Proc. Natl. Acad. Sci. USAVol. 92, pp. 11652-11656, December 1995Plant Biology

Purification, cloning, and functional expression of sucrose:fructan6-fructosyltransferase, a key enzyme of fructan synthesis in barley

(assimilate partitioning/invertase/transient expression/Nicotiana plumbaginifolia protoplasts)

NORBERT SPRENGER*, KARLHEINZ BORTLIK*, ANDERS BRANDTt, THOMAS BOLLER*, AND ANDRES WIEMKEN*t*Department of Botany, University of Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland, and tPhysiology Department, Carlsberg Laboratory, GI Carlsbergvej 10,DK-2500 Valby, Denmark

Communicated by Diter von Wettstein, Carlsberg Laboratory, Copenhagen Valby, Denmark, August 25, 1995

ABSTRACT Fructans play an important role in assimi-late partitioning and possibly in stress tolerance in many plantfamilies. Sucrose:fructan 6-fructosyltransferase (6-SFT), anenzyme catalyzing the formation and extension of P-2,6-linkedfructans typical of grasses, was purified from barley (Hordeumvulgare L.). It occurred in two closely similar isoforms withindistinguishable catalytic properties, both consisting of twosubunits with apparent masses of 49 and 23 kDa. Oligonu-cleotides, designed according to the sequences of tryptic peptidesfrom the large subunit, were used to amplify correspondingsequences from barley cDNA. The main fragment generatedwas cloned and used to screen a barley cDNA expressionlibrary. The longest cDNA obtained was transiently expressedin Nicotiana plumbaginifolia protoplasts and shown to encodea functional 6-SFT. The deduced amino acid sequence of thecDNA comprises both subunits of 6-SFT. It has high similarityto plant invertases and other 3-fructosyl hydrolases but onlylittle to bacterial fructosyltransferases catalyzing the sametype of reaction as 6-SF]r.

Fructans are a class of highly water-soluble polysaccharidesconsisting of linear or branched fructose chains attached tosucrose. They are produced extracellularly by many bacteriagrowing on sucrose (1), and they also represent a majornonstructural carbohydrate in many plant species, includingmajor crop plants such as wheat and barley (2-9). In plants,fructans accumulate in the vacuole (6) and have importantfunctions in the temporary storage and partitioning of assim-ilates (3, 4) and in osmoregulation (7). They may also play arole in resistance to drought and cold stress (4, 5), as supportedby the finding that tobacco, a species normally incapable offorming fructans, shows improved drought resistance upontransformation with a gene encoding a bacterial fructan-forming enzyme (8).The principal fructans formed by bacteria, called levans, con-

sist of long 03-2--*6-linked chains of fructosyl units attached to the6-fructosyl position of sucrose. They are synthesized by levansu-crase (sucrose:fructan 6-fructosyltransferase, EC 2.4.1.10), whichin repeated catalytic cycles cleaves sucrose, releases glucose, andattaches the remaining fructosyl residue initially to anothermolecule of sucrose and then to the growing fructan chain (1).

Plant fructans, in contrast, have more varied structures, andtheir biosynthesis is not well understood (1-3). It is generallythought to occur in two steps (9): First, sucrose:sucrose1-fructosyltransferase (1-SST; EC 2.4.1.99) catalyzes the for-mation of the trisaccharide 1-kestose§ and glucose from twomolecules of sucrose, an essentially irreversible step. Second,fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) revers-ibly transfers fructosyl residues from one fructan with a degreeof polymerization (DP) of -3 to another of DP 2 2, producinga mixture of fructans with different chain lengths.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

We have chosen to study fructan synthesis in barley, whichaccumulates branched fructans, so-called graminans, contain-ing both 13-2-1 and ,B-2->6 fructosyl linkages (1). Youngbarley leaves normally lack fructans but rapidly accumulatethem when detached and put under continuous illumination,with the concomitant induction of sucrose:sucrose fructosyl-transferase (6, 10, 11). In previous work, we discovered thatthis activity is due to two distinct enzymes, 1-SST and 6-SST(12). We noticed that 6-SST preferred as fructosyl acceptors1-kestose and other fructans over sucrose but that it used asfructosyl donor only sucrose and no fructans (12, 13), just likethe bacterial levansucrases. We- thus renamed the enzymesucrose:fructan 6-fructosyltransferase (6-SFT), and we postu-lated a model for fructan biosynthesis in barley in whichtransfer on the f3-2--6-linked fructosyl residues is broughtabout by 6-SFT rather than by a fructan:fructan fructosyl-transferase (13). To examine this model at the molecular level,we have set out to purify and clone these enzymes. Here wedescribe the purification characterization, and cloning of6-SFTV and its functional expression in protoplasts of Nicoti-ana plumbaginifolia, a plant that lacks fructans.

MATERIALS AND METHODSPlant Material and Enzyme Extraction. Young primary

leaves of barley plants (Hordeum vulgare L. cv. Express) wereexcised and continually illuminated [350 ,uE ms-2.s1 (1 einstein= 1 mol of photons)] for up to 72 h to induce the accumulationof fructan-related enzymes, and the enzymes were extracted asdescribed (12). The extract was adjusted to pH 4.75 with 0.1 MHCl, kept at 4°C for 3 h, and centrifuged at 17,000 x g for 30 min.The clarified supematant was dialyzed overnight at 4°C against 10mM methylpiperazine hydrochloride, pH 5.75, containing 1 mMdithiothreitol, 1 mM benzamidine, 1 mM EDTA, and 0.1 mMphenylmethane sulfonyl fluoride (buffer A).

Purification of 6-SFT. The dialyzed enzyme preparation wasloaded onto a 50-ml column of blue Sepharose 6 fast flow

Abbreviations: 6-SFT, sucrose:fructan 6-fructosyltransferase; 1-SST,sucrose:sucrose 1-fructosyltransferase; DP, degree of polymerization;RT, reverse transcription.tTo whom reprint requests should be addressed.§Nomenclature: We use the following names for small fructans:1-kestose for the trisaccharide 0-f3-D-fructofuranosyl-(2-*1)-f3-D-fructofuranosyl-(2*->1)-a-D-gluco-pyranoside; 6-kestose for thetrisaccharide O-3-D-fructofuranosyl-(2--> 6)-13-D-fructofuranosyl-(2<->1)-a-D-glucopyranoside; bifurcose for the branched tetrasaccha-ride O-f3-D-fructofuranosyl-(2--*1)-O-(f3-D-fructofuranosyl-(2-. 6))-,B-D-fructofuranosyl-(2'-4 )-a-D-glucopyranoside. With respect tolarger fructans, we follow Lewis (1): 6b kestin for the tetrasaccharideO-/3-D-fructofuranosyl-(2--*6)-D-fructofuranosyl-(2->6)--D-fructofuranosyl-(2*-l)-a-D-glucopyranoside; 6,6a isokestin for thebranched pentasaccharide O-f3-D-fructofuranosyl-(2- >l)-O-[f3-D-fructofuranosyl-(2-> 6)-/-D-fructofuranosyl-(2- >6)]-13-D-fructo-furanosyl-(2-*1l)-a-D-glucopyranoside.IThe sequence reported in this paper has been deposited in theGenBank data base (accession no. X83233).

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Proc. Natl. Acad. Sci. USA 92 (1995) 11653

(Pharmacia), washed with 150 ml of buffer A and 75 ml of 0.2M NaCl in buffer B (10 mM methylpiperazine hydrochloride,pH 5.75), and eluted with a linear gradient of 0.2-0.5 M NaClin buffer B. Fractions containing 6-SFT activity were pooled,dialyzed against buffer A, and loaded onto a 6-ml Resource Qcolumn (Pharmacia). After the column had been washed withbuffer B, the bound protein was eluted with a linear gradientfrom 0 to 0.15 M NaCl in buffer B. Fractions containing 6-SFTactivity were pooled and chromatographed on alkyl Superosecolumn HR5/5 (Pharmacia) as previously described (12).Fractions containing 6-SFT activity were concentrated andchromatographed on a Superdex 75 HR 10/30 gel filtrationcolumn (Pharmacia) as described (14). Fractions containing6-SFT were pooled, desalted by five sequential concentrationand dilution steps in centrifugal microconcentrator tubes(Centricon-30, Amicon) with buffer B, and rechromato-graphed on the 6-ml Resource Q column under the sameconditions as the first time. Fractions containing 6-SFT activitywere collected separately in pools I and II.Enzyme Assay and Analysis of Soluble Carbohydrates.

During purification, enzyme activities forming glucose fromsucrose (SST, SFT, and invertase) were routinely measured asdescribed (12). For detailed analysis, enzyme samples wereincubated in 25 mM methylpiperazine hydrochloride, pH 5.75,at 27°C for up to 3 h, either with 200 mM sucrose alone or withsucrose and 1-kestose (100 mM each) combined. The reactionwas stopped by incubation at 95°C for 3 min. The samples wereanalyzed for soluble carbohydrates as described below.

Soluble carbohydrates were extracted from leaf material(100-200 mg fresh weights at 80°C in two 0.6-ml portions ofethanol and three 0.6-ml portions of 25% (vol/vol) ethanol (20min each time). The pooled extracts were freeze dried, redis-solved in water, and analyzed by HPLC as described (12), usingresponse factors and retention times determined with pureoligofructans (13).

Gel Electrophoresis and Sequencing of Tryptic Peptides.Purified proteins were separated by SDS/PAGE on a 7.5-12%(wt/vol) gradient gel (15), electrotransferred to a polyvinyl-idene difluoride membrane (Immobilon PVDF, Millipore) inthe CAPS buffer system (16), visualized with 0.2% Ponceau Sin 1% acetic acid, cut out, and digested with trypsin. Trypticpeptides were separated by reversed-phase HPLC and se-quenced by automated Edman degradation.

Cloning and Molecular Analysis of 6-SFI7. Extraction andpreparation of poly(A)+ RNA by poly(U)-Sepharose chroma-tography followed a published protocol (17). For reverse tran-scription (RT)-PCR, single-stranded cDNA was synthesized frompoly(A)+ RNA by using a synthetic oligo(dT) primer (23-mer)and reverser transcriptase from Moloney murine leukemia virusand subjected to PCR according to the Perkin-Elmer protocol,using degenerate primers designed according to two partialsequences of peptides obtained after tryptic digestion of 6-SFT.This procedure yielded a single PCR product of about 400 bp.The PCR product was cloned with the TA-cloning kit (In-vitrogen), sequenced, and labeled with [a-32P]dATP by usinga randomly primed labeling kit (Boehringer Mannheim) ac-cording to the manufacturers' instructions. A cDNA librarywas prepared with poly(A)+ RNA from leaves that had beenilluminated for 48 h, using the ZAP-cDNA Synthesis Kit(Stratagene), and screened with the a-32P-labeled fragment of6-SFT at 60°C according to the Stratagene protocol. Positiveclones were sequenced with the dideoxynucleotide chain-termination method (Sequencing PRO kit, Toyobo, Osaka).RNA blots were hybridized with the a-32P-labeled fragment of6-SFT and washed under high-stringency conditions; radioactiv-ity was visualized by storage phosphorescence imaging in aMolecular Imager (Bio-Rad). Unless otherwise stated, standardprotocols were used for molecular analysis (18). Sequences wereanalyzed with the GCG software package, version 7.2 (1992).

Expression of 6-SFI in N. plumbaginifolia Protoplasts. The6-SFIT cDNA clone was subcloned in a derivative of thepUC119 plasmid (18) under the control of the expressionsignals of the cauliflower mosaic virus 35S transcript (19).Protoplasts of N. plumbaginifolia were isolated and trans-formed as described (20), using 10 ,ug of the plasmid for 106protoplasts. Transformed protoplasts were incubated at 27°Cin K3 medium (20) for 0-27 h. At the end, 2 ml of W5osmoticum (20) was added, and the protoplasts were pelletedat 1000 x g for 10 min and resuspended in 0.1 M citricacid/Na2HPO4 buffer (pH 5.75) before analysis.

RESULTSPurification and Cloning of 6-SFT. Barley 6-SFT was puri-

fied by pursuing and extending earlier work (12). With fivechromatographic steps, an -100-fold purification was achieved,and the molecular mass of the enzyme was determined to be67 kDa by size-exclusion chromatography (data not shown).The last step yielded two overlapping peaks of 6-SFT activitythat matched the A280 trace (Fig. 1A), indicating the presence

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FIG. 1. Separation of two isoforms of 6-SFT. (A) Chromatographyof the purified enzyme on a Resource Q column. A280 indicates proteinconcentration (continuous trace). Enzyme activity was assayed withsucrose (200 mM) as the only substrate (0). Bars indicate pools usedfor further analysis. (B and C) Analysis of products generated by the6-SFT isoforms in pools I and II with sucrose alone (B) or with sucroseand 1-kestose (C). Structures of the substrates and main products aredrawn schematically. Trehalose, internal standard; c, contaminant of1-kestose substrate; g, residual glycerol from enzyme preparation; p,unidentified product resulting from 1-kestose contaminant.

Plant Biology: Sprenger et al.

11654 Plant Biology: Sprenger et al.

B PCR product

A peptide ~ N-terminus ofA peptide 49 kDa subunit

sequences AGGFPW....

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FIG. 2. Cloning of 6-SFT. (A) Peptide sequences, determined by Edman degradation of the subunits of 6-SFT and their tryptic fragments. (B)PCR product obtained by RT-PCR using degenerate primers based on the amino acid sequences shown in italics inA. Black ends, primers; hatchedpart, portion not defined by primers, encoding the expected peptide portion. (C) Structure of the cDNA clone encoding 6-SFT. Amino acidsequences deduced from hatched or black parts perfectly match the peptide sequences in A.

of two essentially pure isoforms. The purest fractions of eachpeak were analyzed further. The two isoforms differed slightlyin their isoelectric points (4.9 and 5.1), as determined byisoelectric focusing, but they were indistinguishable on SDS/PAGE, each yielding two subunits of 49 and 23 kDa (data notshown). These findings, and the almost complete identity ofthe pattern of fragments generated by tryptic digestion of thecorresponding subunits (data not shown), indicate that the twoisoforms are highly similar in structure and sequence.The two 6-SFT isoforms had indistinguishable catalytic

properties. With sucrose (200 mM) as the sole substrate, bothisoforms produced 6-kestose as the main oligomer, but theyalso hydrolyzed sucrose to glucose and fructose (Fig. 1B). Thepatterns of side products were identical as well, including atrace of 1-kestose and small amounts of bifurcose and 6bkestin, the 6-fructosylation product of 6-kestose. With sucroseand 1-kestose (100 mM each), both isoforms produced pre-dominantly bifurcose, and some 6,6a isokestin, the product ofbifurcose 6-fructosylation (Fig. 1C). Under these conditions,both isoforms produced much less fructose and 6-kestose thanwith sucrose alone.The amino acid sequences of the N-termini of the two

subunits were determined, and both were digested with trypsinto obtain internal peptides for sequencing (Fig. 2A). Thesequences of two internal peptides of the larger subunit wereused to design DNA primers (Fig. 2A). RT-PCR with theseprimers yielded a single fragment of -400 bp (Fig. 2B). ThePCR product was cloned, sequenced, and used to screen a

barley cDNA library. After repeated screenings, seven clonesscored as positive under high-stringency conditions. Partialsequencing showed that they were all identical with each otherand with the PCR product.

Nucleotide and Deduced Amino Acid Sequence of the Cloned6-SFT and Comparison with Known Sequences of f3-Fructosi-dases and Fructosyltransferases. One of the longest cDNAswas fully sequenced on both strands.1 This cDNA contains onelong open reading frame (ORF) of 626 codons, comprisingboth subunits (Fig. 2C). The first 21 codons of the ORF havethe characteristics of a signal peptide, but the sequence of the49-kDa subunit begins only at the 68th codon, indicating thatthe enzyme is formed as a larger precursor which is posttrans-lationally processed. The ORF contains six potential glycosyl-ation sites (Asn-Xaa-Ser/Thr). Indeed, 6-SFT binds to con-canavalin A-Sepharose and can be released by 250 mMa-methyl mannoside (data not shown), indicating that it is a

glycoprotein.The deduced amino acid sequence of the cDNA encoding

6-SFT has clear homologies to 13-fructosyl hydrolases andtransferases from various organisms (Fig. 3). As illustrated bythe dendrogram (Fig. 3A), 6-SFT is most related to invertases(,3-fructosidases), and it has the highest similarity to thesoluble (vacuolar) invertases from mung bean (22), carrot (23),and tomato (24). All the well-conserved domains of microbial

13-fructosyl hydrolases (21) are also found both in plantinvertases and in 6-SFT from barley (Fig. 3B). Interestingly,the bacterial levansucrases are much less related to 6-SFTalthough they catalyze a similar 6-fructosyl transfer reaction.Their homology to 6-SFT and to the ,B-fructosyl hydrolases isrestricted to domains C and D (Fig. 3B).

Transcript Levels of 6-SFT and Enzyme Activities uponInduction ofFructan Accumulation. Transcript levels of 6-SFTwere studied in excised primary leaves subjected to continuousillumination (Fig. 4A). There was no hybridization signal in theRNA preparation from untreated leaves; a hybridizing band of1800 bp appeared after 8 h (the first time point examined),

steadily increased during the first 24 h of treatment, and thenremained approximately constant (Fig. 4A).Enzyme activities were determined in the same experiment

(Fig. 4B). 6-SFT activity was low from the first 8 h and thenincreased strongly. 6-SST increased in parallel but had a muchlower activity. In contrast, 1-SST was present already at thebeginning but then was also increased about 4-fold (Fig. 4B).

Carbohydrate levels were analyzed in the same experimentas well (Fig. 4C). Fructans were virtually absent initially butsteadily increased to very high levels after the initial lag phase.The level of sucrose increased about 3-fold within 8 h andthereafter remained approximately constant. 1-Kestose was thefirst fructooligosaccharide to be detected; it accumulatedrapidly and reached a plateau within 16 h. In contrast, both6-kestose and bifurcose remained at a low level during the first8 h and started to accumulate only thereafter, in parallel to thelevel of total fructans.

Transient Expression of SFT cDNA in N. plumbaginifoliaProtoplasts. The 6-SFIT cDNA was transiently expressed inNicotiana protoplasts under the control of the cauliflowermosaic virus 35S mRNA promoter. After an initial lag phaseof about 3 h, the protoplasts steadily accumulated an activitythat formed 6-kestose from sucrose (Fig. 5A) and bifurcosefrom sucrose and 1-kestose (Fig. SB). Control protoplasts hada low, constant background of 6-kestose-forming activity andwere virtually devoid of bifurcose-forming activity. Productionof bifurcose from sucrose and 1-kestose was about 4 timeshigher than production of 6-kestose from sucrose, as alsofound for purified 6-SFT. These results directly demonstratethat the cDNA encodes a functional 6-SFT.

DISCUSSION

Invertases are well known to act as fructosyltransferases undercertain conditions, forming 6-kestose or 1-kestose from su-crose, and it has therefore been suggested that the SSTactivities in plants might be carried out by invertases (3). Theresults presented here show that at least one of the fructosyl-transferases in barley, namely the enzyme initially called 6-SST(12) and now renamed 6-SFT (13), indeed has close relation-ships to invertase at the biochemical and molecular level.

1 46 247 358-402

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Proc. Natl. Acad. Sci. USA 92 (1995)

Proc. Natl. Acad. Sci. USA 92 (1995) 11655

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IDPNG HLYFQ WGH ySGSm qFRDP qYECP GKDYYA FIG. 4. Induction of 6-SFT and fructan accumulation in excised

IDPcl HvgFl ss FdGS. aFRDP tWagr GfssYA barley leaves under continuous illumination. Samples were harvestedInad. HivFa Yqk WSGSA tLRDP k. rtA ymlGYV and extracted after the time periods indicated (note that abscissa is notIDakt qLvva Ynk WSGSA aMRDP mYnrA vinlGYV a linear scale). (A) RNA blot, hybridized with the PCR fragment ofA B B' C D E F 6-SFT. (B) Enzyme activities in extracts. Activities of 1-SST (-) and

6-SST (0) were measured simultaneously as 1-kestose and 6-kestoseFIG. 3. Comparison of the deduced amino acid sequence of barley

(H.v.) 6-SFT with the sequences of various 03-fructosyl hydrolases(f3-fructosidases = invertases, fructan hydrolases = inulinases andlevanases) and fructosyltranferases (levansucrases). (A) Dendrogramshowing relatedness of sequences. Order of branching indicates sta-tistical similarity. (B) Alignment of the sequence parts correspondingto well-conserved regions of microbial f3-fructosyl hydrolases (21),including an additional region, B'. Boldface capital letters, residuesalmost perfectly conserved in ,B-fructosyl hydrolases; lightface capitalletters, residues corresponding to the consensus of either ,B-fructosylhydrolases and 6-SFT or of levansucrases; lowercase letters, deviatingresidues; dots, gaps introduced for better alignment. The followingsequences were included: Soluble (vacuolar) invertases of plants: V.r.Inv, from mung bean (Vigna radiata) (22); D.c. Inv, from carrot(Daucus carota) (23); L.e. Inv, from tomato (Lycopersicon esculentum)(24). Cell wall invertase of plants: D.c. cwlnv, from carrot (25).Bacterial invertases: E.c. Inv, from Escherichia coli, product of generafD (26); S.m. Scrb, from Streptococcus mutans, gene scrB (27).Microbial fructan hydrolases: B.s. SacC, levanase from Bacillus subtilis(28); K.m. Inu, inulinase from Kluyveromyces marxianus (29). Fungalinvertases: S.c. Invl, from baker's yeast (Saccharomyces cerevisiae),gene SUC1 (30); A.n. Inv, from Aspergillus niger (31). Bacteriallevansucrases: B.s. SacB, from B. subtilis (32); S.m. SacB, from S.mutans (33).

In biochemical terms, the purified barley enzyme retainsconsiderable invertase activity when incubated with sucrose

alone while it acts as 6-SST. However, when it is incubated withsucrose and 1-kestose, it acts mainly as a 6-SFT formingbifurcose, and its 6-SST and invertase activities are suppressed(12, 13).

In molecular terms, the cloned enzyme has the highestsimilarity with plant vacuolar invertases and is only distantlyrelated to levansucrases. It has been shown that bacteriallevansucrases can be converted into invertases by single pointmutations (34). Similarly, in evolution, plant invertases may

proucutLiU iromi sucruose alone. o-br i i/) was mieisureU sepaadtDley asbifurcose production from sucrose and 1-kestose. (C) Soluble carbo-hydrates in extracts. Total fructan (0), sucrose (o), 1-kestose (-),6-kestose (0), and bifurcose (0) were measured. FW, fresh weight.

have been recruited for fructosyltransferase activities by smallmutational changes. It will be interesting to find out whetherspecific amino acid changes can convert a plant invertase into6-SFT.We have verified the identity of the cloned enzyme by

transient expression in Nicotiana protoplasts. Nicotiana doesnot form fructans and therefore is an ideal system for heter-ologous expression of fructan-metabolizing enzymes. Thetransgene-encoded 6-SFT is located within the protoplasts,probably in the vacuoles, indicating that it is correctly targeted.Nevertheless, the protoplasts that express 6-SFT do not accu-

mulate fructans: this may be due to the low concentration ofsucrose and to the absence of the preferred acceptor, 1-kes-tose, in the vacuoles of Nicotiana.We have used the 6-SFT clone as a probe to study expression

of its mRNA in excised illuminated barley leaves. There is anexcellent correlation between induction of 6-SFT mRNAaccumulation, enzyme activity, and accumulation of bifurcoseand fructans of higher DP. In physiological terms, the pre-ferred substrates for 6-SFT, sucrose and 1-kestose, accumulatefirst, followed by induction of 6-SFT, which then causes

accumulation of bifurcose and larger fructans as well as ofsome 6-kestose.

In conclusion, barley 6-SFT is closely related to vacuolarinvertase but transfers the fructosyl residue from sucrose

preferentially to 1-kestose or larger fructans rather than towater. The next question to address is whether the otherenzymes involved in fructan synthesis, and in particular 1-SST,are similarly related to invertase. It will also be interesting to

0-fructosidase 1-fructosidaseIl

cell wall vacuoleL JI

Plant Biology: Sprenger et al.

11656 Plant Biology: Sprenger et al.

0A sucrose + sucrose 6-kestose0.6

0.4

E 0.2

B sucrose + 1-kestose bifurcose

0 -

0 9 18 27Expression time Ihi

FIG. 5. Expression of barley 6-SFT in N. plumbaginifolia proto-plasts. Protoplasts were transformed with vector without insert (w) or

with the 6-SFT cDNA (0), incubated for the indicated time periods,and then used for enzyme assays. Error bars, SEM. (A) 6-Kestoseformation with sucrose as sole substrate. (B) Bifurcose productionwith sucrose and 1-kestose as substrates.

see whether expression of 6-SFT, in conjunction with 1-SST,can cause accumulation of fructans in nonfructan plants andlead to an increased resistance to water stress in a way similarto expression of bacterial levansucrase (8).

We thank P. Jeno (Biozentrum, University of Basel) and R. Mathies(Friedrich Miescher-Institut, Basel) for peptide sequencing, N. Burck-ert and U. Hochstrasser (Botanisches Institut, University of Basel) forDNA sequencing and technical assistance, M. Muller and T. Hohn(Friedrich Miescher-Institut, Basel) for production of protoplasts, and

M. Hirayama for a gift of 1-kestose. This work was supported, in part,by the Swiss National Science Foundation, Grant 31-27923.89.

1. Cote, G. L. & Ahlgren, J. A. (1993) in Science and Technology ofFructans, eds. Suzuki, M. & Chatterton, N. J. (CRC, Boca Raton,FL), pp. 141-168.

2. Lewis, D. H. (1993) New Phytol. 124, 583-594.3. Pollock, C. J. & Cairns, A. J. (1991)Annu. Rev. Plant. Physiol. 42,

77-101.4. Hendry, G. A. F. (1993) New Phytol. 123, 3-14.5. Suzuki, M. & Chatterton, N. J. (1993) Science and Technology of

Fructans (CRC, Boca Raton, FL).

6. Wagner, W., Keller, F. & Wiemken, A. (1983) Z. Pflanzenphysiol.112, 359-372.

7. Bieleski, R. L. (1993) Plant Physiol. 103, 213-219.8. Pilon-Smits, E. A. H., Ebskamp, M. J. M., Paul, M. J., Jeuken,

M. J. W., Weisbeek, P. J. & Smeekens, S. C. M. (1995) PlantPhysiol. 107, 125-130.

9. Edelman, J. & Jefford, T. G. (1968) New Phytol. 67, 517-531.10. Wagner, W., Wiemken, A. & Matile, P. (1986) Plant Physiol. 81,

444-447.11. Obenland, D. M., Simmen, U., Boller, T. & Wiemken, A. (1991)

Plant Physiol. 97, 811-813.12. Simmen, U., Obenland, D., Boller, T. & Wiemken, A. (1993)

Plant Physiol. 101, 459-468.13. Duchateau, N., Bortlik, K., Simmen, U., Wiemken, A. & Bancal,

P. (1995) Plant Physiol. 107, 1249-1255.14. Obenland, D. M., Simmen, U., Boller, T. & Wiemken, A. (1993)

Plant Physiol. 101, 1331-1339.15. Laemmli, U. K. (1970) Nature (London) 227, 680-685.16. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038.17. Brandt, A. & Ingversen, J. (1978) Carlsberg Res. Commun. 43,

451-469.18. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press.,Plainview, NY), 2nd Ed.

19. Neuhaus, J.-M., Sticher, L., Meins, F. & Boller, T. (1991) Proc.Natl. Acad. Sci. USA 88, 10362-10366.

20. Goodall, G. J., Wiebauer, K. & Filipowicz, W. (1990) MethodsEnzymol. 181, 148-161.

21. Gunasekaran, P., Karunakuran, T., Cami, B., Mukundan, A. G.,Preziosi, L. & Baratti, J. (1990) J. Bacteriol. 172, 6727-6735.

22. Arai, M., Mori, H. & Imaseki, H. (1992) Plant Cell Physiol. 33,245-252.

23. Unger, C., Hardegger, M., Lienhard, S. & Sturm, A. (1994) PlantPhysiol. 104, 1351-1357.

24. Elliott, K. J., Butler, W. O., Dickinson, C. D., Konno, Y., Ved-vick, T. S., Fitzmaurice, L. & Mirkov, T. E. (1993) Plant Mol. Biol.21, 515-524.

25. Sturm, A. & Chrispeels, M. J. (1990) Plant Cell 2, 1107-1119.26. Aslanidis, C., Schmid, K. & Schmitt, R. (1989) J. Bacteriol.

6753-6763.27. Sato, Y. & Kuramitsu, H. K. (1989) Infect. Immun. 56, 1956-

1960.28. Martin, I., Debarbouille, M., Ferrari, E., Klier, A. & Rapoport,

G. (1987) Mol. Gen. Genet. 208, 177-184.29. Laloux, O., Cassart, J.-P., van Beeumen, J., Delcour, J. &

Vandenhaute, J. (1991) FEBS Lett. 289, 64-68.30. Hohmann, S. & Gozalbo, D. (1988) Mol. Gen. Genet. 211,

446-454.31. Boddy, L. M., Berges, T., Barreau, C., Vainstein, M. H., Dobson,

M. J., Ballance, D. J. & Pederby, J. F. (1993) Curr. Genet. 24,60-66.

32. Steinmetz, M., LeCoq, D., Aymerich, S., Gonzy-Treboul, G. &Gay, P. (1985) Mol. Gen. Genet. 200, 220-228.

33. Shiroza, T. & Kuramitsu, H. K. (1988)J. Bacteriol. 170, 810-816.34. Chambert, R. & Petit-Glatron, M.-F. (1991) Biochem. J. 279,

35-41.

Proc. Natl. Acad. Sci. USA 92 (1995)