Modificados en Plantas Transgénicas

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Summary

In recent years the focus of interest in theproduction of transgenic plants has movedfrom agronomically important features likeherbicide resistance or male sterility to theso-called output traits, which affect thequality of the harvested material.Besidemodifications in fatty acid composition [1]and protein quality [2,3],tailoring of car-bohydrates is one of the main fields formolecular breeding programs.As carbohy-drates are not only important storage com-pounds but the primary energy fixation pro-ducts and main transport metabolites,threedifferent aspects have to be considered incarbohydrate modification research:(1.) at-tempts to increase primary production,(2.)analysis and modification of allocation ofphotosynthates,and (3.) engineering of sto-rage carbohydrates.The scope of this articleis to give some examples on recent advancesin the field of carbohydrate modification and

then to present in more detail the transfer ofa new metabolic pathway to transgenic po-tato that leads to the production of inulin,afructose-based carbohydrate polymer thatincreases nutritional quality of the transge-nic plants.

Key words

Carbohydrates Inulin Fructan Geneticallymodified potato Nutritional quality

Introduction

Primary production

In most agricultural habitats, biomassproduction is not limited at the level ofmetabolism but by abiotic factors ofwhich water is the most important one.Biotechnological concepts to optimizeproduction are therefore of limited val-ue. Improving salt tolerance could be-come an important goal, because irriga-tion of farm land is often accompaniedby increased salt concentrations [4].Only under certain conditions, e.g. ele-vated carbon dioxide or low tempera-tures,primary production could be lim-ited because of the accumulation of end-products of photosynthesis.

One of the transgenic approaches toprevent end-product accumulation in-volved heterologous expression of Su-crose-Phosphate Synthase (SPS) frommaize in tomato plants to improve par-titioning of photoassimilates into thetransport sugar that is delivered to thefruits [5]. Despite the observation ofchanges in the flowering time of thetransgenic plants, an effect on yieldcould not unequivocally be demonstrat-ed [6] and seemed strongly dependenton growth conditions. In a recent reviewon biotechnological attempts to increaseyield of crop plants, Herbers andSonnewald argued that because of regu-latory networks acting on carbohydrate

metabolism, the alteration of single en-zyme activities could principally be in-adequate to improve the storage of pho-toassimilates in transgenic plants [7].

Allocation of photosynthates

Heterotrophic organs,i.e.non-green tis-sues like tubers or roots,as well as grow-ing leaves that consume more photosyn-thates than they produce are defined as,,sinks in contrast to the mature leavesand stem, which are net exporters of as-similates and are therefore termed,,sources. The transport of carbohy-drates from sources to sinks is not onlydepending on the productive capacity ofsources, but also on the competitiveability of sink organs to attract assimi-lates,which is defined as ,,sink strength[7]. The sink strength of harvestable or-gan is of prime importance, as it deter-mines the proportion of biomass that isaccessible to human consumption, inother words the ,,harvest index. Har-vest index has been the focus of classi-cal breeding for long time, and majoradvances in the 1970s are rememberedas the ,,green revolution.

Leitthema: Perspektiven zur Entwicklung der ,,Grnen GentechnikBundesgesundheitsbl -Gesundheitsforsch - Gesundheitsschutz2000 43:9498 Springer-Verlag 2000

A.G.HeyerMax Planck Institute,Golm

Production of modified

carbohydrates in transgenicplants

Arnd G.Heyer

Max Planck Institute of Molecular Plant

Physiology, Am Mhlenberg 4,D-14476 Golm

(Germany),E-mail:heyer@mpimp-

golm.mpg.de


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Bisherige Versuche, Ernteertrgedurch gentechnische Vernderungen

zu steigern, haben noch nicht zu ver-wertbaren Ergebnissen gefhrt.

The contribution of biotechnology is notyet significant in this field, and the crit-

ical remarks of Herbers and Sonnewaldconcerning the restrictions of singlegene transfer are in fact targeted at sinkstrength modification in general.Never-theless,there are examples of successfulalteration of carbohydrate allocation intransgenic plants.One of them was giv-en by Sonnewald and colleagues: expres-sion of yeast invertase as an apoplasticenzyme in tubers of potato leads to aprofound change in tuber size and num-ber [8]. The transgenic plants produceless tubers with an increased size of up

to 1 kg. The total yield is not changedand therefore total sink capacity of thetubers is unchanged. Nevertheless, theability of a single tuber to attract assim-ilates seems strongly increased.

Storage carbohydrates

Starch is by far the most important stor-age carbohydrate isolated from plants.2030106 tons of starch are producedannually, serving a wide range of indus-trial applications such as coating of tex-tiles and paper,or as a thickening or gel-ling agent in the food industry. Most ofthe starch is isolated from corn, but alsocassava,potato,and wheat are sources ofstarch. While there are still open ques-tions, the biosynthesis of starch is suffi-ciently understood to allow bioengi-neering of plants for optimized produc-tion of the raw material (for recent re-view, s. [9]).

Four different starch synthases areinvolved in starch synthesis,one of thembeing tightly bound to the growingstarch granule,the others are soluble en-zymes [10]. The a-(1,4)-glucan chainproduced by the synthases is subject tomodification by branching enzymes thatintroduce a-(1,6) branches that charac-terize the amylopectin portion of starch.Besides branching enzymes, debran-ching enzyme and disproportioning en-zyme as well as starch phosphorylasesand the degradative amylases are in-volved in the metabolism of starch. Allthese enzymes have been characterizedand the genes are cloned from differentspecies.By means of antisense reduction

of enzyme activity of single or multipleenzymes, a set of starches with differentproperties has been generated. Reduc-tion of the activity of granule boundstarch synthase leads to an amylose-freestarch that has excellent co-polymeriza-tion capability and is already a very in-

teresting raw material for uses as foodadditive in production of soups andsauces [11].

The antisense repression of the so-called R1-enzyme, which is involved instarch modification in a way not yetcompletely understood, yields a starchthat has a strongly reduced phosphatecontent and an altered gelation capacity[12],which might make it superior in theproduction of films.

Die Strkezusammensetzung vonPflanzen kann bereits gentechnischverndert werden und erffnet neueund verbesserte Anwendungs- undVerarbeitungsmglichkeiten.

A combination of different antisense-in-hibition strategies will lead to the pro-duction of different starches with vary-ing physico-chemical properties that fitthe consumers needs.

Fructans are soluble polymeric car-bohydrates composed of fructosyl units(for review,see [13]).During recent yearsthey attracted growing interest as func-tional food ingredients because of ben-eficial effects on human health [14]. Inour own research work on fructan me-tabolism, we focused on artichoke (Cy-nara scolymus) as an example of inulinsynthesis in plants, and on Streptococcusmutans, which is the only bacterial spe-cies known so far that produces a highmolecular weight inulin.

Results and discussion

Plant oligomeric fructans

According to a model of fructan synthe-sis proposed by Edelman and Jefford[15], at least two enzymes are needed tosynthesize inulin type fructans, whichare the most common fructans of dicot-yledonous plants. The first enzyme inthe pathway should be a sucrose-depen-dent sucrose fructosyltransferase (SST)that produces the trisaccharide 1-kesto-se.1-Kestose would then be the substratefor the fructan-dependent fructan fruc-tosyltransferase (FFT) that catalyzes a

Bundesgesundheitsbl - Gesundheitsforsch - Gesundheitsschutz 22000 95

A.G.Heyer

Produktion modifizierter Kohlenhydratein transgenen Pflanzen

Zusammenfassung

In den vergangenen Jahren hat sich derSchwerpunkt der Arbeiten mit transgenenPflanzen gewandelt.Anstelle der agrono-misch wichtigen Eigenschaften wie Herbi-zidtoleranz oder mnnliche Sterilitt sind diesogenannten ,,output traitsins Zentrum ge-rckt,die die Qualitt der erntebaren Pflan-zenteile betreffen.Neben einer Vernderungder Fettsurezusammensetzung [1] oder derProteinqualitt [2,3],ist die Optimierungvon Kohlenhydraten ein Hauptgebiet dermolekularbiologischen Bearbeitung vonPflanzen.Da Kohlenhydrate nicht nur wichti-ge Speicherstoffe,sondern auch die Primr-produkte der Photosynthese und wichtigeTransportmetabolite sind,mssen dreiAspekte der Vernderung von Kohlenhydra-ten beachtet werden:1) Versuche zur Steige-rung der Primrproduktion;2) Analyse undModifikation von sink/source Wechselwir-kungen; 3) Bearbeitung von Speicherkohlen-hydraten.In diesem Beitrag sollen Beispielefr Fortschritte auf dem Gebiet der Koh-lenhydratmodifikation gegeben und einge-hender ein Ansatz vorgestellt werden,beidem ein neuer Stoffwechselweg in Kartoffel-pflanzen etabliert wurde,der zur Produktionvon Inulin,einem Kohlenhydratpolymer aufFruktosebasis,fhrt und den ernhrungs-physiologischen Wert der Pflanze steigernkann.

Schlsselwrter

Kohlenhydrate Inulin Fruktan Gen-technisch vernderte Kartoffel Ernhrungs-physiologischer Wert

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Leitthema: Perspektiven zur Entwicklung der ,,Grnen Gentechnik


reversible chain elongation of fructanmolecules. To find out whether the mo-del is sufficient to describe inulin syn-thesis, we used the SST and FFT genesfrom artichoke to produce transgenicpotato plants expressing either one orboth genes and analyzed the carbohy-drate composition of these plants.

Plants expressing the 1-SST geneonly under the control of the constitu-tive 35S promoter of the cauliflower mo-saic virus (CaMV) accumulated oligo-fructans in leaves and tubers [16]. Byhigh performance liquid chromatogra-phy (HPLC) we could show synthesis ofthe trisaccharide 1-kestose (GF2),the tet-rasaccharide 1,1-nystose (GF3) and thepentasaccharide (GF4) in transgenicplants, but also higher inulin homologsup to GF6 could be detected in somelines (Fig. 1). The production of mole-cules of a higher degree of polymeriza-tion (DP) than 3 had already been re-ported for purified SST, but a DP ofmore than 5 was never observed in vitro.Whether the capability of SST to pro-duce higher DP oligomers is of physio-logical relevance or just a lack of sub-strate specificity has yet to be demon-strated, but it seems very interesting,considering that inulin oligomers are of-ten discussed as relevant in responses ofplants to abiotic stress like cold anddrought [17].

Production of oligo-fructans hadno measurable influence on other solu-ble sugars in leaf tissue: no differencesin glucose, fructose and sucrose content

could be observed between wild typeand transgenic plants. In tubers, 1-kes-tose was the most abundant oligosac-charide, and the sucrose concentrationwas again not significantly altered in thetransgenic lines. In contrast to leaves,transgenic tubers contained significant-

ly higher amounts of glucose, whereasthe oligo-fructan accumulation had nosignificant influence on the starch con-tent. This result is astonishing for tworeasons. First, it is in contrast to the re-sults of Sevenier et al., who reported adramatic reduction of sucrose concen-tration in SST-expressing sugar beet[18]. In tap roots the sucrose leveldropped down to about 5%. Second, assucrose is unchanged in the potato tu-bers and fructan oligomers accumulate,the total amount of soluble sugars is el-

evated more than three-fold without anyadverse effect on the phenotype of theplants. For potato plants that showed astrong increase in soluble sugar concen-tration of tubers because of a repressionof starch synthesis [19], a strong reduc-tion of tuber size was reported that coin-cided with an increased tuber numberand an overall loss of biomass, whichwas not observed for the fructan potato.

Plant inulin

The next and very important questionis,whether additional expression of FFTin the SST lines would lead to the pro-duction of the full set of inulin mole-

cules naturally occurring in the plantthat was taken as a source for the fruc-tosyltransferase genes. By comparingthe FFT genes of Jerusalem artichoke(Helianthus tuberosus) and artichoke(Cynara scolymus) in a transient expres-sion system (tobacco protoplasts), we

obtained evidence that substrate prefer-ences of this enzyme might define thechain length distribution of the inulinsynthesized [20].The 1-FFT of artichokehas a low affinity to 1-kestose as fruc-tosyl acceptor in vitro and prefers oligo-mers of higher DP, whereas the homolo-gous enzyme of Helianthus tuberosusproduces a set of inulin oligomers of aDP of up to 10, more or less irrespectiveof the substrate offered.

Expression of SST and FFT genes ofartichoke (Cynara scolymus) in trans-

genic potato yielded results that strong-ly support the model of Edelman andJefford. Analysis of the fructan compo-sition of transgenic tubers by HPLC re-vealed a pattern that was undistinguish-able from that of artichoke (Fig. 2) andgel permeation chromatography (GPC)of extracts taken from artichoke root,wild type potato tubers and transgenictubers confirmed that the inulin patternof artichoke and transgenic potato isprincipally the same with inulin mole-cules of a maximum size of more than100 units (data not shown).

Like for 1-SST expressing transgenicplants,activity of both fructosyltransfer-ases has no significant impact on other

Fig.1HPLC chromatogram of fructose oligo-mers extracted from leaves of transgenic pota-to plants expressing 1-SST from artichoke.Theanalysis was performed as described in [16].Numbers indicate DP of Sucrose (2) and fructo-oligosaccharides (37).X-axis: time in minutes.Y-axis:relative detector signal in nCoulomb

Fig.2 Expression of 1-SST and 1-FFT in transgenic potato: HPLC analysis of extracts from artichokeroot (B) and transgenic potato tubers expressing artichoke fructosyltransferase genes (A).The analy-sis was performed as described in [16].X-axis: time in minutes.Y-axis:relative detector signal innCoulomb

A

B


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It is obvious that such a polymer wouldbe a very interesting raw material for in-dustrial purposes. Unfortunately all at-tempts to produce the material intransgenic plants failed, because theyield,reaching only 0.1% of the dry mat-ter of potato tubers,was too low for eco-

nomic production. Nevertheless, theremight still be a chance for high molecu-lar weight inulin production, since firstattempts to immobilize the isolated en-zyme on membranes in order to use it ina bio-reactor like procedure have provenfeasible [31].

Outlook

It seems clear that modification of car-bohydrates in transgenic plants has al-ready become a valuable tool to produce

high-quality food, feed, and industrialraw material, whereas convincing con-cepts for increasing plant yield have notyet been presented. The molecular toolsto modify plants in order to produce tai-lored carbohydrates like starch or fruc-tan are available,but problems concern-ing the extraction and marketing of themany different products at reasonablecosts still have to be solved. Innovativeideas as well as the clear decision to pro-mote renewable resources to make themcompetitive with petrochemical prod-ucts are needed to make the next stepsin molecular breeding.

References

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Plant desaturases: harvesting the fat ofthe land. Current Opinion in Plant Biology2:123127

2. Bagga S,Adams H,Kemp JD,Senguptagopalan

C (1995) Accumulation of 15-Kilodaltonzein in novel protein bodies in transgenictobacco. Plant Physiology 107:1323

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Imamura J (1995) Improvement in thequality of seed storage protein by trans-formation of Brassica napus with anantisense gene for cruciferin. Theoretical &Applied Genetics 91:627631

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Bundesgesundheitsbl - Gesundheitsforsch - Gesundheitsschutz 22000 97

soluble sugars (Table 1). The inulin con-tent is about 3-fold higher than sucrose,which seems high regarding the inulinas additional soluble sugar,but it is low

considering starch, which is 20-foldhigher concentrated.The transgenic po-tato lines display a reduction in starchcontent that lies within natural variationbut is consistently found amongtransgenic lines. Therefore we concludethat fructan synthesis does not lead toan increase in total carbohydrate contentof the tubers.The inulin does not estab-lish an additional,but an alternative sinkfor storage carbohydrates.

Mit ,,prbiotischen Nahrungsmittelnwie Kartoffeln, die einen erhhtenInulingehalt aufweisen, knnte dasWachstum probiotischer Bakterien imDickdarm stimuliert werden. Damitwrden vermehrt kurzkettige Fettsu-ren produziert und die Aktivittreduktiver Enzyme und krebsfrdern-der Substanzen vermindert.

The inulin potatoes can be regarded as,,pre-biotic foodthat should have a pos-itive effect in human nutrition becauseof a stimulation of ,,pro-bioticbacteri-al species in the colon.Inulin containingdiets selectively stimulate bifidobacteriaand make them the predominant bacte-rial species in the large intestine [21].Bifidobacteria are classified ,,pro-biot-ic, because their presence in the coloncause an increase in concentration ofshort-chain fatty acids and a decrease ofthe activity of reductive enzymes and oftumor promoting substances like am-monia [22, 23, 24]. Fructans are naturalfood ingredients being present in wheat,onion, garlic, bananas, asparagus, andothers (for Review, see [25]). The aver-

age daily consumption is estimated to be14 g in the United States and 311 g inEurope [14].In order to increase the dai-ly intake up to the physiologically rele-

vant threshold of 10 to 15 g/day,it wouldbe useful to transfer the fructan synthe-sis capacity to other crop plants like po-tato or vegetables.

Bacterial high molecular weightinulin

Fructans synthesized by bacteria aregenerally high molecular weight poly-mers and in almost all cases of the levantype and show a high proportion ofbranching [26]. The only bacterial spe-cies known so far that produces an inu-lin type fructan is Streptococcus mutans,a human pathogen involved in dentalcaries [27]. This inulin, despite having amolecular mass of several million, ishighly water soluble and less branchedthan levans [28, 29]. A water-solublepolymer of high molecular weight couldbe an interesting raw material for indus-trial purposes such as production of sur-factants. We have expressed the Strepto-coccus fructosyltransferase gene as a fu-sion protein in Escherichia coli, and withthe purified protein we performed invitro synthesis of high molecular weightinulin to study the enzymatic propertiesof the fructosyltransferase.

The enzyme displayed very interest-ing kinetic features.Only for the first sixhours of reaction time we could measurean increase of the polymer synthesized.Between 24 and 48 h, the average molec-ular weight of the polymer remains con-stant at 70106, whereas the amount ofinulin still increases.The polydispersityof the polymer is very small, i.e.all mol-ecules virtually have the same size [30].

Table 1

Sugar content of transgenic potato plants expressing 1-SST and1-FFT from artichoke.The values represent the mean of 10samplesstandard deviation for mol hexose per g fresh weight.n.d.: not detectable

[mol/g fwt] suc glc Inulin Starch

wild-type 9,111,77 2,121,62 n.d. 770113

22/30 10,66,41 5,633,31 334,08 645132

22/19 14,675,21 3,874,24 34,14,71 631144

22/34 9,712,17 7,214,14 37,775,95 62075


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G.Tariverdian,M.PaulGenetische Diagnostik in Geburtshilfeund GynkologieBerlin, Heidelberg,New York:Springer,1999.538 S., 229 Abb.,(ISBN 3-540-65328-7),geb.,DM 198,

C.Wagener

Molekulare OnkologieStuttgart, New York: Thieme,1999. 337 S.,101 Abb., 63 Tab., (ISBN 3-13-103512-9),geb.,DM 148,

W.Spann,E.Rauch

Sexualdelikt und rztlicheBegutachtungLandsberg:ecomed, 1999.216 S.,(ISBN 3-609-51880-4), geb.,DM 58,

A. Faller

Der Krper des Menschen13.kompl.berarb.u.neu gestaltete.Aufl.;

Stuttgart, New York: Thieme,1999.736 S., 290 Abb.,12 Tab.,1 Falltaf.,(ISBN 3-13-329713-9/692),flex. Tb,DM 39,90

U.Amon,Ruckriegel

Qualittsmanagement in der ArztpraxisBerlin, Heidelberg,New York:Springer,2000.

154 S.25 Abb.,10 Tab.,(ISBN 3-540-66210-3),brosch.,DM 129,

D.Ganten,K.Ruckpaul

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geb.,DM 298,

W.Eiff et al.

Der Krankenhaus-managerBerlin, Heidelberg,New York:Springer,2000.784 S., 94 Abb.,(ISBN 3-540-65735-5),

mit CD-ROM,DM 298,

H.Drfler,W.Eisenmenger,H.D.Lippert

Das medizinische GutachtenBerlin, Heidelberg,New York:Springer,1999.470 S., 11 Abb., 55 Tab., (ISBN 3-540-64774-0),DM 148,

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