is thereforedone by ultrarapid freezing at atmospheric pressure or by high-pressure freezing.Recrystallrzation of amorphous ice during the follow-up procedure must be avoided. Forfreezing potato tuber tissue we have used high-pressure freezing. With this method, inprinciple, tissue blocks up to 500 pm in thickness may be vitrified. The frozen tissue wasthen substituted according to different protocols. Unfortunately the tissue was badlydamaged, due to ice crystal formation. Whether crystallization of ice had occurred duringfreezing or the follow-up procedure, is not known. It is probable, however, that the largewatery cells of potato tubers cannot be frozen without freezing-damage.For cryosectioning, fresh tissue is chemically fixed, infused with sucrose, frozen inliquid nitrogen and cut at -100'C. The sucrose works as a cryoprotectant and facilitatesultrathin sectioning at low temperatures. The frozen sections are subsequently thawed,labeled, and embedded on-grid. This method does not involve dehydration and is therefore,in theory, ideal for starch. It proved to be impossible, however, to cut intact cryosectionsof potato tuber tissue. The cells, consisting of amyloplasts surrounded by a large vacuole,are probably unsuitable for cryosectioning. Cryosections of isolated starch granules didstay intact and could be used for ultrastructural research.Tissue embedded at low temperature in Lowicryl K4M resulted in suitable sections.Tissue was fixed in glutaraldehyde, dehydrated in alcohol under progressive lowering ofthe temperature to -30oC, an embedded in Lowicryl K4M at that temperature. Theultrastructure of the cells was well preserved, although the sections were fragile. Thestarch granules tended to detach from the surrounding tissue. This problem could belargely overcome by cutting slightly thicker sections, i.e. 150 nm in thickness, andretrieving the sections quickly form the water surface. Tissue embedded in LR White at4oC was also suitable for ultrathin sectioning. Structural details were, however, less wellpreserved in LR White than in Lowicryl. LR White, on the other hand, is user friendly,easy to section, and the starch does not detach as easily. For localization of starch granulebound proteins, therefore, LR White may be a better choice.Chapter 3 describes the localization of branching enzyme and AGPase in potato tubers,embedded in Lowicryl K4M, with antibodies raised against denatured potato branchingenzyme (kindly supplied by dr. G. Vos-Scheperkeuter and dr. A.S. Ponstein) and nativespinach leaf AGPase (kindly supplied by dr. J. Preiss). Branching enzyme was located inthe stroma of the amyloplasts, concentrated at the surface of the growing starch granule.107AGPase, on the other hand, was evenly distributed over the stroma. Branching enzymewas also found in a membrane-bounded inclusion body in the stroma. The specificlocation of branching enzyme at the granule surface indicates that branching enzyme isactive at that surface.In chapter 4 the localization of GBSS is described. The crude antiserum, raised againstdenatured GBSS (kindly supplied by dr. R. Visser), reacted strongly with GBSS on aWestern blot. The corresponding preimmune serum showed no reaction. On sections ofwild-type tubers a strong labeling of the starch was seen and, to a lesser extent, also of thecytoplasm and the phytoferritin in the stroma. Furthermore, a strong labeling of acrystalline structure in the cytoplasm was found. However, the crude antiserum alsoreacted strongly with the starch granules of amylose-free tubers. The starch of this mutantdoes not contain GBSS and therefore labeling of amylose-free starch, and possibly also ofwild-type starch, could not be specific. To check whether labeling of wild-type starch wasspecific for GBSS, the crude antiserum was purified. Affinity-purified antibodies, specificfor GBSS, did not react with the starch, although labeling in the rest of the cell wassimilar to that found with crude antiserum. This result suggested that the crude antiserumcontained antibodies with affinity for another granlule bound protein or the starch matrix.Since the crude antiserum did not react with other proteins on a Western blot, the secondpossibility was more likely. This hypothesis was then tested by incubating crude antiserumwith amylose-free starch. After pelletting the starch granules, the supernatant was used forlabeling experiments. The results with starch-purified antiserum were comparable to thosefound with affinity-purified antibodies. We concluded, therefore, that the crude antiserumcontained antibodies with affinity for starch as well as GBSS, leading to a false positiveresult. The implications of these results for the work of others is discussed. We have notbeen able to locate GBSS with purified antibodies.In chapter 5 the localization of AGPase in barley endosperm is described. Recentpublications indicate that AGPase in the endosperm of cereals is located in the cytoplasm,although it was assumed that all AGPases are located in plastids. Barley seeds of differentdevelopmental stages were embedded in Epon. It proved that barley seeds harvested l5days after flowering (15 DAF) were good material for electron microscopy. For immuno-localizations 15 DAF barley seeds were embedded in Lowicryl K4M at -30'C with the useof progressive lowering of the temperature. Sections were labeled with antibodies specificfor the small subunit of spinach leaf AGPase (kindly supplied by dr. J Preiss and dr. L.A.Kleczkowski). Label was found in the stroma of amyloplasts in the endosperm but also inthe cytoplasm of endosperm cells. Since the corresponding preimmune serum was notavailable, other controls were sought. For comparison, cells in the pericarp of barley seeds,present in the same section as the endosperm cells, were used as controls. In addition, theantibodies were tested on potato tuber cells. Only in cells of the endosperm, label wasfound in the stroma as well as the cytoplasm. In cells of the pericarp and in potato tubercel ls, labelbarley seedmeans thatamyloplastsIn chaptrusing the rultrastructuthat the sygranule. Tlprobably alAmylopechains witattributed tstarch mutstarch syntlAlthouglGBSS is  nhowever, iglucose isgranule. Tlble to branWe prolis one prtherefore ,synthesisproper grtaffected.108Branching enzymeoma. The specificanching enzyme isrum, raised againstwith GBSS on arn. On sections of'extent, also of the)ng labeling of ade antiserum alsolrch of this mutantnd possibly also ofild{ype starch wasmtibodies, specificst of the cell waste crude antiserumthe starch matrix.n blot, the secondtg crude antiserumatant was used forrmparable to thosere crude antiserumto a false positive;sed. We have notdescribed. Recentin the cytoplasm,seeds of different:eds harvested 15lpy. For immuno-30"C with the usetntibodies specificreiss and dr. L.A.sperm but also ine serum was notp of barley seeds,i. In addition, thesperm, label wasrd in potato tubercells, label was confined to the plastids. We conclude, therefore, that in the endosperm ofbarley seeds AGPase is located in the amyloplast stroma as well as in the cytosol. Thismeans that ADPglucose is probably formed in the cytosol and transported into theamyloplasts.In chapter 6 a preliminary model for the biosynthesis of starch in vivo is presentedusing the results of the localization experiments and our present knowledge of starchultrastructure and biosynthesis. From the localization of branching enzyme we concludethat the synthesis of amylopectin occurs predominantly at the surface of the growinggranule. The soluble starch synthases, providing the substrate for branching enzyme, aÍeprobably also active at the granule surface.Amylopectin has a polymodal chain length distribution, which means that it containschains with specific lengths. The formation of different chain lengths was alwaysattributed to the specific action of branching enzymes. However, recent publications onstarch mutants of the green alga Chlamydomonas reinhardtii, indicate that the solublestarch synthases are responsible for a polymodal chain length distribution.Although we were not able to localize GBSS in the starch granule it is probable thatGBSS is more or less evenly distributed throughout the granule. The synthesis of amylose,however, is likely to be confined to the outer layer of the growing granule, since ADp-glucose is formed in the stroma. GBSS may elongate amylopectin molecules inside thegranule. The granule bound location of GBSS makes the newly formed amvlose inaccessi-ble to branching enzyme.We propose that the synthesis of amylopectin and the crystallization of the linear chainsis one process. The concerted action of the different starch synthesizing enzyme istherefore directly responsible for the formation of starch ultrastructure. Changes in thesynthesis of amylopectin influences the structure of starch. When the formation of aproper granule is disturbed, the synthesis of amylose and amylopectin is probably alsoaffected.109

Structure and biosynthesis of starch. The localization of starch synthesizing enzymes with the use of immunoelectron microscopy.

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Structure and biosynthesis of starch. The localization of starch synthesizing enzymes with the use of immunoelectron microscopy.

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