unexpected presence of graminan- and levan-type fructans in the

Unexpected Presence of Graminan- and Levan-TypeFructans in the Evergreen Frost-Hardy EudicotPachysandra terminalis (Buxaceae): Purification, Cloning,and Functional Analysis of a 6-SST/6-SFT Enzyme1[W]

Wim Van den Ende*, Marlies Coopman, Stefan Clerens, Rudy Vergauwen, Katrien Le Roy,Willem Lammens, and Andre Van Laere

Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, K.U.Leuven, B–3001 Leuven,Belgium (W.V.d.E., M.C., R.V., K.L.R., W.L., A.V.L.); Aquatic Biology, KULAK IRC, 8500 Kortrijk, Belgium(M.C.); and Protein Quality and Function Team, AgResearch Limited, Lincoln Research Centre, Christchurch8140, New Zealand (S.C.)

About 15% of flowering plants accumulate fructans. Inulin-type fructans with b(2,1) fructosyl linkages typically accumulate inthe core eudicot families (e.g. Asteraceae), while levan-type fructans with b(2,6) linkages and branched, graminan-typefructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- andlevan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate inPachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructansas accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherentsucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series ofcereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purifiedby classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-basedcDNA cloning. The functionality of the 6-SST/6-SFT cDNAwas demonstrated after heterologous expression in Pichia pastoris.Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiartemperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructansaccumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our datasuggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connectionsbetween abiotic stress and fructans are discussed.

About 45,000 species of angiosperms, approximately15% of the flowering plants, store fructans, Fru-basedoligo- and polysaccharides derived from Suc. Fructansare known to occur in the highly evolved orders of thePoales (Poaceae), Liliales (Liliaceae), Asparagales, As-terales (Asteraceae and Campanulaceae), and Dip-sacales as well as within the Boraginaceae (Hendry,1993). Fructans are believed to accumulate in the vac-uole (Wiemken et al., 1986), although fructans andfructan degrading enzymes (fructan exohydrolases[FEHs]) have also been reported in the apoplast(Livingston and Henson, 1998; Van den Ende et al.,

2005). To explain this observation, it was hypothesizedthat fructans can be transferred from the vacuole to theouter side of the plasma membrane by vesicle-medi-ated exocytosis (Valluru et al., 2008, and refs. therein),especially under stress. Fructans might protect plantsagainst freezing/drought stresses (Valluru and Vanden Ende, 2008) by stabilizing membranes (Vereykenet al., 2001; Hincha et al., 2002, 2003). Recent studies ontransgenic plants carrying fructan biosynthetic genes(Parvanova et al., 2004; Li et al., 2007; Kawakami et al.,2008) suggest that the enhanced tolerance of theseplants is associated with the presence of fructans.Their reduced lipid peroxidation levels indicate thatfructans, similar to raffinose family oligosaccharides(Nishizawa et al., 2008), might directly act as reac-tive oxygen species scavengers, too (Van den Endeand Valluru, 2009; Bolouri-Moghaddam et al., 2010;Stoyanova et al., 2010).

While dicotyledonous species were believed to ex-clusively store inulin-type fructan consisting of linear b(2,1)-linked fructofuranosyl units, b(2,6) levan-type andmixed-type fructans predominate in monocots (Vijnand Smeekens, 1999). Different types of fructan biosyn-thetic enzymes (also termed fructosyltransferases [FTs])

1 This work was supported by the Fund for Scientific Research,Flanders, and K.U.Leuven (funds to W.V.d.E., M.C., R.V., K.L.R.,W.L., and A.V.L.).

* Corresponding author; e-mail [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Wim Van den Ende ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.162222

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have now been characterized that can readily explainthe diversity of fructans in plants (Lasseur et al., 2006;Tamura et al., 2009). They can be classified in S-type FTs(using Suc as donor substrate) and F-type FTs (usingfructans as donor substrate; Schroeven et al., 2009).Two different enzymes (Suc:Suc 1-fructosyltransferase[1-SST] and fructan:fructan 1-fructosyltransferase[1-FFT]) are required to synthesize the most commonand best studied inulin-type fructans occurring inAsteracean species (Edelman and Jefford, 1968; Vanden Ende and Van Laere, 2007). Depending on thespecies, a more complex cocktail of FTs (1-SST, 1-FFT,Suc:fructan 6-fructosyltransferase [6-SFT], and 6G-FFT)is needed within the monocots (Prud’homme et al.,2007; Yoshida et al., 2007). Structure-function relation-ships have been found explaining the evolutionarydifferences between vacuolar invertases and FTs onthe one hand (Schroeven et al., 2008; Altenbach et al.,2009) and between different types of FTs on the otherhand (Lasseur et al., 2009; Schroeven et al., 2009). Clearly,the capacity for fructan biosynthesis arose manytimes in the course of evolution, in bacteria, fungi,and higher plants (Ritsema et al., 2006; Altenbach andRitsema, 2007). In plants, FTs evolved from vacuolarinvertases (Wei and Chatterton, 2001; Schroeven et al.,2008), while FEHs probably evolved from cell wallinvertases (Le Roy et al., 2007). Like other Suc splittingenzymes, S-type FTs can fulfill important roles inregulating source/sink balances in plants (Ji et al.,2010).

Pachysandra terminalis (Japanese spurge; Buxaceae)is a frost-hardy evergreen plant originating from Japanand China but became increasingly popular as a ground-cover plant in Europe and North America (Zhu andBeck, 1991; Zhou et al., 2005). Pachysandra can survivefreezing temperatures up to 233�C (plants.usda.gov).Together with Sarcococca, Styloceras, Buxus, and Noto-buxus, the genus Pachysandra belongs to the Buxaceaefamily within the basal eudicots (Hoot et al., 1999; vonBalthazar et al., 2000; Anderson et al., 2005; Jiao and Li,2009). Pachysandra and other members of the Buxaceaecontain alkaloids with antibacterial, antiviral, andanticancer properties (Kinghorn et al., 2004; Devkotaet al., 2008). Moreover, P. terminalis was found to be aneffective vole repellent (Curtis et al., 2002).

To the best of our knowledge, so far the presence offructans has not been reported in P. terminalis or anyother species within the basal eudicots. To date, thepresence of graminan- and levan-type fructans withpredominant b(2,6) linkages was thought to be re-stricted to monocot plant species only. In this article,we demonstrate for the first time, to our knowledge,that cereal-type levans and graminans are presentin P. terminalis, a representative of the primitive eudi-cots. We report on the purification and characteriza-tion of the main FT involved and on the cloning andheterologous expression of the cDNA encoding thisenzyme, showing peculiar substrate specificity char-acteristics as a function of the incubation temperatureand time.

RESULTS AND DISCUSSION

P. terminalis Accumulates Both Graminan- andLevan-Type Fructans

Linear b(2,1) inulin-type fructans with 1-kestotrioseas backbone, as occurring, for example, in chicoryroots (Fig. 1), are the most studied fructans and be-come more and more popular as prebiotics in food(Roberfroid and Delzenne, 1998; Roberfroid, 2007).Linear levan-type fructans (backbone: 6-kestotriose)accumulate in a few grass species, including Dactylisglomerata (Fig. 1). Graminan-type fructans, with 1&6-kestotetraose (also termed bifurcose) as backbone, arepredominant in cereals such as wheat (Triticum aesti-vum in Fig. 1; Yoshida et al., 2007). As a result of amajor screening of .100 plant species belonging todifferent families in the plant kingdom, by means ofhigh-performance anion-exchange chromatographyand pulsed-amperometric detection (HPAEC-PAD),the rhizomes of P. terminalis were found to contain acomplicated mixture of so far unidentified oligo- andpolysaccharides in Figure 1. When a neutral, carbohy-drate-containing fraction of P. terminalis rhizomes wassubjected to mild acid and enzymatic hydrolysis withchicory (Cichorium intybus) 1-FEH IIa (Van den Endeet al., 2001) and sugar beet (Beta vulgaris) 6-FEH (Vanden Ende et al., 2003a), a massive production of Fruwas observed (data not shown). A closer inspection ofthe oligosaccharides from P. terminalis on the HPAEC-PAD chromatograms revealed that it contained (1) aseries of peaks corresponding to the graminan-type

Figure 1. HPAEC-PAD chromatograms of the neutral carbohydratefractions derived from P. terminalis, wheat, D. glomerata, and chicory.A reference with sugar standards is also included. G, Glc; F, Fru; S, Suc;R, raffinose; St, stachyose; 1K, 1-kestotriose; M,maltose; 6K, 6-kestotriose;nK, neokestose or 6G-kestotriose; N, 1,1-nystose; B, 1&6-kestotetraoseor bifurcose; F2, inulobiose; F3, inulotriose; IX, inulin with a DP of X;LX, levan with a DP of X; F2, inulobiose; F3, levantriose. Peaks a to mfrom P. terminalis were manually collected and further characterized(see Table I).

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fructans as occurring in wheat and (2) another serieswith elution times identical to the linear levan-typefructans as accumulating inD. glomerata (Fig. 1). Thesefindings already suggested that P. terminalis accumu-lates a mixture of both graminan- and levan-typefructans, as well as some 1-kestotriose. High degreeof polymerization (DP) levan-type fructans occur bothin Pachysandra and in Dactylis (arrows in Fig. 1). Hardproof of the putative fructan identities was generatedby manually collecting individual compounds (a–m inFig. 1) during elution from a preparative HPAEC-PADcolumn. Enzymatic and mild acid hydrolysis treat-ments indeed confirmed the unique presence of thesame graminan- and levan-type fructan structures(Table I) as earlier described in wheat (Bancal et al.,1993; Van den Ende et al., 2003b; Yoshida et al., 2007).However, the levan to graminan ratio is higher inPachysandra compared to wheat (Fig. 1) but lower thanthe one observed in D. glomerata (Maleux and Van denEnde, 2007) and in Phleum pratense (Tamura et al.,2009). At the end of a long cold period, rhizomes of P.terminalis accumulated up to 12.3% of fructans on afresh weight basis. For comparison, commercial chic-ory root cultivars can accumulate up to 18% inulin-type fructans (VanWaes et al., 1998; Van den Ende andVan Laere, 2007).

Purification and Properties of a 6-SST/6-SFT Involved inGraminan and Levan Synthesis

The dominant character of graminan- and levan-type fructans in rhizomes of P. terminalis suggested

the presence of a prominent enzymatic activityresponsible for the formation of b(2,6) Fru-Fru link-ages. Preliminary in vitro incubations with Suc asa single substrate indeed showed a prominent for-mation of 6-kestotriose compared to 1-kestotrioseand 6G-kestotriose (neokestose) and the formation of1&6-kestotetraose from Suc and 1-kestotriose (data notshown). Therefore, the enzyme was provisionallytermed a 6-SST/6-SFT. The b(2,6)-forming enzymewas purified by monitoring its 6-SST activity (quanti-fication of 6-kestotriose production from Suc byHPAEC-PAD). The results of a typical purificationare presented in Table II. Although considerable activ-ity losses occurred on concanavalin A, Mono S pH 4.5,and Mono S pH 4.0, a maximal purification of about88-fold and a specific activity of 2.1 units mg21 proteinwere found (Table II), similar to the purified 1-SSTfrom chicory roots (Van den Ende et al., 1996a).

Similar to all other plant FTs purified so far (Van denEnde and Van Laere, 2007), the retention on conca-navalin A confirmed that this enzyme is also a glyco-protein. The molecular mass of the native protein wasabout 70 kD, as determined by gel filtration (data notshown). The most pure fraction (Mono S pH 4.0; TableII), resulted in 53- and 22-kD subunits after SDS-PAGE(Fig. 2). Such heterodimeric nature is a typical charac-teristic for FTs derived both from dicot and monocotspecies (Altenbach et al., 2004; Van den Ende and VanLaere, 2007), although a few monomeric FTs have alsobeen reported (Koops and Jonker, 1994). Within GH32plant enzymes, the large subunit contains the catalytic

Table I. Characterization of fructans in P. terminalis

Peak1-FEH-Generated

Products

6-FEH-Generated

Products

Fru to Glc Ratio

after Mild Acid HydrolysisFructan Structure

a Suc; Fru None 2 1-Kestotriose (1K)b Fru None No Glc Inulobiose (F2)c None Suc; Fru 2 6-Kestotriose (6K)d 1K; Suc; Fru None 3 1,1-Kestotetraose/1,1-nystose (N)e 6K; Fru (low) 1K; Fru (low) 3 1&6-Kestotetraose/ bifurcose (B)f F2; Fru None No Glc Inulotriose (F3 in Fig. 1)g 6K; Fru None 3 6,1-Kestotetraose (6,1K)h None 6K; Suc; Fru 3 6,6-Kestotetraose (6,6K)i B; 6,1K; Fru N; Fru 4 6&1,1-Kestopentaose + 1&6,1-kestopentaosej 6,6K; Fru B; 1K; Fru 4 1&6,6-Kestopentaose (1&6,6K)k None 6,6K; 6K, Fru 4 6,6,6-Kestopentaose (6,6,6K)l 6,6,6K; Fru 1&6,6K; B; Fru 5 1&6,6,6-Kestohexaosem None 6,6,6K; 6,6K; 6K; Fru 5 6,6,6,6-Kestohexaose

Table II. A typical purification of the 6-SST/6-SFT from P. terminalis rhizomes

Fraction Protein Activity RecoverySpecific

ActivityPurification

mg units % units (mg protein)21 fold

Crude extract 162 3.9 100 0.024 1.0(NH4)2SO4 30%–80% 151 3.7 95 0.025 1.0Concavanalin A 5.3 2.5 64 0.48 20Mono S 4.5 0.92 1.5 38 1.6 67Mono S 4.0 0.17 0.35 8.8 2.1 88

b(2,6)-Linked Fructans in Pachysandra

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triad (Lammens et al., 2009), but the small subunit isalso essential to preserve the biologic activity of FTs(Altenbach et al., 2004). The pH optimum of the 6-SST/6-SFT was between 5.0 and 5.5 (Fig. 3A), consistentwith other Suc-splitting plant FTs (Van den Ende andVan Laere, 2007) and a presumptive vacuolar locali-zation. The temperature optimum was close to 20�C(Fig. 3B), which is considerably lower than the 30�C to35�C of the 1-kestotriose- and 6-kestotriose-formingenzymes in barley (Hordeum vulgare; Simmen et al.,

1993) but comparable with the 20�C to 25�C of the1-SST from Helianthus tuberosus (Koops and Jonker,1996). The high activity at 0�C (40% of the maximum)might represent a specific adaptation to increase fruc-tan accumulation at lower temperatures, as occurringwithin the Pooideae (Sandve and Fjellheim, 2010),including cereals (Yoshida et al., 2007) and othergrasses (Hisano et al., 2004, 2008).

Mass Fingerprint of the Purified Pt 6-SST/6-SFT

The two subunits were excised and trypsinized in-gel, and the masses of ZipTip-eluted tryptic peptideswere determined by quadrupole time-of-flight (Q-TOF) mass spectrometry. A theoretical tryptic digestof the cDNA-derived 6-SST/6-SFT sequence (obtainedlater; see Fig. 4) yielded 50 peptides (designated T1–T50 from N to C terminus). Most masses matched,within the acceptable mass measurement error of60.2D, with one of the theoretical fragments (SupplementalTable S1). Two fragments, representing the N- andC-terminal parts of the enzyme, did not match with themasses of the theoretical fragments, demonstratingthe existence of an alternative N-terminal (VPYPWS-NAQLSWQR and YPWSNAQLSWQR) and C-terminal(IWEMNSAFIQPFH) processing in planta. To the bestof our knowledge, such (differential) N- or C-terminalprocessing has not been reported before for plant FTs.

Cloning a cDNA Encoding the Pt 6-SST/6-SFT and

Phylogenetic Analysis

A full 6-SST/6-SFT cDNA from P. terminalis was ob-tained by a combination of reverse transcription (RT)-PCR, PCR, and 5# and 3# RACE RT-PCR. On the onehand, the Pt 6-SST/6-SFT cDNA-derived amino acidsequence (EMBL accession no. FN870376) is aligned(Fig. 4) with the most identical sequence available inthe literature (65% identity with Pyrus pyrifolia vacu-olar invertase; Yamada et al., 2007). On the other hand,it is compared to the functionally related 6-SFT en-zymes from barley and P. pratense (Sprenger et al.,1995;Tamura et al., 2009). Five potential glycosylation sites

Figure 2. SDS-PAGE (12%) of 3 mg native 6-SST/6-SFT enzyme fromP. terminalis stained with Coomassie Brilliant Blue R 250. The size ofthe molecular mass markers in kilodaltons is indicated to the left.

Figure 3. Effect of pH (A) and incubation temper-ature (B) on the 6-SST activity of the purified6-SST/6-SFT enzyme from P. terminalis. Activitiesare expressed as a percentage of the maximalactivity. Vertical bars indicate SE for n = 3.

Van den Ende et al.




[N-X-(S/T)] were detected (Fig. 4). The estimatedmole-cular mass of the derived polypeptide, without takinginto account putative glycosylations, was predicted at60.7 kD. Its predicted pI was 5.2, which is typical for allvacuolar FTs and invertases (Van den Ende and VanLaere, 2007). The NWMNDPNG (or b-fructosidase)

motif is changed into SWMSDPDG (Fig. 4). An alteredb-fructosidase motif is typical for all FTs described sofar (Altenbach et al., 2009; Van den Ende et al., 2009).Another characteristic of FTs is the presence of analtered W(A/G)W motif (Altenbach et al., 2009). Pt6-SST/6-SFT is an exception since it contains a full

Figure 4. Alignment of the P. termina-lis 6-SST/6-SFT (FN870376), P. pyrifo-lia vacuolar invertase (the mostidentical GH32 member, BAF35859),barley 6-SFT (X83233), and P. pratense6-SFT (BAH30252). Potential glycosy-lation sites are underlined. The threecarboxylic acids that are crucial forenzyme catalysis (Lammens et al.,2009) are in bold and underlined.Boxes indicate motifs involved in do-nor and acceptor substrate specificity(Van den Ende et al., 2009). The arrowindicates the N terminus of the mature6-SST/6-SFT protein. Consensus line:Asterisks indicate identical residues,colons indicate conserved substitu-tions, and periods indicate semicon-served substitutions.





WAWmotif (Fig. 4). Intriguingly, in Pt 6-SST/6-SFT, anAsp/Gln couple (DEEQ) is observed instead of anAsp/Arg couple as observed in P. pyrifolia VI (DDDR)and in the P. pratense and barley 6-SFTs (DDER; Fig. 4).Perhaps the Asp/Gln couple can fulfill a similar rolefor stabilizing Suc (Lasseur et al., 2009).

An unrooted phylogenetic tree was constructedcontaining the Pt 6-SST/6-SFT cDNA-derived aminoacid sequence (see arrow, No. 1 in Fig. 5) and many

other vacuolar types of plant invertases and FTs. Threedistinct groups can be clearly distinguished (Fig. 5).Groups I and II contain dicotyledonous enzymes,and group III contains monocotyledonous enzymes.Hitherto, it was believed that all dicot FTs (see 1-SSTsand 1-FFTs in subgroup IIb, Fig. 5) evolved from TypeII vacuolar invertases (Van den Ende et al., 2002) bychanges in their WMNDPNG and WGW motifs(Schroeven et al., 2008; Altenbach et al., 2009). Here,

Figure 5. Unrooted phylogenetic tree of vacuolar invertases and FTs. Three main groups can be discerned. First group (I): Type Ivacuolar invertases and a single FT from (basal and core) eudicots. 1, P. terminalis 6-SST/6-SFT (FN870376); 2, Vitis vinifera INV(Q9S943); 3, sugar beet INV (AJ277455); 4, Daucus carota INV (Q42722); 5, chicory INV (AJ419971); 6, P. pyrifolia INV(BAF35859); 7, Citrus unshiu INV (AB074885); 8, Phaseolus vulgaris INV (O24509); 9, Vicia faba INV (Q43857); 10,Arabidopsis INV (AY039610); 11, Arabidopsis INV (AY046009); 12, Brassica oleracea INV (AF274298). Second group, subgroupIIa: Type II vacuolar invertases from core eudicots. 13, V. vinifera INV (QS944); 14, D. carota INV (X75352); 15, Lycopersiconesculentum INV (P29000); 16, Capsicum annuum INV (P93761); 17, Ipomoea batatas INV (AY037937); 18, I. batatas INV(AF017082). Second group, subgroup IIb: core eudicot fructan biosynthetic enzymes 1-SST and 1-FFT. 19, Cynara scolymus1-SST (Y09662); 20, H. tuberosus 1-SST (AJ009757); 21, Taraxacum officinale 1-SST (AJ250634); 22, chicory 1-SST (U81520);23, Lactuca sativa 1-SST (ABX90019); 24, Echinops ritro 1-FFT (AJ811624.1); 25, chicory 1-FFT (U84398); 26, Cynara scolymus1-FFT (AJ000481); 27, H. tuberosus 1-FFT (AJ009756); 28, Viguiera discolor 1-FFT (AJ811625). Third group: subgroup IIIa, FTsand a putative INV from Poaceae. 29, Barley 6-SFT (X83233); 30, wheat 6-SFT (AB029887); 31, Lolium perenne putative 6-FT(AF494041); 32, Poa ampla 6-SFT (AF192394); 33, P. pratense 6-SFT (BAH30252); 34, L. perenne putative INV (AF481763); 35,L. perenne 6G-FFT (AF492836); 36, Festuca arundinacea 1-SST (AJ297369); 37, L. perenne 1-SST (AY245431); 38, wheat 1-SST(AB029888); 39, wheat 1-FFT (AB088409). Subgroup IIIb: monocot vacuolar invertases. 40, Oryza sativa INV; 41, L. perenneINV (AY082350); 42, wheat INV (AJ635225). Subgroup IIIc: FTs from Asparagales. 43, Allium cepa 6G-FFT (AY07838); 44,Asparagus officinalis 6G-FFT (AB084283); 45, A. officinalis INV (AF002656); 46, A. cepa INV (AJ006067); 47, A. cepa 1-SST(AJ006066); 48, Agave tequiliana 1-SST (DQ535031). Subgroup IIId, monocot vacuolar INV. 49, Tulipa gesneriana putative INV(X97642); 50, Zea mays INV (P49175); 51, O. sativa INV (AF276703). The P. terminalis 6-SST/6-SFT is indicated with an arrow.The bar indicates a distance value of 0.1.

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we demonstrate for the first time that an FT (Pt 6-SST/6-SFT; No. 1 in Fig. 5) shows higher identity to Type Ivacuolar invertases (52%–65% identity to 2–12 in Fig.5) than to Type II vacuolar invertases (51%–57% iden-tity to 13–18 in Fig. 5), suggesting that the basal eudicotPt 6-SST/6-SFT evolved from a Type I vacuolar inver-tase or, alternatively, from an ancestral vacuolar in-vertase before the separation of the two vacuolarinvertase subgroups by duplication. Further sequenc-ing of vacuolar invertases of lower plants and anumber of basal eudicot species is necessary to dis-criminate between these two possibilities. However,care should be taken since gene losses might furthercomplicate the interpretation of such phylogeneticanalyses. For instance, the two only vacuolar inver-tases in the genome of Arabidopsis (Arabidopsis thali-ana; Nos. 10 and 11 in Fig. 5) belong to Type I vacuolarinvertases, suggesting that the group II gene was lostduring evolution and the group I gene was duplicatedin this species. Four subgroups (IIIa–IIId) can be dif-ferentiated within monocots. The subgroups IIIb andIIId contain vacuolar invertases. Subgroup IIIc harborsboth vacuolar invertases and FTs from Asparagales.

Subgroup IIIa contains mainly FTs, but the presence ofa genuine invertase in this subgroup (No. 34 shows anintact WMNDPNG and WGW motif; Schroeven et al.,2008; Lasseur et al., 2009) cannot be excluded. Thissubgroup further splits up in enzymes that biosynthe-size b(2,1) fructosyl linkages (1-SSTs) and those thatmake b(2,6) linkages (6-SFTs and a putative 6-FT).Taken together, the phylogenetic tree (Fig. 5) suggeststhat the capability to create fructans was gained atleast four times throughout plant evolution: within thebasal eudicots and within the Asterales, Poaceae, andAsparagales. In this scenario, it can be speculated thatdifferent FTs were created by a small number of mu-tations (Schroeven et al., 2008, 2009). It was recentlyspeculated that this evolution occurred in two steps:first, the development of S-type FTs (1-SST and 6-SFT;still using Suc as a donor substrate) from vacuolarinvertases followed by the development of F-type FTs(1-FFT and 6G-FFT; using fructan as donor substrate)from S-type FTs (Schroeven et al., 2009).

Heterologous Expression of the Pt 6-SST/6-SFT cDNA inPichia pastoris: Comparison of the Native andHeterologously Produced Enzymes

Heterologous expression in P. pastoris is consideredas a reliable method to study the functionality withinthe GH32 family (Van den Ende et al., 2009). We areconfident that the native 6-SST/6-SFT enzyme was.99% pure since the same enzyme preparation wasused for enzyme crystallization and three-dimensionalstructure determination (W. Lammens, K. Le Roy, R.Vergauwen, A. Rabijns, A. Van Laere, S.V. Strelkov,and W. Van den Ende, unpublished data). Short-termincubations with the native and recombinant Pt6-SST/6-SFT and Suc showed the production of Glcand 6-kestotriose (Fig. 6). When using 1-kestotriose asa single substrate (data not shown), or when combin-ing Suc and 1-kestotriose, no 1,1-nystose was pro-duced, showing that the (native and recombinant)

Figure 6. In vitro fructan synthesis by the purified native and heterol-ogous 6-SST/6-SFT (1 mg/80 mL) when incubated with 300 mM Suc andwith 300 mM Suc + 300 mM 1-kestotriose. Incubation times, 0 and 13 h;incubation temperature, 0�C. A reference with sugar standards isincluded. G, Glc; F, Fru; S, Suc; 1K, 1-kestotriose; 6K, 6-kestotriose;N, 1,1-nystose; B, 1&6-kestotetraose or bifurcose.

Figure 7. Substrate velocity plots for the production of 1&6-kestote-traose (abbreviated Bif) from 150 mM Suc and a range of 1-kestotrioseconcentrations. Reaction temperatures: 0�C (diamonds) and 30�C(squares). The corresponding linear Hanes plots are shown in Supple-mental Figure S1. Vertical bars indicate SE for n = 3.





enzymes lack 1-FFT activity. By combining Suc and1-kestotriose as substrates, 1&6-kestotetraose (bifur-cose) is the main fructan product formed by both thenative and heterologous enzyme (Fig. 6). Only lowamounts of 6-kestotriose were produced, indicatingthat 1-kestotriose is a more preferential acceptor sub-strate than Suc (Fig. 6). This was further corroboratedby kinetic studies (both at 0�C and 30�C) on the recom-binant enzymes at varying Suc concentrations (6-SSTactivity) and at a constant Suc concentration (150 mM)combined with varying 1-kestotriose concentrations(6-SFT activity). With Suc as single substrate, no satu-ration was observed, not even at 1.2 M Suc (data notshown). The Suc to 1-kestotriose transfer reaction wasclearly saturable (Fig. 7; Supplemental Fig. S1). Theapparent Km values for 1-kestotriose as an acceptor sub-strate were estimated at 35 mM (0�C) and 76 mM (30�C),respectively. These apparent Km values are surpris-ingly low compared to those derived for other plantFTs (Schroeven et al., 2008). Inhibition of 1&6-kestote-traose formation was observed at 0�C but not at 30�C(Fig. 7). The native enzyme shows only minor 1-SSTside activities at 0�C but higher 1-SST side activities at30�C (Supplemental Fig. S2), probably contributing tothe difference in fructan patterns (graminan to levanratio) generated from Suc as a single substrate (Sup-plemental Fig. S2). However, this temperature-depen-dent difference in the graminan to levan ratio was foundto be less prominent for the recombinant enzyme (Fig. 8).

Hydrolytic Activities of 6-SST/6-SFT: A MultifunctionalPremature FT?

Evolutionary Considerations

Besides limited sucrolytic activities (Fig. 6), pro-longed incubation times (substrate depletion) surpris-ingly demonstrated that the Pt 6-SST/6-SFT alsoshowed intrinsic 6-FEH activities (Fig. 8). Typically,the hydrolase to transferase ratio increases with in-creasing temperature, as observed for the chicory1-SST and 1-FFT (Van den Ende et al., 1996a, 1996b).Therefore, it seems reasonable to assume that theintrinsic 6-FEH activity of Pt 6-SST/6-SFT might in-crease at higher temperatures, explaining why higherDP levans do not accumulate at 30�C (Fig. 8B) com-pared to 0�C (Fig. 8A). This finding is in line withprevious observations that some vacuolar invertasesshow intrinsic FEH activities (Van den Ende et al.,2003a; De Coninck et al., 2005; Ji et al., 2007), suggest-ing that perhaps many vacuolar invertases have suchcharacteristics. This new point of view sheds a differ-ent light on some former data and conclusions. Indeed,leaves of many grasses, typically showing huge solu-ble invertase activities, also show high in vitro FEHactivities, even during periods of active fructan syn-thesis (Lothier et al., 2007, and refs. therein). Therefore,it should be reinvestigated whether these prominentFEH activities represent genuine FEH activities ratherthan FEH side activities of vacuolar invertases. Per-

haps these vacuolar invertase side activities are strongenough to play a role in planta. Whether genuine6-FEH-type enzymes occur in the vacuoles of fructanplants is still not clear since the characterized 6-FEHfrom wheat is believed to be apoplastic (Van Riet et al.,2006).

We propose here that intrinsic FEH activities invacuolar invertases can have important physiologicaland evolutionary implications. In Poaceae, it is believedthat the fructan syndrome was initiated shortly after aglobal supercooling period at the Eocene-Oligoceneboundary (Sandve and Fjellheim, 2010). When vacuo-lar invertase develops transferase capacity (by creatingextra space and interaction capacity for binding thesugar acceptor substrate; Altenbach et al., 2009), thefructans that are produced need to be broken down ata later time point, consistent with the idea that fructansmainly act as reserve compounds. Therefore, coevolu-tion of a cell wall invertase into specific FEHs (Le Royet al., 2007) is needed as well as the recruitment of avacuolar targeting signal to translocate the FEHs fromthe cell wall to the vacuole (Van den Ende et al., 2001).

Figure 8. Long-term in vitro fructan synthesis by the native 6-SST/6-SFTat 0�C (A, 2.5 mg/ 80 mL) and at 30�C (B, 1 mg/80 mL) with 300 mM Suc.A reference with sugar standards, phlein, and the carbohydrate profileof P. terminalis are also included for comparison. G, Glc; F, Fru; S, Suc;1K, 1-kestotriose; 6K, 6-kestotriose; N, 1,1-nystose; B, 1&6-kestote-traose or bifurcose; LX, levan with a DP of X.

Van den Ende et al.




However, maturation of vacuolar invertases to genu-ine FTs as well as the subsequent development ofspecific FEHs probably require extended time periodsthroughout evolution. This means that there is prob-ably an evolutionary window in which fructans areaccumulating, while specific vacuolar FEHs are not yetavailable. We speculate that the Pt 6-SST/6-SFT, prob-ably representing a premature or evolutionary youngFTstill containingmany side activities (invertase, FEH,and 1-SST), might be involved in vacuolar fructanpolymerization reactions as long as the Suc concen-tration is high enough. However, when the Suc con-centration decreases, e.g. after defoliation or arrest ofphotosynthesis, the enzyme might play a role in fruc-tan degradation as well. In chicory, in which thefructan syndrome developed a very long time ago(Fig. 5; Schroeven et al., 2009), a similar kind of Suc-based regulation is observed bymeans of very specific,Suc-inhibited 1-FEHs (Van den Ende et al., 2001). Suchdepolymerization reactions might provide hexoses forregrowth, contribute to osmotic adjustments, or adjustthe DP of the fructans to create a sugar mix that isoptimal for membrane and protein stabilization understress (Livingston et al., 2009, and refs. therein).It is well known that graminan- and levan-type (2,6

linkage forming) FT genes are induced by cold ingrasses (del Viso et al., 2009; Tamura et al., 2009; delViso et al., 2010). We demonstrate here that the b(2,6)linkage-forming Pt 6-SST/6-SFT enzyme shows anexcellent activity even at lower temperatures (Fig.3B) and a good levan polymerization rate at 0�C (Fig.8A), and both might represent important physiologicaladaptations to lower temperatures. Particularly, thepresence of high DP fructans with b(2,6) linkages, asoccurring in Pachysandra (Fig. 1), might play an im-portant role in these processes, as suggested before(Volaire et al., 1998). A recent extensive screening of 42annual bluegrass ecotypes, accumulating levan-typefructans, highlighted a strong correlation between theconcentrations of high DP fructans and adaptation tosubfreezing temperatures (Dionne et al., 2009). Note-worthy, the results also revealed that other knowncryoprotectants, such as Suc and Pro, were not closelycorrelated with variations in cold tolerance in thisspecies. Combined with the results presented here, thedata bring a new contribution to the current debate onthe adaptive value of fructans for cold adaptation ingrasses and fructan accumulating basal eudicots.

CONCLUSION

So far, graminan- and levan-type fructans withpredominant b(2,6) linkages were believed to be ab-solutely restricted to monocot plant species. The dis-covery of cereal-type fructans in P. terminalis, a basaleudicot cold-tolerant species, changed this point ofview and confirms the polyphyletic origin of fructanbiosynthesis. Enzyme characterization of the nativeand recombinant versions of an important 6-SST/

6-SFT type of FT involved in the synthesis of fructansin Pachysandra revealed some remarkable properties,consistent with the point of view that it represents apremature FT involved in the regulation of fructanlevels in this species. The enzyme shows peculiar,temperature-dependent properties, urging deeper re-search into the side activities of vacuolar invertasesand premature FTs of other fructan plants.

MATERIALS AND METHODS

Plant Material and Carbohydrate Analyses

Pachysandra terminalis (Buxaceae) was grown on a private field in the

Leuven area (Belgium) and harvested in the winter period (February). Soluble

sugars were extracted from its rhizomes and analyzed by analytical HPAEC-

PAD (DX-300; Dionex) as described (Vergauwen et al., 2000). Individual

fructans were collected manually using preparative HPAEC-PAD. For some

compounds (e.g. peaks f and g, Fig. 1), repeated injections were necessary to

collect enough material for further analysis. All the isolated fructans were

subjected to enzymatic incubations with sugar beet (Beta vulgaris) 6-FEH and

chicory (Cichorium intybus) 1-FEH IIa (10 units) in reaction mixtures buffered

at pH 5.0 with 100 mM NaAc. After several time periods, the reaction was

stopped by boiling for 5 min. All assays were analyzed by analytical HPAEC-

PAD. In parallel, the isolated fructans were subjected to mild acid hydrolysis

as described by Vanhaecke et al. (2006). The DP was determined by the Fru to

Glc ratio (Table I).

Purification of the 6-SST/6-SFT and Q-TOF

Mass Spectrometry

P. terminalis leaves and stems were removed. Rhizomes were washed with

cold tap water, and 0.7 kg was homogenized in 0.9 liters of 50 mM NaAc buffer,

pH 5.0, containing 1 mM phenylmethylsulfonylfluoride, 1 mM mercaptoeth-

anol, 10 mM NaHSO3, and 0.1% (w/v) Polyclar AT. The homogenate was

squeezed through cheesecloth.

Ammonium sulfate was added to a saturation of 30% and stirred on ice for

30 min. After centrifugation for 20 min at 40,000g, the pellet was discarded.

Ammonium sulfate was further added to the supernatant up to a final

concentration of 80% and stirred on ice for 30 min. This time, the supernatant

was discarded after centrifugation for 20 min at 40,000g. The precipitate was

collected and redissolved in 50 mM NaAc buffer (pH 5.0) supplemented with

1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. Undissolved material was spun

down for 10 min at 40,000g. The supernatant was applied to a concavanalin

A-Sepharose column equilibrated with 50 mM NaAc buffer (pH 5.0) supple-

mented with 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. The column was

washed with the same buffer. Bound proteins were eluted with 0.5 M methyl

a-D-mannopyranoside in 50 mM NaAc buffer. The eluate was collected and

proteins were loaded onto a Mono S (HR5/5; Pharmacia Uppsala) cation-

exchange column equilibrated with 50 mM NaAc buffer, pH 4.5. Bound

proteins were eluted using a linear gradient from 0.0 to 0.3 M NaCl in 30 min.

Fractions of 1 mL were collected. The most active fractions were reapplied

onto the Mono S column at pH 4.0 and eluted as described above. SDS-PAGE

of the most active fraction occurred in 12.5% (w/v) polyacrylamide gel, and

staining was performed with Coomassie Brilliant Blue R 250. The purification

procedure was performed at 0�C to 4�C, and 0.02% NaN3 (w/v) was added to

all buffers to prevent microbial growth.

The SDS-PAGE protein bands (Fig. 2) were subjected to mass spectrometric

identification. The Coomassie Brilliant Blue-stained protein bands were

excised, trypsinized, extracted, desalted, and analyzed on Q-TOF as earlier

described (Van den Ende et al., 2001). Sequence information was derived from

the tandem mass spectrometry data with the aid of MaxEnt 3 (deconvoluting

and deisotoping of data) and PepSeq software of the Micromass BioLynx

software package.

Enzyme Assays

During purification of the 6-SST/6-SFT enzyme, aliquots (10–50 mL) were

incubated in 50 mM NaAc buffer at pH 5.0 supplemented with 300 mM Suc





(final concentration) and 0.02% (w/v) NaN3. After 30 min incubation, the

reaction was stopped by heating at 95�C for 5 min. Samples were diluted

3-fold with 0.02% (w/v) NaN3. From these, 25 mL was automatically injected

onto the HPAEC-PAD column. Enzymatic activity is expressed in units,

defined as the amount of enzyme producing 1 mmol of 6-kestotriose per min

with 300 mM Suc as a single substrate. The same assay was used to determine

temperature and pH optima of the enzyme. The 6-kestotriose standard was a

generous gift of Dr. M. Iizuka (Kobe Shoin Women’s University, Japan) and

was used as a standard. The 1&6-kestotetraose (bifurcose) is not commercially

available. Small amounts were obtained from wheat (Triticum aestivum) crown

tissue by preparative HPAEC-PAD. Phlein consists of levan-type fructans

ranging from DP4 to DP12, a kind gift of Dr. J. Chatterton (Utah State

University, Logan, UT).

Enzyme Kinetics

For the kinetical analyses, great care was taken to select time points in the

linear region, ensuring that,10% of the original substrate was consumed. On

the one hand, the recombinant 6-SST/6-SFTwas incubated at 0�C and 30�C in

50 mM sodium acetate buffer (pH 5.0) containing 0.02% (w/v) NaN3 and

different Suc concentrations ranging between 50 and 1200 mM. Similarly, the

enzyme was incubated together with 150 mM Suc (donor substrate) and a

range of 1-kestotriose concentrations (12.5–800 mM) as acceptor substrate. The

incubation reactions were stopped by heating at 90�C for 5 min. The reaction

products were analyzed by HPAEC-PAD as described (Vergauwen et al.,

2000). By comparing the peak areas of 1&6-kestotetraose (6-SFT activity) and

6-kestotriose (6-SST activity) with known amounts of standard compounds,

the products were quantified. In the case of 6-SFT activity, an apparent Km

could be estimated based on the linear Hanes plots (Supplemental Fig. S1) as

described before (Schroeven et al., 2008).

RNA Preparation, Cloning, Sequencing, and Phylogeny

Fresh rhizomes were washed, peeled, cut in small pieces, and mixed. Total

RNA was isolated using the RNeasy plant mini kit (Qiagen). The peptide

sequence YPWSNAQ (part of the N terminus; Supplemental Table S1) was

used to create the sense primer PTN (5#-TAYCCNTGGWSNAAYGCNCA-3#),while part of the C terminus (WEMNSAF; Supplemental Table S1) was used to

design the antisense primer PTC (5#-AANGCNSWRTTCATYTCCCA-3#).One-step RT-PCR with these primers (Access RT-PCR System; Promega)

was performed. The RT reaction was performed at 48�C. PCR conditions were

as follows: 94�C for 3 min, followed by 37 cycles of 94�C for 30 s, 50.5�C for

30 s, and 72�C for 2 min. Final extension was at 72�C for 10 min. A smear

was detected after agarose gel electrophoresis. Therefore, a second nested

PCR was performed on this reaction mixture using conserved GH32-

specific primers HFQP (5#-GSWTWYCAYTTYCARCC-3#) and CTERMINV

(5#-CCNGTNGCRTTGTTRAA-3#). PCR conditions were as follows: 94�C for 3

min, followed by 30 cycles of 94�C for 30 s, 48�C for 30 s, and 72�C for 2 min.

Final extension was at 72�C for 10 min. A clear 1,500-bp band was detected.

The PCR mixture was ligated in the TOPO-XL vector and transformed to

Escherichia coli (TOPO-XL cloning kit; Invitrogen). Plasmids were extracted

using the Qiaprep Spin Miniprep kit (Qiagen), and the insert was sequenced.

Two specific forward primers PAF1 (5#-GTGTGGGCTTGGACTAAGGA-3#)and PAF2 (5#-AGCTTTGCTCAGGGTGGAAG-3#) were selected. In a first RT-

PCR, PAF1 was combined with an oligo(dT)-based primer. The RT reaction

was performed at 48�C. PCR conditions were as follows: 94�C for 3 min,

followed by 37 cycles of 94�C for 30 s, 60�C for 30 s, and 72�C for 2 min. Final

extension was at 72�C for 10 min. Since multiple bands were still visible, a

seminested PCR was set up with PAF2 and an oligo(dT) primer with identical

PCR conditions except that an annealing temperature of 57�Cwas used and 30

cycles were accomplished. The obtained 500-bp PCR product was amplified,

cloned in the TOPO-TA vector (TOPO-TA cloning kit; Invitrogen), and

sequenced. To clone the 5# part of the cDNA, the SMART PCR cDNA

synthesis kit (Clontech) was used. Total RNAwas isolated as described above,

and single-stranded cDNA was produced using the Takara Bio PrimeScript

reverse transcriptase. Further manipulations were as described in the man-

ufacturer’s manual. The advantage 2 polymerase kit (Clontech) was used for

DNA amplification. In the process, the 5# SMART primer 5#-CAACGCA-

GAGTACGCGGG-3# was combined with the specific 6-SST/6-SFT antisense

primer PtR1 (5#-GGATAGGCGAGGCTCAACATCTC-3#). The obtained PCR

product (approximately 750 bp) was cloned in the TOPO-TA vector and

sequenced. The phylogenetic tree was created with TREEVIEW as described

before (Van den Ende et al., 2005).

Heterologous Expression in Pichia pastoris

Because the N-terminal protein part was found (Supplemental Table S1),

the exact mature protein encoding region could be selected. An RT-PCR was

performed by using the primers PicPtF (5#-AGCTGCAGTTCCGTATCCAT-

GGAGCAACGCT-3#) and PicPtR (5#-CATCTAGATCAACGATGGAAGG-

GCTGAATG-3#). The Pfu Proofreading polymerase (Promega) was used

(annealing temperature 57�C for 35 cycles). Three independently amplified

PCR fragments were first subcloned in the TOPO-XL vector and fully

sequenced (TOPO-XL cloning kit; Invitrogen). After vector amplification in

E. coli, the DNA fragment was cut out with PstI and XbaI and ligated in the

pPICZa B vector (Invitrogen). The resulting expression plasmid pPt 6-SST/

6-SFT contains the mature protein part in frame behind the a-factor signal

sequence. Further handlings and transformation were as described by Van

den Ende et al. (2006).

Sequence data from this article can be found in the EMBL data library

under accession number FN870376.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Linear Hanes plots derived from the data

presented in Figure 7.

Supplemental Figure S2. Long-term in vitro fructan synthesis by the

purified native 6-SST/6-SFT at 0�C and at 30�C with 300 mM Suc.

Supplemental Table S1. Peptide masses of the native 6-SST/6-SFT from P.

terminalis.

ACKNOWLEDGMENTS

We thank Veerle Cammaer and Ingeborg Millet for technical assistance.

Received July 2, 2010; accepted October 29, 2010; published October 29, 2010.

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unexpected presence of graminan- and levan-type fructans in the

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