169

The cloning of a gene responsible for the phosphorylation ofglucans has made it possible to genetically engineer thephosphorylation level of starches in higher plants. Through themanipulation of starch synthase activity, it is now also possible togenetically tailor the chain-length distribution in the amylopectin.Both findings will lead to the development of novel starchesutilized as a renewable resource. The production of fructans on alarge scale can also be envisioned for the near future.

AddressesMax-Planck-Institute of Molecular Plant Physiology, Karl-Liebknecht-Strasse24–25, D-14476 Golm, Germany*e-mail: heyer@mpimp-golm.mpg.de†e-mail: lloyd@mpimp-golm.mpg.de‡e-mail: kossmann@mpimp-golm.mpg.de

Current Opinion in Biotechnology 1999, 10:169–174

http://biomednet.com/elecref/0958166901000169

© Elsevier Science Ltd ISSN 0958-1669

Abbreviations1-SST sucrose:sucrose fructosyltransferase6-SFT sucrose:fructan 6-fructosyltransferaseFFT fructan:fructan fructosyltransferaseGBSS granule-bound SSSS starch synthase

IntroductionStarch and fructans are both polymeric carbohydrates inplants, for which the biosynthesis is sufficiently understoodto allow the bioengineering of their properties, or to engi-neer crops to produce polysaccharides not normally present.

Annually, 20–30 × 106 tons of starch are isolated to serve awide range of industrial applications, such as the coatingof textiles and paper, or as a thickening or gelling agent inthe food industry [1]. Most of the starch is isolated fromcorn, but cassava, potato and wheat are also sources ofstarch. Potato starch is produced mainly in countries of theEuropean Union. For a wide range of applications starchis treated chemically or physically in order to adapt itsproperties to optimally serve its purpose. It is a major chal-lenge to engineer the biosynthesis of starch to make thesetreatments obsolete. One of the first crop plants amenableto genetic transformation was the potato. As it is also amajor source of isolated starch, most of the advances withrespect to the production of modified polymers will bediscussed in the context of this species.

Fructans are of growing interest as functional food ingredi-ents because they are beneficial for human health [2••]. Ashuman enzymes cannot digest fructans, they reach the colonand serve as a substrate for enterobacterial growth. Inulin-containing diets selectively stimulate bifidobacteria andmake them the predominant species [3,4]. Consequently, anincreased fecal content of short-chain fatty acids and a

decreased concentration of of tumor-promoting substances,such as ammonia, is observed [5–7]. Fructans are normallyisolated from crop plants with low agronomic value, such asthe Jerusalem artichoke (Helianthus tuberosus) and chicory.There are, however, other fructan producers which areimportant in human nutrition (e.g. wheat, onion, garlic,banana, artichoke, asparagus; for review, see [8]). The aver-age daily consumption is estimated to be 1–4 g in the UnitedStates and 3–11 g in Europe [2••]. In order to make the pro-duction of fructans economically more feasible, the ability totransfer the biochemical capacity for the synthesis of fruc-tans to plants with higher agronomic value is of majorinterest. The crop of predominant interest is the sugar beet,because the major storage compound of this species issucrose, the direct precursor for fructan biosynthesis.

This review will cover the progress made in the fields ofbioengineering starch and fructan metabolism in higherplants from the end of 1997 until the end of 1998.

Starch: structure and biosynthesisStarch is deposited as granular material in the plastidic com-partment of plant cells, for example, in the amyloplast ofplant storage organs. Starch is constituted of 20–30% of theessentially linear polymer amylose, in which glucose ispolymerized via α-1,4-glycosidic linkages. To a lesserextent, α-1,6-glycosidic linkages (branchpoints) occur(0.1%). 70–80% of the starch is accounted for by amy-lopectin, which has a higher molecular weight than amylose(107–108 Da in contrast to 105–106) and is more frequentlybranched (4–5%). In contrast to amylose, amylopectin issemi-crystalline in nature, which is probably due to the factthat the branchpoints do not occur randomly, but rather thebranches are arranged in clusters allowing the formation ofα-helices [9]. Another unique feature of amylopectin is thepresence of covalently linked phosphate monoesters.These can be linked either to the C3- or C6-position of theglucose monomers, and occur to a higher extent in starchfrom tuberous species, especially in potato starch [10].

The initial step in starch synthesis is the conversion of glu-cose-1-phosphate to ADP-glucose by the enzymeADP-glucose pyrophosphorylase. ADP-glucose serves as asubstrate for the starch synthases. The starch synthases(SSs) catalyze the chain elongation through transferring theglucose moiety from ADP-glucose to α-1,4-glucans. At leastfour isozymes are known and, depending on plant species,different isozymes contribute different amounts to theincorporation of glucose into starch. One isoform, the gran-ule-bound SS (GBSS), however, is known to be responsiblefor the synthesis of amylose. Numerous mutants have beendescribed that accumulate an amylose-free starch due to amutation in the respective gene. The branchpoints intostarch are introduced by branching enzymes. For one iso-form (branching enzyme A) a range of mutations have been

Production of modified polymeric carbohydratesArnd G Heyer*, James R Lloyd† and Jens Kossmann‡

described resulting in the synthesis of high-amylose-con-taining starch. The role of the other isozyme (branchingenzyme B) has yet to be determined. The crystalline natureof amylopectin is probably dependent on the action ofdebranching enzymes. In one model of discontinuous syn-thesis of amylopectin, these are needed to removeexcessive branchpoints introduced by the branchingenzymes [9,11,12]. Certain aspects of starch synthesis arestill unclear. These relate to the questions on how chainelongation is initiated, how granule formation is triggered,and how the incorporation of phosphate monoesters occurs.

Fructans: structure and biosynthesisFructans are a diverse group of polysaccharides that con-tain one or more β-linked fructose units. In the mostprominent structural types, inulin and levan, the fructosechain emerges from the fructose part of a sucrose molecule,proceeding via β-2,1- and β-2,6-linkages, respectively.Besides inulin and levan, the so-called neo-kestose serieshas been described where chain elongation occurs at theglucose portion of sucrose or in both directions [13].Branches occur in all types of fructans, but are more fre-quent in fructans of Gramineae [14,15].

According to a model of fructan synthesis proposed byEdelman and Jefford in 1968 [16], biosynthesis of fructansstarts from two molecules of sucrose. One of them is the

acceptor of a fructosyl residue that is transferred from theother by the action of the enzyme sucrose:sucrose fructo-syltransferase (1-SST). The trisaccharide 1-kestose that isproduced by 1-SST serves — like all higher fructans — asdonor and acceptor of fructosyl residues for the secondenzyme, the fructan:fructan fructosyltransferase (FFT) inthe production of inulin. Levan production is initiated bythe enzyme sucrose:fructan 6-fructosyltransferase (6-SFT)that transfers a fructose residue from sucrose preferential-ly to the F6 position of 1-kestose, but can also use sucroseas the fructosyl acceptor [17]. Because 1-kestose is the pre-ferred fructosyl acceptor for 6-SFT, the enzyme 1-SST isbelieved to be important in the biosynthesis of levan aswell as in inulin accumulation [18].

The first report of the isolation of a plant fructosyltrans-ferase gene was on 6-SFT from barley [19]. This wasfollowed by the cloning of fructan:fructan 6-glucose-fructo-syltransferase from onion [20], which catalyzes the first stepin the production of neo-kestose type fructans typical forspecies of the genus Allium. The current knowledge on theenzymology of fructan synthesis is outlined in Figure 1.

Manipulation of the amylose content inpotato starchThe presence or absence of amylose greatly influences thephysico-chemical properties of starch [21,22]. Mutants

170 Plant biotechnology

Figure 1

Enzymology of fructan synthesis in plants.Sucrose (G-F) is a substrate for 1-kestose(G-F2-1F) and 6-kestose (G-F2-6F) synthesis.The former is catalyzed by 1-SST [39••]. 6-SFTcatalyzes 6-kestose synthesis in barley [19]and can introduce branches into longer chains,but the chain elongating activity that produceslevan, a putative fructan:fructan 6-fructosyltransferase (6-FFT), has not yet beenisolated. For species accumulating unbranchedlevan (e.g. Poa ampla), a 6-SST is postulated[15]. 1-kestose is a substrate for inulinsynthesis, as well as for the fructan neoseries,which requires the enzyme fructan:fructan6-glucose-fructosyltransferase (6-SFT) [17].Chain elongation for inulin and the inulinneoseries is catalyzed by fructan:fructan1-fructosyltransferase (1-FFT) [45•].

2 G-FG-F 2-1F

F2-6G-F

F2- 1F2-6G-F

G-F 2

F 6

G-F 2- 1F

F 6

F2-6G-F

F6

1-SST

6-SFT

1-FFT

6G-FFTG-F

G-F

6-SFT

6-SFT6-SFT

Inulin

Graminan Levan (phlein)

Inulin neoseries

Levan neoseries1-FFT

6-FFT ?

6-FFT ?

6-SST

Current Opinion in Biotechnology

lacking amylose have been described for a long time forspecies such as maize, rice and sorghum [23,24]. Othercomponents of starch, such as the covalently linked phos-phate, which is found in extremely high levels in potatostarch, also determine its properties and uses. The produc-tion of potato starch exclusively composed of amylopectin,therefore, was regarded as being of extreme value.Amylose-free potatoes were developed using both muta-tion induction and genetic engineering [25,26]. In bothcases, GBSS activity is largely abolished, similar to mutantsof other plant species lacking amylose. In the Netherlands,600 ha of these plants were grown in 1996; this area wasincreased to 2000 ha in 1997 [27•]. In the near future, theamylose-free potato starch will be introduced into com-mercialization. Along with the performance of thesegenotypes in breeding programs, the physico-chemicalproperties of these starches have now been intensivelycharacterized [27•,28•,29]. The most prominent changewas observed with respect to the clarity of starch pastesafter storage at low temperatures. In contrast to pastesformed using amylose-containing potato starch, thesepastes stay clear for prolonged storage periods, indicatingthat they could serve as a thickening agent in food appli-cations, where turbidity is unwanted.

In another approach it was intended to increase the amy-lose content of potato starch by downregulating theexpression of the major branching enzyme isozyme(branching enzyme B) [30•]. Surprisingly, no effects on theamylose content were observed. A 50–100% increase of thephosphorous content was observed, however, which mightbe useful for specific applications of potato starch, such asin the wet end during papermaking.

The in vitro synthesis of amyloseThe in vitro synthesis of amylose with isolated starch gran-ules has only been achieved when high concentrations ofmalto-oligosaccharides were added to the incubationmedium with ADP-glucose [31]. In a novel approach usingstarch granules devoid of amylose, but containing GBSSand hence having the capacity to synthesize starch, it waspossible to achieve the synthesis of amylose after pro-longed incubation periods with labeled ADP-glucosewithout the addition of primer molecules [32••]. Theauthors propose that longer linear glucans are first generat-ed by the elongation of the reducing ends of theamylopectin and released subsequently by hydrolysis. Theenzyme responsible for hydrolysis is unknown. If themechanism underlying the cleavage of amylose from amy-lopectin becomes understood, this would enable noveltechnologies for the manipulation of starch structure.

The determination of chain-length distributionin the amylopectinFor two mutants, dull1 in maize and rugosus5 in pea, it waspossible to show that the mutation is associated with theinactivation of one isozyme of SS [33•,34•,35]. This is thefirst time that mutations for SS isozymes other than GBSS

have been identified in higher plants. In the case of dull1,a novel isozyme of SS is affected, whereas in the case ofrugosus5 the SS II isozyme is absent. Both mutations causesimilar effects on the starch content and structure. Thestarch content is greatly lowered, leading to a wrinkledseeded phenotype in the case of the pea mutant, and theapparent amylose content of the starch seems to beincreased. Most interestingly, the amylopectin structurealso is greatly affected, as a shift in the chain-length distri-bution is observed. In both of the mutants, a shift fromintermediate-size glucans towards shorter and extra-longchains is observed. These findings are surprising, becausedifferent classes of starch synthase are affected in each ofthe mutants. It is possible, therefore, to speculate that thedifferent starch synthases interact in the production ofintermediate-size glucans. If this interaction is disturbedby the absence of one isozyme, the production of theseglucans is largely arrested.

The industrial use of these polysaccharides with altered struc-ture is hindered by the decreased starch yield. This might beovercome by the generation of transgenic potato plants pro-ducing starch with similar modifications, which was achievedby the simultaneous reduction of the expression of twoisozymes of SS [36•]. In this case, no drastic reduction of thestarch content in the transgenic plant is observed.

The incorporation of phosphate monoestersinto starchThe presence of relatively high amounts of phosphatemonoesters in potato starch was described almost thirtyyears ago [37]. The biochemical mechanism underlyingthe phosphorylation of starch still remains elusive; howev-er, it was possible to isolate a novel cDNA from potatoencoding a 160 kDa protein that is probably responsible forthe phosphorylation of starch [38••]. Antisense suppressionof this protein leads to a drastic reduction of the phospho-rylation level of potato starch. Conversely, it is possible tostimulate the phosphorylation of glycogen, a polysaccha-ride very similar to amylopectin, if this protein is expressedin Escherichia coli. This protein seems to influence thephosphorylation of α-1,4-glucans at both the C3- and C6-position of the glucose monomers, because both areequally affected in the transgenic plants.

It now seems possible, therefore, to achieve the phospho-rylation of cereal starches through the ectopic expressionof this protein. This is of economic and ecological impor-tance, because maize starch is often chemicallyphosphorylated to make it applicable for the paper indus-try. The respective gene is not unique to solanaceousgenomes, but seems to be universally present in higherplant genomes, as corresponding sequences can be foundin databases from Arabidopsis and rice. The different phos-phorylation levels of the starches derived from varyingstorage organs has to be explained, therefore, either by theexpression levels of this protein, or by the substrate avail-ability of the putative phosphoryl-donor.

Production of modified polymeric carbohydrates Heyer, Lloyd and Kossman 171

Production of fructans in potatoThe first 1-SST gene sequence was published byHellwege et al. [39•] and was isolated from the inulin-pro-ducing artichoke (Cynara scolymus). As the SST-catalyzedreaction that yields 1-kestose and glucose from two mole-cules of sucrose is essentially irreversible, this enzymecould be the controlling factor in fructan synthesis.

Expression of the 1-SST gene in potato led to the accu-mulation of 1-kestose and nystose (glucose–fructose3) andan overall increased content of soluble sugars. It could bedemonstrated that the enzyme is capable of kestose pro-duction even at low substrate concentrations. This is animportant requirement for the production of fructans invegetables and other edible plants under the objective ofraising their nutritional value.

Production of fructans in sugar beetA major advance in agricultural fructan production was thetransformation of sugar beet, this time with a 1-SST geneform Jerusalem artichoke (Helianthus tuberosus) as reportedby Sévenier et al. [40••]. In sugar beet, vacuolar sucrose con-centrations between 350 and 600 mM are idealpreconditions for high-yield fructan production.Nevertheless, a conversion of more than 90% of sucroseinto fructan is remarkable.

The authors claim that the fructo-oligosacchrides pro-duced in the so-called fructan beet could replace sucrose asa sweetener, but detailed studies show that the sweeteningability of the trisaccharide kestose is only 31% as comparedto sucrose and decreases with increasing chain length [41].Besides, the caloric value of fructans is not negligible. Dueto bacterial fermentation and resorption of fermentationproducts, the available energy content of fructans is about4.13 kJ/g (1 kcal/g) reaching 40% of the value for free hex-oses [42]. It is therefore unlikely that fructans can competewith sweeteners such as maltitol (the disaccharide α-1,4-glucosylsorbitol), but longer chain fructans could serve asbulking agents in combination with sweeteners or as a fatreplacement [43].

The expression of both SST and FFT activities thatshould lead to high yields of inulin in an agronomicallyimportant crop plant might be even more attractive thanshort chain fructan synthesis in sugar beet as reported bySévenier et al. [40••]. Expression of both activities intransgenic plants was reported by van der Meer et al. [44•].Interestingly, the expression of SST and FFT in petuniadid not yield the same fructan pattern as found in theendogenous system, Jerusalem artichoke. There are twopossible explanations for this finding. One would implythat the model of fructan synthesis as proposed forJerusalem artichoke by Edelman and Jefford [16] waswrong and additional enzymes are needed to reach thecomplete set of fructans. Alternatively, the differencecould be due to FFT substrate concentrations in thetransgenic plants. It has been shown that Jerusalem arti-

choke FFT prefers short chain fructans as acceptors offructosyl residues and synthesizes longer chains onlywhen threshold concentrations of precursors are achieved.Hellwege et al. [45•] demonstrated that FFT enzymes ofdifferent species show characteristic differences in theirsubstrate affinities when expressed in heterologous sys-tems. It can be speculated, therefore, that expression ofthe artichoke FFT in the ‘fructan beet’ described bySévenier et al. [40••] might be the most promisingapproach towards agricultural fructan production.

Engineering fructan synthesis infructan-storing cropsApproaches to improve quality and/or quantity of fructanby expressing heterologous fructosyltransferases in fruc-tan-producing species have already been demonstrated byVijn et al. [46] and Sprenger et al. [47]. In the first case, thepreviously mentioned 6-glucose-fructosyltransferase ofonion was expressed in chicory, thereby leading to the pro-duction of neo-kestose-type fructans in parallel to inulin[46]. In the second, the barley 6-SFT was expressed inchicory and led to the synthesis of a branched fructan typ-ical for Graminean species [47].

ConclusionsIn the past two years, large amounts of genetically modi-fied potatoes producing an amylose-free starch have beenproduced. This will be the first example of geneticallyengineered starch with superior quality over traditionalstarches entering the markets. It is foreseeable thatstarches with other alterations will follow in the next fewyears. Examples will be starches with an altered amy-lopectin chain length distribution or a modifiedphosphate content, as it is possible now to specificallyengineer these traits. It can also be envisioned that abroad range of novel starches will be produced throughcombining the downregulation or overexpression of sev-eral genes.

It is now also possible to produce fructans in crops withsuperior agronomic performance. After additional engi-neering, this will allow the utilization of fructans as a foodingredient on a larger scale. The cloning of further geneswill enable the production of a larger diversity of fructanswith different structures, such as a FFT catalyzing thepolymerization of α-2,6-linked fructose units.Furthermore, the cloning of a fructanexohydrolase willlead to the engineering of fructan-storing crops to pro-duce fructans with an increased chain length.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Lillford PJ, Morrison A: Structure/function relationship of starchesin food. In Starch Structure and Functionality. Edited by Frazier PJ,Richmond P, Donald AM. Cambridge: The Royal Society of Chemistry;1997:1-8

172 Plant biotechnology

2. Roberfroid MB, Delzenne NM: Dietary fructans. Annu Rev Nutr•• 1998, 18:117-143. This excellent review on the physiological effects and potential health bene-fits of fructans gives a critical overview of what is known on the fermentationof inulin by bacteria in the large intestine and the effects on the host of pro-duction of short-chain fatty acids by these bacteria. Physiological conse-quences on mineral absorption, lipid metabolism and blood glucose andinsulin are summarized, and the potentials in risk reduction of diseases arediscussed. It distinguishes between inulin, oligofructoses produced byhydrolysis of inulin, and synthetic fructans.

3. Gibson GR, Beatty ER, Wang X, Cummings JH: Selectivestimulation of bifidobacteria in the human colon by oligofructoseand inulin. Gastroenterol 1995, 108:975-982.

4. Roberfroid MB, Vanloo JAE, Gibson GR: The bifidogenic nature ofchicory inulin and its hydrolysis products. J Nutr 1998, 128:11-19.

5. Gallagher DD, Stallings WH, Blessing LL, Busta FF, Brady LJ:Probiotics, cecal microflora, and aberrant crypts in the rat colon.J Nutr 1996, 126:1362-1371.

6. Reddy BS, Hamid R, Rao CV: Effect of dietary oligofructose andinulin on colonic preneoplastic aberrant crypt foci inhibition.Carcinogenesis 1997, 18:1371-1374.

7. Rowland IR, Rumney CJ, Coutts JT, Lievense LC: Effect ofBifidobacterium longum and inulin on gut bacterial metabolismand carcinogen-induced aberrant crypt foci in rats. Carcinogenesis1998, 19:281-285.

8. Vanloo J, Coussement P, Deleenheer L, Hoebregs H, Smits G: Onthe presence of inulin and oligofructose as natural ingredients inthe western diet. Crit Rev Food Science Nutr 1995, 35:525-552.

9. Smith AM, Denyer K, Martin C: The synthesis of the starch granule.Annu Rev Plant Physiol Plant Mol Biol 1997, 48:67-87.

10. Jane J, Kasemsuwan T, Chen JF, Juliano BO: Phosphorus in rice andother starches. Cereal Foods World 1996, 41:827-832.

11. Ball S, Guan HP, James M, Myers A, Keeling P, Mouille G, Buleon A,Colonna P, Preiss J: From glycogen to amylopectin: a model forthe biogenesis of the plant starch granule. Cell 1996,86:349-352.

12. Nelson O, Pan D: Starch synthesis in maize endosperms. AnnuRev Plant Physiol Plant Mol Biol 1995, 46:475-496.

13. Ernst MK, Chatterton NJ, Harrison PA, Matitschka G:Characterization of fructan oligomers from species of the genusAllium l. J Plant Physiol 1998, 153:53-60.

14. Carpita NC, Housley TL, Hendrix JE: New features of plant-fructanstructure revealed by methylation analysis and carbon-13 NMRspectroscopy. Carbohydr Res 1991, 217:127-136.

15. Chatterton NJ, Harrison PA: Fructan oligomers in poa ampla. NewPhytol 1997, 136:3-10.

16. Edelman J, Jefford TG: The mechanism of fructosan metabolism inhigher plants as exemplified in Helianthus tuberosus. New Phytol1968, 67:517-531.

17. Duchateau N, Bortlik K, Simmen U, Wiemken A, Bancal P: Sucrose-fructan 6-fructosyltransferase, a key enzyme for diverting carbonfrom sucrose to fructan in barley leaves. Plant Physiol 1995,107:1249-1255.

18. Housley TL, Pollock CJ: The metabolism of fructan in higher plants.In Science and Technology of Fructans. Edited by Suzuki M,Chatterton NJ. Boca Raton: CRC Press; 1993:192-223.

19. Sprenger N, Bortlik K, Brandt A, Boller T, Wiemken A: Purification,cloning, and functional expression of sucrose-fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.Proc Natl Acad Sci USA 1995, 92:11652-11656.

20. Vijn I, Vandijken A, Luscher M, Bos A, Smeets E, WeisbeekI P, WiemkenA, Smeekens S: Cloning of sucrose-sucrose 1-fructosyltransferasefrom onion and synthesis of structurally defined fructan moleculesfrom sucrose. Plant Physiol 1998, 117:1507-1513.

21. Sanders EB, Thompson DB, Boyer CD: Thermal behaviour duringgelatinization and amylopectin fine structure for selected maizegenotypes as expressed in four inbred lines. Cereal Chem 1990,67:594-602.

22. Lii CY, Tsai ML, Tseng KH: Effect of amylose content on therheological property of rice starch. Cereal Chem 1996,73:415-420.

23. Mayer A: Ueber Stärkekörner, welche sich mit Jod rot färben. Berd deutsch bot Ges 1986, 4:337-362. [Title translation: About starchgranules which stain red with iodine.]

24. Collins GN: A new type of Indian corn from China. USDA Bur PlantIndus Bull 1909, 161:1-30.

25. Hovenkamp-Hermelink JHM, Jacobsen E, Ponstein AS, Visser RGF,Vos-Scheperkeuter GH, Bijmolt EW, De Vries JN, Witholt B,Feenstra WJ: Isolation of an amylose-free starch mutant of thepotato (Solanum tuberosum L.). Theor Appl Genet 1987,75:217-221.

26. Visser RGF, Somhorst I, Kuipers GJ, Ruys NJ, Feenstra WJ,Jacobsen E: Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. MolGen Genet 1991, 225:289-296.

27. Visser RGF, Suurs LCJM, Bruinenberg PM, Bleeker I, Jacobsen E:• Comparison between amylose-free and amylose-containing

potato starches. Starch/Staerke 1997, 49:438-443.In this paper, the authors analyze the chemical properties of amylose-freecompared to amylose-containing potato starch. They are able to showthat next to the lack of amylose no other changes occur to the starch, indi-cating that the superior properties of potato starch, such as the low lipidor the high phosphate content, are maintained within the genetically mod-ified starch.

28. Visser RGF, Suurs LCJM, Steeneken PAM, Jacobsen E: Some• physicochemical properties of amylose-free potato starch.

Starch/Staerke 1997, 49:443-448.The authors analyze the physico-chemical properties of amylose-free com-pared to amylose-containing potato starch. It becomes evident that the envi-ronmental benefits will be high if the amylose-free starch is introduced intothe market. Because it displays a reduced viscosity and a increased gel sta-bility and clarity, derivatisations which are normally needed to achieve thisbecome obsolete.

29. Heeres P, Jacobsen E, Visser RGF: Behaviour of geneticallymodified amylose free potato clones as progenitors in a breedingprogram. Euphytica 1997, 98:169-175.

30. Safford R, Jobling SA, Sidebottom CM, Westcott RJ, Cooke D, • Tober KJ, Strongitharm BH, Russell AL, Gidley MJ: Consequences of

antisense RNA inhibition of starch branching enzyme activity onproperties of potato starch. Carbohyd Polym 1998, 35:155-168.

The almost complete suppression of branching enzyme activity down toless than 5% of wild-type levels in transgenic potato tubers is described.Surprisingly, no changes in the amylose content of the starches derivedfrom these transgenic lines are found when compared to wild-type starch.Differences in the gelatinization properties (an increase of up to 5°C in thepeak temperature and viscosity onset temperature) of the different starch-es as determined by differential scanning calorimetry are reported. Theauthors speculate that these changes are correlated with the branchingpattern of the starch that results in changes of double helix lengths.Conversely, it is also possible that the increased phosphate content mea-sured in the genetically modified starches results in the observed elevationof the gelatinization temperature.

31. Denyer K, Clarke B, Hylton C, Tatge H, Smith AM: The elongation ofamylose and amylopectin chains in isolated starch granules.Plant J 1996, 10:1135-1143.

32. Van de Wal M, D´Hulst C, Vincken JP, Buleon A, Visser R, Ball S:•• Amylose is synthesized in vitro by extension and cleavage from

amylopectin. J Biol Chem 1998, 273:22232-22240.For the first time, amylose is synthesized in vitro without the addition ofhigh amounts of maltodextrins. A model that amylose is synthesized byGBSS attached to amylopectin and released by subsequent hydrolysis isproposed. It is speculated that GBSS might harbor a second catalytic(hydrolytic) activity, or that the branching enzymes catalyze an intramolec-ular transglycosylation, or that a hitherto unknown endoamylase entrappedin starch granules releases the amylose from amylopectin. If the model iscorrect, the first possibility seems most probable because a knockoutmutation should abolish both activities of GBSS. This would explain whya mutation in an amylose-releasing activity has never been identified.

33. Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL, • Martin C, Hedley CL, Smith AM: Mutations in the gene encoding

starch synthase II profoundly alter amylopectin structure in peaembryos. Plant Cell 1998, 10:413-426.

The authors conclusively demonstrate that the rugosus5 gene from peaencodes the major starch synthase expressed in pea embryos. Biochemicalstudies indicate that the protein is not detectable in some rugosus5 mutants.Furthermore, one mutated allele was cloned that carries a basepair substi-tution which introduces a stop codon into the open reading frame. The analy-sis of the starch from the mutated plants reveals that the effects are similarto those observed in dull1 mutants from corn.

Production of modified polymeric carbohydrates Heyer, Lloyd and Kossman 173

34. Gao M, Wanat J, Stinard PS, James MG, Myers AM: Characterization• of dull1, a maize gene coding for a novel starch synthase. Plant

Cell 1998, 10:399-412.A portion of the dull1 locus from maize was cloned by transposon taggingand a nearly full-length cDNA was isolated and subsequently sequenced.Sequence alignments indicate that dull1 encodes a novel class of starchsynthase with a predicted molecular mass of 188 kDa. The high molecularmass is unique among the starch synthases known so far.

35. Taylor CB: Synthesizing starch: roles for rugosus5 and dull1. PlantCell 1998, 10:311-314.

36. Lloyd JR, Landschütze V, Kossmann J: Simultaneous antisense• inhibition of two starch synthase isoforms in potato tubers leads

to accumulation of grossly modified amylopectin. Biochem J1999, in press.

The authors describe the effects on the chain length distribution of the amy-lopectin if starch synthase (SS) II and SS III are reduced solely or simultane-ously. A shift from longer chains to shorter chains is observed if SS II and SSIII are suppressed on their own. The effects are only moderate if SS II isreduced in activity and are more pronounced if SS III is reduced, which cor-relates with the amount of activity they each contribute to the total starch syn-thase activity in potato tubers. This is the first report, however, on a change instarch structure that was observed in plants with reduced activity of SS II. IfSS II and SS III are suppressed simultaneously, the effects are similar to othermutants with a defect in a starch synthase isozyme [33•, 34•,35]. An increasein extra long chains is observed and a even more pronounced shift towardschains of the shortest fraction in the amylopectin is observed.

37. Hizukuri S, Tabata S, Nikuni Z: Studies on starch phosphate. Part 1.Estimation of glucose-6-phospate residues in starch and thepresence of other bound phosphate(s). Starch/Staerke 1970,10:338-343.

38. Lorberth R, Ritte G, Willmitzer L, Kossmann J: Inhibition of a starch•• granule-bound protein leads to modified starch and repression of

cold sweetening. Nat Biotechnol 1998, 16:473-477.The authors describe the cloning of a novel cDNA that was isolated on thebasis that its product binds to potato starch granules. Using antisense sup-pression, they are able to show that this protein is probably responsible forthe phosphorylation of starch. Intriguingly, there were side effects observedin the potato plants with a lowered level of starch phosphorylation. The trans-genic plants display a starch excess phenotype in leaves, as they do notdegrade their starch even after prolonged periods of darkness. Similarly, thedegradation of starch and the accumulation of reducing sugars during coldstorage of potato tubers are largely eliminated. Both effects could be inter-preted as non-degradability of the non-phosphorylated starch in vegetativeplant organs; however, experiments have to be undertaken to prove thisassumption. These observations are both of agronomic and economic impor-tance. The accumulation of starch in green photosynthesizing organs ofplants makes them more suitable as a fodder crop for ruminants, because itultimately increases the C:N ratio of the substrate and prevents `bloat´. Theaccumulation of reducing sugars in potato tubers is a long-standing problemin the potato processing industry, as it causes the Maillard reaction to occurleading to an undesired dark coloring of fried products.

39. Hellwege EM, Gritscher D, Willmitzer L, Heyer AG: Transgenic• potato tubers accumulate high levels of 1-kestose and nystose —

functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossomdiscs. Plant J 1997, 12:1057-1065.

This paper gives the first report of cloning and heterologous expression of asucrose:sucrose1-fructosyltransferase (1-SST) cDNA. The cDNA wascloned from an artichoke blossom disk library by homology to thesucrose:fructan 6-fructosyltransferase from barley that was cloned bySprenger and co-workers in 1995 [19]. The cDNA was expressed tran-siently in tobacco protoplasts and also in transgenic potato. As a character-

istic difference, the enzyme produced only 1-kestose in vitro, but also thenext higher homologs nystose and fructosyl-nystose in vivo. In contrast toexperiences with bacterial fructosyltransferases, expression of the artichoke1-SST gene in potato under the control of the constitutive cauliflower mosa-ic virus 35S promoter did not cause a visible phenotype and did not influ-ence tuber yield.

40. Sévenier R, Hall RD, van der Meer IM, Hakkert HJC, van Tunen AJ,•• Koops AJ: High level fructan accumulation in a transgenic sugar

beet. Nat Biotechnol 1998, 16:843-846. This paper describes the transformation of sugar beet with a cDNA of 1-SSTfrom Jerusalem artichoke. Sugar beet is highly recalcitrant to genetic modifi-cation, and therefore a special protocol for the transformation of protoplastshas been developed. Expression of the 1-SST cDNA under the control of theconstitutive cauliflower mosaic virus 35S promoter leads to a nearly com-plete conversion of sucrose into short chain oligofructoses. The plants showno visible phenotype and have no yield reduction. The high conversion ratefor sucrose is accompanied by an eightfold increase in glucose, which mightraise problems in isolation and purification of fructan.

41. Yun JW: Fructooligosaccharides — occurrence, preparation, andapplication. Enzyme Microb Technol 1996, 19:107-117.

42. Roberfroid M, Gibson GR, Delzenne N: The biochemistry ofoligofructose, an approach to calculate its caloric value. Nutr Rev1993, 51:137-146.

43. Rapaille A, Gonze M, Vanderschueren F: Formulating sugar-freechocolate products with maltitol. Food Technol 1995, 49:51-54.

44. van der Meer IM, Koops AJ, Hakkert JC, van Tunen AJ: Cloning of the• fructan biosynthesis pathway of Jerusalem artichoke. Plant J

1998, 15:489-500.The paper describes transgenic petunia plants that express both fructosyl-transferases from the Jerusalem artichoke that are believed to be necessaryfor inulin production: 1-SST and 1-FFT. Only in senescent leaves, fructansup to GF25 could be detected, whereas the maximal degree of polymeriza-tion of inulin is about 50 hexose units in Jerusalem artichoke. Additionally, theratios of the fructan levels ranging from GF2 to GF9 were different in thepetunia as compared to Helianthus tuberosus.

45. Hellwege EM, Raap M, Gritscher D, Willmitzer L, Heyer AG:• Differences in chain length distribution of inulin from Cynara

scolymus and Helianthus tuberosus are reflected in a transientplant expression system using the respective 1-FFT cDNAs. FEBSLett 1998, 427:25-28.

The authors compared two fructan:fructan 1-fructosyltransferase (1-FFT)cDNAs from different species by transient expression in tobacco protoplas-ts. They expressed an FFT cDNA of Jerusalem artichoke and artichoke andfound that the latter enzyme was poorly active on 1-kestose as substrate ascompared to the Jerusalem artichoke counterpart. Nevertheless, it producedlonger chain fructans than the other when incubated with a mixture of medi-um length fructo-oligosaccharides (GF2 to GF4). The results can explain thedifference in fructan patterns found among different species of theAsteraceae. It is also obvious that the 1-FFT of artichoke offers advantagesfor the production of fructans in transgenic plants because of the high meandegree of polymerization of inulin produced by this enzyme.

46. Vijn I, Vandijken A, Sprenger N, Van Dun K, Weisbeek P, Wiemken A,Smeekens S: Fructan of the inulin neoseries is synthesized intransgenic chicory plants (Cichorium intybus l) harbouring onion(Allium cepa l) fructan-fructan 6g-fructosyltransferase. Plant J1997, 11:387-398.

47. Sprenger N, Schellenbaum L, van Dun K, Boller T, Wiemken A:Fructan synthesis in transgenic tobacco and chicory plantsexpressing barley sucrose-fructan 6-fructosyltransferase. FEBSLett 1997, 400:355-358.

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