6-SFT - Oxford Journals - Oxford University Press

F. del Viso1, A. F. Puebla1,2, C. M. Fusari1, A. C. Casabuono3, A. S. Couto3, H. G. Pontis2, H. E. Hopp1,4 and R. A. Heinz1,4,* 1Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA, Castelar), (1686), Hurlingham, Buenos Aires, Argentina 2Centro de Investigaciones Biológicas, Fundación para Investigaciones Biológicas Aplicadas (FIBA), Vieytes 3103 (7600), Mar del Plata, Argentina 3CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (1428), C.A. Buenos Aires, Argentina 4Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

Fructans are fructose polymers synthesized from sucrose in the plant vacuole. They represent short- and long-term carbohydrate reserves and have been associated with abiotic stress tolerance in graminean species. We report the isolation and characterization of a putative sucrose: fructan 6-fructosyltransferase (6-SFT) gene from a Patagonian grass species, Bromus pictus, tolerant to drought and cold temperatures. Structural and functional analyses of this gene were performed by Southern and Northern blot. Sugar content, quality and fructosyltransferase activity were studied using HPAEC-PAD (high-pH anion-exchange chromatography with pulsed amperometric detection), enzymatic and colorimetric assays. The putative 6-SFT gene had all the conserved motifs of fructosyl-transferase and showed 90% identity at the amino acid level with other 6-SFTs from winter cereals. Expression studies, and determination of sugar content and fructosyl-transferase activity were performed on five sections of the leaf. Bp6SFT was expressed predominantly in leaf bases, where fructosyltransferase activity and fructan content are higher. Bp6-SFT expression and accumulation of fructans showed different patterns in the evaluated leaf sections during a 7 d time course experiment under chilling treatment. The transcriptional pattern suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions, in contrast to previous reports in other graminean species. Low temperatures

Regular Paper

Molecular Characterization of a Putative Sucrose:Fructan 6-Fructosyltransferase (6-SFT) of the Cold-Resistant Patagonian Grass Bromus pictus Associated With Fructan Accumulation Under Low Temperatures

caused an increase in Bp6-SFT expression and fructan accumulation in leaf bases. This is the first study of the isolation and molecular characterization of a fructosyltransferase in a native species from the Patagonian region. Expression in heterologous systems will confirm the functionality, allowing future developments in generation of functional markers for assisted breeding or biotechnological applications. Keywords: Abiotic stress • Bromus pictus • Fructan • Low temperatures • Patagonia • 6-SFT. Abbreviations DMSO, dimethylsulfoxide; DP, degree of polymerization; FEH, fructan exohydrolase; 1-FFT, fructan:fructan 1-fructosyltransferase; FT, fructosyltransferase;HPAEC-PAD,high-pHanion-exchangechromatography with pulsed amperometric detection; Indels, insertions/ deletions; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; ORF, open reading frame; PAR, photosynthetically active radiation; PMSF, phenylmethylsulfonyl fluoride; RACE, rapid amplification of cDNA ends; RT–PCR, reverse transcription– PCR; 6-SFT, sucrose:fructan 6-fructosyltransferase; SNP, single nucleotide polymorphism; 1-SST, sucrose:sucrose 1-fructosyltransferase; TBA, thiobarbituric acid; TLC, thinlayer chromatography; UTR, untranslated region; WSC, water-soluble carbohydrate.

*Corresponding author: E-mail, [email protected]; Fax, +54-11-621-0199.

Plant Cell Physiol. 50(3): 489–503 (2009) doi:10.1093/pcp/pcp008, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Plant Cell Physiol. 50(3): 489–503 (2009) doi:10.1093/pcp/pcp008 © The Author 2009.

489

F. del Viso et al.

The nucleotide sequence of Bp6-SFT, reported in this paper has been submitted to GenBank under accession number FJ424612. A list of the accession numbers used for phylogenetic analyses is available on Supplementary Table S2.

Introduction Fructans are storage carbohydrates present in a wide range of bacteria and fungi (van Hijum et al. 2003, Martinez-Fleites et al. 2005) and in about 15% of the angiosperm plant species (Hendry 1993). Among these plant species there are many important cereals, such as wheat and barley; vegetables such as chicory, onion and lettuce; and forage grasses such as perennial ryegrass and Festuca (Hendry and Wallace 1993). Fructans are linear or branched fructose polymers that are synthesized from sucrose in the plant vacuole (Wagner et al. 1983), although the vacuolar model of fructan synthesis has recently been questioned and a vesicular model was proposed (Cairns et al. 2008). Plant fructans can be divided into five classes (inulin, levan, mixed levan–graminan, inulin neoseries and levan neoseries) according to the type of linkage between adjacent fructose units, the presence of branches and the position of the sugar residue. Dicotyledonous species accumulate mostly inulin fructan series (Van den Ende and Van Laere 2007), whereas monocotyledonous species predominantly store a mixture of the different fructan types (Bancal et al. 1992, Livingston et al. 1993, Luscher and Nelson 1995, Wei et al. 2002). The health-promoting effects of fructans have been reported (Roberfroid 2007), and several food industry applications were developed taking advantage of their use as prebiotics. Regarding their role in plants, particularly in grasses, fructans of the graminan and levan type have an important role as short-term reserves (Maleux and Van den Ende 2007) and also as long-term storage compounds to enable survival of winter dormancy. In dicotyledonous species, inulin is mostly accumulated in underground storage organs as long-term reserve carbohydrates (Van den Ende and Van Laere 2007). Fructans can also be an important source of carbon for rapid regrowth after defoliation when energy is needed. Fructanaccumulating species are predominant in cold and dry environments and almost absent in tropical or aquatic environments (Hendry 1993). In wheat, graminan- and levantype fructans accumulate during the period of cold acclimation, and this accumulation has been associated with freezing tolerance and with over-wintering ability (Yoshida et al. 2007). Fructan metabolism has been associated with cold and drought stress tolerance in plants based on physiological and biochemical evidence (Pontis 1988, Tognetti et al. 1990, Santoiani et al. 1993, Yoshida et al. 1998, De Roover et al. 2000, Hincha et al. 2002, Vereyken et al. 2003). This association was further reinforced based on studies with transgenic

490

plants involving fructan metabolism (Pilon-Smits et al. 1995, Konstantinova et al. 2002, Hisano et al. 2004, Li et al. 2007, Kawakami et al. 2008, Li et al. 2008). One of the mechanisms involved in this protection might be membrane stabilization by direct interaction of fructans with membrane lipids (Valluru and Van den Ende 2008). Fructans are synthesized by the action of two or more different fructosyltransferases (FTs) depending on the species. In dicotyledonous species, the combination of sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT) produces the fructan inulin series. In the monocotyledonous Poaceae family, sucrose:fructan 6-fructosyltransferase (6-SFT) plays an important role in fructan synthesis, mainly through the production of β2–6 linkages. 6-SFT biosynthesizes bifurcose (a tetrasaccharide) and 6-kestose from sucrose and 1-kestose. Several 6-SFT genes have been isolated and functionally characterized in wheat, barley and Agropyrum cristatum (Sprenger et al. 1995, Wei and Chatterton 2001, Kawakami and Yoshida 2002). Bromus pictus is a perennial graminean species native to Patagonia, Argentina, that grows under extreme drought, chilling and freezing conditions. Bromus pictus accumulates large amounts of fructans and has an increasing FT activity when exposed to low temperatures (Puebla et al. 1997, Puebla 1999). Considering that it is one of the main forage sources of the region, the study of B. pictus fructan metabolism through the molecular characterization of its FTs, together with gene regulation analyses, will contribute to the actual knowledge of fructan-mediated abiotic stress tolerance mechanisms in a graminean species highly adapted to growing conditions of extreme cold and drought. Here, we report the cloning and molecular characterization of the first putative FT of B. pictus, its pattern of expression in different leaf sections and in response to low temperatures, with simultaneous analysis of the fructan accumulation profile.

Results Cloning and sequencing of B. pictus 6-SFT cDNA First, a partial cDNA of 760 bp of B. pictus with high similarity to wheat and barley 6-SFTs was isolated by reverse transcription–PCR (RT–PCR) using oligonucleotide priming on highly conserved boxes of plant FTs (U2–L2) (Fig. 1A and Supplementary Table S1). Based on this sequence, primers A and B were designed (Supplementary Table S1) to perform 5′ and 3′ RACE (rapid amplification of cDNA ends), respectively, in order to obtain the complete cDNA. 3′ RACE performed with primer B yielded a 1,100 bp fragment overlapping with the 760 bp fragment (Fig. 1A). Subsequent attempts to clone the complete cDNA by 5′ RACE failed, possibly due to the rather high GC content present in the 5′ end of FT genes (Luscher et al. 2000, Chalmers et al. 2003). The complete


Cold response of a putative 6-SFT in Bromus pictus

B

760 bp

A 156 bp 150 bp

5’

1100 bp

polyA L19

L57

L260

UTR2

B

DEG2

A

ATG

TAA

3’ 2151 bp

Probe

C

5’ 61

E1

I1

E2

289

154

9

I2 578

E3 854

I3 707

E4 702

3’ 236 (bp)

Fig. 1 Schematic representation of Bp6-SFT cDNA and its genomic structure. (a) Partial fragments obtained in the cloning of Bp6-SFT. Primers used for amplification are indicated with arrows. (b) Bp6-SFT cDNA. (c) Bp6-SFT genomic structure. Black and white boxes indicate exons (E) and introns (I), respectively. The length in bp of exons, introns and UTRs is indicated. The probe used for Southern and Northern analyses is shown.

5′ end of B. pictus cDNA was obtained by a combination of PCR and RT–PCR strategies, including the use of degenerate primers and 5′ RACE (see the Materials and Methods) (Fig.1A). The full-length cDNA, Bp6-SFT, (2,151 bp) was obtained by RT–PCR using primers UTR2 and L19 (Supplementary Table S1). The overall GC content of this cDNA is 60.12%, with values surpassing 70% in the first 250 bp. This high GC content could explain the difficulties encountered in the amplification of the mRNA (Fig. 1B). Since B. pictus has a decaploid genome, putative differences between alleles and homeoalleles were explored. Thus, nucleotide sequencing of 20 independent full-length cDNA clones was carried out. Although >75% were identical clones, others exhibited nucleotide variability that ranges from 1 to 5% single nucleotide polymorphisms (SNPs). Though most of the differences were SNPs, small insertions and/or deletions (Indels) were also detected. The majority of the nucleotide substitutions found were synonymous, leading to no changes in the amino acid sequence (data not shown). The most frequent clone was used for further characterization. The Bp6-SFT cDNA contains a single open reading frame (ORF) of 618 amino acids starting at position 62 and ending at position 1,918 of the cDNA. The amino acid sequence predicts the occurrence of seven putative N-glycosylation sites (N-XS/T) (Fig. 2). As reported for most FTs in plants, the ORF starts with a signal sequence that guides the protein into the secretory pathway, probably ending in the vacuole where fructans are putatively synthesized (Wagner et al. 1983). Based on sequence comparison with other graminean 6-SFTs and using signal peptide prediction software, the

mature protein is predicted to start at either residue 49 or residue 58 (Fig. 2). Consequently, the predicted molecular weight of the mature protein will be approximately 62 kDa without considering glycosylations, and its pI may range between 5.0 and 5.13. Bp6-SFT mature protein contains an active aspartate (D) residue in the sucrose-binding box NDPNG, and a glutamate (E) residue in the conserved block WECXD. Furthermore, it also contains the third active site residue, aspartate (D) in the FRDP box (Fig. 2). The functionality of these three conserved regions was tested previously by site-directed mutagenesis for bacterial and plant enzymes (Altenbach et al. 2005, Ritsema et al. 2005, Ritsema et al. 2006). In addition, these three boxes are essential for enzymatic activity and are conserved in all FTs, invertases and fructan exohydrolases (FEHs) from plants species (Chalmers et al. 2005). Comparison of 6-SFTs showed the highest identity, 90%, at the nucleotide and amino acid level with wheat and barley 6-SFTs (accession Nos. AB029887 and X83233, respectively). Agropyrum cristatum 6-SFT (accession No. AF211253) showed 82% identity with Bp6-SFT (Fig. 2). At the amino acid level, identity with 1-SSTs ranged from 58% with wheat 1-SST (accession No. AF029888) to 42.8% with 1-SST of Taraxacum officinale (accession No. AF250634). Regarding identity with monocotyledonous vacuolar invertases or with plant FEHs, it ranged from 48 to 55% and from 30 to 34%, respectively. Phylogenetic analyses were performed with the amino acid sequence information of Bp6-SFT together with protein sequences of available FTs, vacuolar and cell wall invertases, and FEHs. The tree shown in Fig. 3 can be divided into two

Plant Cell Physiol. 50(3): 489–503 (2009) doi:10.1093/pcp/pcp008 © The Author 2009

491

F. del Viso et al.

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300

310

320

330

340

350

360

370

380

390

400

410

420

430

440

450

460

470

480

490

500

510

520

530

540

550

560

570

580

590

600

610

620

630

Bp6-SFT Ta6-SFT Hv6-SFT Ac6-SFT








Fig. 2 Multiple alignment of Bp6-SFT with other 6-SFTs. Bromus pictus 6-SFT (Bp6-SFT), wheat 6-SFT (Ta6-SFT, AB029887), barley 6-SFT (Hv6SFT, X83233) and A. cristatum 6-SFT (Ac6-SFT, AF211253). Identical amino acids are highlighted in gray. Asterisks, colons and periods indicate identical residues, conserved substitutions and semi-conserved substitutions, respectively. The arrows indicate the putative N-terminus of B. pictus mature protein. Conserved domains among invertases and FTs: SDPNG (sucrose-binding box), the RDP motif and the EC domain are boxed. Inverted triangles above the sequence alignments indicate the three carboxylic acids that are crucial for enzyme catalysis. Potential glycosylation sites are underlined.

492



6SFTPs 6SFTBp 6GFFTLp 1SSTLp 6SFTAc 1SSTHv 1SSTFa 6SFTHv 1SSTTa 97 6KEHTa 1FFTTa 6SFTTa 6KEHTa1 100 100 Zmlvr2 1FFTTab 6-1FEHTa 100 100 1SSTAc Osvacinv3 Zmlvr 100 100 100 100 Lpinv 100 Hvinv 1FEHLp? Tainv 1FEHLp 100 91 1FEHTaW3 75 100 100 1FEHTaW1 100 73 Tainv1 63 1FEHTaW2 84 98 68 1FEHHv100 100 100 100

6GFFTAc

6GFFTAo

I 100

Zmlncw4

93

99

Osvacinv2 Zmlvr1

89

98

Zmlncw3 100

II 100 1FFTVd

85

63

100

100

100

63

Talvr1

72 1FFTHt

100 Zmlncw1 100 Zmcwinv OsCIN1

100

96 99

1FEHICi

Zmlncw2 Zmcwinv3 Hvinccw1

1FFTEr

58

100

100

1FFTCs

6FEHBv

1FEHllaC 1FEHllbCi

1SSTHt 1SSTCi

1SSTTo

Fig. 3 Unrooted phylogenetic tree of protein sequences of plant fructan metabolism genes and some vacuolar and cell wall invertases. (I) Fructosyltransferases and vacuolar invertases group, (II) Fructan exohydrolases and cell wall invertases group. FTs that have been functionally characterized by heterologous expression are underlined. Bp6-SFT (6SFTBp) is shown in bold. Numbers below branches indicate bootstrap values (100 replicates). Accession numbers of the nucleotide sequences of proteins used are detailed in Supplementary Table S2.

main groups: (I) FTs and vacuolar invertases; and (II) FEHs and cell wall invertases. Bp6-SFT clustered with monocotyledonous FTs and vacuolar invertases, clearly separated from their homologs in dicotyledonous species (Fig. 3 and Supplementary Table S2). As expected from similarity analysis, it grouped with 6-SFTs from wheat, barley and A. cristatum.

Genomic structure of the B. pictus 6-SFT gene Genomic PCR was used to isolate intron sequences. Considering that B. pictus is a decaploid species, the optimization of this technique required a strict set up of amplification conditions as described in the Materials and Methods. Using

the primer combination UTR2 and L8 (Supplementary Table S1), a 1,200 bp fragment was obtained. The sequence of this fragment exactly matched the sequence of the cDNA and it contained two introns (introns 1 and 2) separated by a mini exon of only 9 bp (Fig. 1C). The AT content in intron 1 and intron 2 was 48.7 and 61.25%, respectively. Subsequent PCRs using primers U2 and L2 (Supplementary Table S1) yielded a 1,000 bp fragment that was cloned and sequenced. This fragment contained a 707 bp intron (intron 3), with 62.8% AT content. In summary, the Bp6-SFT gene is 3,591 bp long and contains four exons and three introns (Fig. 1C). All introns are bounded by the consensus 5′-GT and AG-3′ splice sites. Several clones from each amplified fragment


493

F. del Viso et al.

EI

P

EV

B

A

D

1

2

3

4

5

2.4 kb-

rRNA

1

2

−4 Kb −2 Kb

FT activity (nmol h−1 (mg prot)−1)

B

−4.8 Kb

−1.6 Kb

C

27 24 21 18 15 12 9 6 3 0 1

2

3

4

5

1

2

3

4

5

80 Fructan (mmol (g FW)−1)

−1 Kb

Blade Tip

Mature blade

Sheath

−5.7 Kb

5

4

3

70 60 50 40 30 20 10 0

Gluc.-Fruct.-Suc. (mmol (g FW)−1)

Fig. 4 Southern blot analysis. Total DNA isolated from B. pictus was digested with EcoRI (EI), PvuII (P), EcoRV (EV), BglI (B) or DraI (D) and was hybridized with a 700 bp probe from exon 3 of Bp6-SFT cDNA (Fig. 1c).

were sequenced. These sequences showed nucleotide variability within the introns as well as differences in intron size.

16 Glucose

14

Fructose

12

Suctose

10 8 6 4 2 0 1

Copy number of B. pictus 6-SFT Southern blot was performed to determine the copy number of the 6-SFT gene in the B. pictus genome. A 700 bp probe, corresponding to an exon 3 fragment with no restriction sites for the five different enzymes, was used for hybridization (Fig. 1C). A single and strong signal band was detected with the majority of the restriction enzymes used. However, when EcoRV was used, two bands of the same intensity were detected (Fig. 4). This result indicates that there are at least two copies of the 6-SFT gene in the haploid B. pictus genome but probably not as many as the number of homeologous chromosomes in this species (5×). Low intensity bands are probably due to cross-hybridization with other FT genes.

494

2

3

4

5

Fig. 5 The Bp6-SFT expression pattern correlates with FT activity and fructan accumulation in B. pictus leaf. (a) Northern blot containing 10 µg of total RNA of different sections of sheaths and blades of mature plants of B. pictus. A schematic leaf is depicted to indicate the sections used. Two independents extractions from different individuals were processed. The 28S rRNA bands stained with ethidium bromide are shown as a loading control. (b) FT activity in the leaf sections. Crude protein extracts were analyzed for FT activity for a 4 h period. The fructan produced was quantified by the TBA method. Means and standard deviations of three independent experiments are shown. (c) Tissue distribution of fructans (open circles), sucrose (filled circles), glucose (filled squares) and fructose (filled triangles). Means and standard deviations of three independents experiments of each section are shown.



A

D

B

E

0.00

5.00

10.00

15.00

20.00

4

C 2

3

25.00

F 5

6 7 8 9 10 11 12 13

0.00

5.00

10.00

15.00

20.00

25.00

min

500

1000

1500

19 20 21 22 23 24 14 15 16 17 18

2000 2500 m/z

3000

3500

Fig. 6 Fructan profile within B. pictus mature leaf. (a–e) HPAEC-PAD elution profiles of sugar extracts of mature sheaths [section 1 (a), section 2 (b), section 3 (c)] and mature blades [section 4 (d) and section 5 (e)]. (f) MALDI-TOF MS analysis of section 2. The numbers in (f) indicate the DP (in hexose units) corresponding to each m/z value. Masses and the probable composition of some of the main ions in (f) are described in Supplementary Table S3.

Gene expression analysis The expression of Bp6-SFT was analyzed in different leaf parts (sheaths and blades of mature leaves) by Northern blot hybridization. RNA was extracted from three sections of 2 cm of mature sheaths, a middle section of the mature blade and the tip of the mature blade. Two independent extractions of different plants were performed. A 700 bp DNA probe (Fig. 1C) detected the presence of the transcript mainly in the first two sections of the sheaths (sections 1 and 2), but not in photosynthetically active tissues (sections 4 and 5) (Fig. 5A). Similar expression patterns were observed in other graminean species such as perennial ryegrass, darnel ryegrass

(Lolium temulentum), tall fescue (Festuca arundinacea), barley and wheat (Luscher et al. 2000, Kawakami and Yoshida 2002, Chalmers et al. 2003, Gallagher et al. 2004).

Enzyme activity and carbohydrate analysis In order to evaluate the association between the Bp6-SFT transcript level, fructan content and enzymatic activity, the amount of fructan and the FT activity were quantified in the same sections previously described. FT activity was higher in the sheaths than in the blades (Fig. 5B). Although the FT activity within B. pictus tissues probably involves the concerted action of at least two different enzymes, a positive


495

F. del Viso et al.

1.97 0.68

1.63 0.43

4

1

1.2

1.4 3.83

0.29

60 50

300 250 200

40

150

30 20

100

10

50

0

0 C

1d

2d

0.2

0.5

Leaf Section 3 350 Glucose Fructose Sucrose Fructan

70 60 50

250 200 150

30 20

100

10

50

0

0 C

1d

2d

0.68 0.16

0.21 0.45 0.2

300

40

2

1

7d

80

3 1

4d

0.1

1

4d

7d

350 Glucose Fructose Sucrose Fructan

70 60 50

300 250 200

40

150

30 20

100

10

50 0

0 C

1d

2d

4d

Fructan (mmol (g FW)−1)

70

Leaf Section 2 80

7d

Leaf Section 4 350

80

Glucose Fructose Sucrose Fructan

70 60 50

300 250 200

40

150

30 20

100

10

50


1


0

0 C

1d

2d

4d

7d

Leaf Section 5 350


70 60 50

300 250 200

40

150

30 20

100

10

50

0


5

Leaf Section 1 80


0.08

B


1.07 0.53 0.12

7d


4d


2d


1

1d


C


A

0 C

1d

2d

4d

7d

Fig. 7 Changes in Bp6-SFT transcription levels and WSCs under cold temperatures. (a) Plants were incubated at 4°C for 1, 2, 4 or 7 d. A Northern blot containing 10 µg of total RNA of sheath and blade sections of control or cold-treated plants of B. pictus. Nylon membranes were hybridized with a 700 bp Bp6-SFT radioactively labeled fragment from exon 3. The 28S rRNA bands stained with ethidium bromide are shown as a loading control. Ratios of the radioactivity intensity normalized with the RNA loaded are indicated above. (b) Tissue distribution of fructans (open squares), sucrose (filled circles), glucose (filled squares) and fructose (filled triangles). Means and standard deviations of three independent experiments of each section for each day of treatment are shown.

correlation between Bp6-SFT expression and FT activity was observed. Water-soluble carbohydrates (WSCs) were extracted and analyzed by the thiobarbituric acid (TBA) method (Percheron 1962) and a derived enzymatic assay (Fig. 5C), by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Fig. 6) and by thin-layer chromatography (TLC). Fig. 5C shows that the highest amount of fructans and sucrose was found in the first two sections of the leaf sheath, in agreement with the Bp6-SFT expression pattern and FT activity. On the other hand, in the upper section of the sheaths and in photosynthetically active tissues

496

(sections 4 and 5), the amount of fructosides was low, also correlating with the low Bp6-SFT transcript level. The glucose and fructose content showed less variation along the B. pictus leaf. The same fructan accumulation pattern was confirmed by sugar extract determinations using HPAEC-PAD chromatography (Fig. 6A–E). The second section of the sheaths showed the highest amount of fructans; however, structures up to DP5 (degree of polymerization) were predominant (Fig. 6B). The first section also showed a considerable amount of fructans, but in this case higher DP fructans were predominant (Fig. 6A). TLC runs confirmed these results (data not shown). When sugar extracts obtained from section



2 were analyzed by UV matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), fructan peaks with different DPs were detected (Fig. 6F, and Supplementary Table S3). In summary, the distribution of fructans and the FT activity in the mature leaf of B. pictus positively correlated with the Bp6-SFT expression pattern.

Alterations in the expression of Bp6-SFT and in the content of WSCs in response to low temperatures The effect of low temperatures on the Bp6-SFT expression pattern was evaluated by Northern blot assays performed with tissue extracted after cold treatment experiments. Bp6SFT mRNA levels changed in response to low temperatures. In the first two sections of the sheath, Bp6-SFT mRNA significantly decreased after 1 d at 4°C, but increased again by the second day of cold treatment when compared with control tissue (Fig. 7A). After 7 d at 4°C, Bp6-SFT transcripts decreased >90% in these sections. In the third section of the sheath, the mRNA level increased gradually, showing almost a 4-fold increase at 4 d of treatment. Yet, after 7 d at 4°C the mRNA level decreased dramatically as in the previous sections of the sheath (Fig. 7A). In the blade sections, the mRNA level presented variations during the cold treatment, but expression levels were significantly lower than those detected in sheath sections (Fig. 7A). The fructan levels in the sheath sections showed a similar pattern to that described for the transcripts, with a gradual increase during the cold treatment, after an initial drop at 1 d at 4°C. Substantially higher levels of fructans were found after 7 d of cold treatment in the sheaths in comparison with the first day of treatment (Fig. 7B). Fructan levels in both sections of the blade were much lower than in sink tissues (sheaths), with a slight increase at 4 d of treatment concomitant with a significant increase in sucrose content (Fig. 7B). The sucrose accumulation pattern was similar to that of fructans in all sections. Glucose and fructose content showed a small variation during cold treatment; however, a significant increase in both monosaccharides was detected by 7 d of treatment in blade tissues.

Discussion Bromus pictus contains fructans of the graminean type, the bifurcose series being the most represented; however, three trisaccharides (1-kestose, 6-kestose and neo-kestose) were detected (Puebla 1999). Previous studies showed that SST activity in B. pictus increased when plants were exposed to low temperatures compared with related species from warmer regions such as Bromus auleticus (Puebla et al., 1997). In the Patagonian species B. pictus, fructans were present under all treatments (cold and drought), whereas in B. auleticus they were not (Puebla et al. 1997).

The complete cDNA and corresponding genome sequences of a putative 6-SFT were isolated from B. pictus as a first step in the characterization of fructan metabolism of this key forage species from the Patagonian region. The inferred protein sequence contains a signal peptide and seven potential N-glycosylation sites, features that suggest that Bp6-SFT is a glycoprotein probably located in the vacuole, or, as recently suggested, in small vesicles, where fructan synthesis occurs (Cairns et al. 2008). Bp6-SFT contains all the important conserved motifs for FT activity: the sucrose-binding box (SDPNG), the WECIDF block and the FRDP box. Recently, a model to predict the enzyme functionality of GH32 enzymes based on the amino acid sequence has been proposed (Lasseur et al. 2009). The presence of the pair D–R (DDER, amino acids D307 and R310 in Bp6-SFT) indicates that Bp6-SFT belongs to the group of enzymes that are able to use sucrose as a donor substrate, the S-type (Lasseur et al. 2009). According to the absence of a proline residue in the WECIDF box (W. Van den Ende, personal communication) and the altered WMNDPNG motif (Ritsema et al. 2006), Bp6-SFT is predicted to be an FT of the SST/SFT group (Lasseur et al. 2009). Furthermore, when compared with other FTs, FEHs, vacuolar and cell wall invertases, Bp6-SFT clustered with other 6-SFTs from graminean species, with 90% identity at the amino acid level (Figs. 2, 3). In agreement with previous reports for other FTs and invertases (Sturm 1999, Francki et al. 2006, Ji et al. 2007), the Bp6-SFT gene presents a mini exon of 9 bp comprising part of the sucrose-binding box (the DPN triplet). Regarding the number of gene copies, there is not much information available for previously isolated FT genes. We found that the Bp6SFT gene is present in at least two copies in the B. pictus genome (Fig. 4). Recently, Hisano et al. (2008) reported that some FT genes had two or three copies in the Lolium perenne genome, with probably more than one allele for each gene, although previous studies of other FT genes suggested that they are usually present as a single copy. Sequence variants of Bp6-SFT have been detected at both the genomic and cDNA level, the sequence described here corresponding to the most abundant one. At the cDNA level, most of the detected changes are SNPs, with the exception of a 9 bp Indel in one of the variants. The occurrence of different gene alleles is not surprising considering that B. pictus is a decaploid species (Naranjo et al. 1990). In several graminean species fructans accumulate in the growth zone, at the base of the leaf and in mature sheaths (Luscher and Nelson 1995, Roth et al. 1997, Morvan-Bertrand et al. 2001, Lasseur et al. 2006). The expression of Bp6-SFT mRNA is highest in sink tissues (sheaths) and almost absent in photosynthetically active tissues, in agreement with other FTs, such as 1-SST of L. perenne (Chalmers et al. 2003), 1-SST of F. arundinacea (Luscher et al. 2000) and an FT of L. temulentum (Gallagher et al. 2004).


497

F. del Viso et al.

The variety of fructans present in B. pictus strongly suggests that there are two or more FTs involved in fructan synthesis; therefore, the FT activity determined in this study probably involved all functional FTs in B. pictus. Nevertheless, the accumulation pattern of the Bp6-SFT transcript closely resembled that of FT activity in the different leaf sections, suggesting that Bp6-SFT is involved in fructan synthesis in B. pictus (Fig. 5A, B). In addition, the larger amounts of fructans and sucrose were found in the same leaf sections where Bp6-SFT is expressed at a high level and where the FT activity is also highest (Figs. 5, 6). In conclusion, the level of the Bp6-SFT transcript, the FT activity, and sucrose and fructan accumulation followed a concerted profile pattern along the evaluated leaf sections. This correlation was also reported for the 6-SFT of A. cristatum (Wei and Chatterton 2001) and barley (Sprenger et al. 1995). In order to characterize the Bp6-SFT gene regulation pattern in response to low temperatures and to evaluate its association with sucrose, glucose, fructose and fructan accumulation, a cold response experiment was conducted including different leaf sections. Bp6-SFT gene expression decreased in the first two sections of the sheaths after 1 d of cold treatment and did not increase significantly in the third section of the sheath (Fig. 7A). This agrees with the fructan pattern of these sections, where fructans only surpassed control levels after 4 or even 7 d of cold treatment. In the third sheath section, a gradual increase in transcript accumulation was detected, reaching the highest levels by 4 d of treatment; a similar course of accumulation was reported for wheatgrass (A. cristatum) and bluegrass (Poa secunda) 6-SFTs under cool temperatures (Wei and Chatterton 2001, Wei et al. 2002). 6-SFT mRNAs from A. cristatum and P. secunda increased during the first days at low temperatures and decreased again after 8 d of treatment. In the present study, Bp6-SFT mRNA decreased during the first day at 4°C (in sections 1 and 2 from the sheaths) and slightly increased in the third section of the sheath; on subsequent days Bp6-SFT continued increasing in all sheath sections. Both the high expression level of Bp6-SFT in control conditions and its immediate decrease upon cold treatment are different from the transcriptional patterns reported for wheatgrass and bluegrass 6-SFTs. By 7 d Bp6-SFT mRNA declined to very low amounts, resembling the decrease seen in A. cristatum and P. secunda 6-SFTs (Wei and Chatterton 2001, Wei et al. 2002). It is worth mentioning that in these previous studies no leaf dissection was carried out; thus the detection of more abrupt increments or reductions in mRNA levels could have been limited. Recently Hisano et al. (2008) showed that several fructan genes in L. perenne presented two different expression patterns at the transcriptional level during cold treatment. The first involved an abrupt increase within the first day of treatment followed by a decrease after several days. The other pattern was a gradual increase over a long period

498

of cold treatment. Putative 6-SFTs from L. perenne followed the second pattern of expression (Hisano et al. 2008) which is different from that reported here for Bp6-SFT, within the first 7 d of cold treatment. Taken together, these results suggest that Bp6-SFT is regulated at the transcriptional level, as was described for other FTs (Sprenger et al. 1995, Kawakami and Yoshida 2002, Nagaraj et al. 2004, Lasseur et al. 2006, Hisano et al. 2008), not excluding the possibility of posttranscriptional regulation as well (Lasseur et al. 2006, Lothier et al. 2007). The highest fructan levels in sink tissues were detected after 7 d of treatment, when the transcript levels had decreased (Fig. 7A, B). This observation might be related to the action of other FTs or to the delay between gene transcription and fructan synthesis. In barley and in other species, the key enzyme regulating the flow of carbon into fructan synthesis is 1-SST, displaying a faster response than 6-SFT (Nagaraj et al. 2004). Future gene expression analysis of 1-SST, as well as 1-SST activity in B. pictus will contribute to unravel fructan regulation under low temperatures. Sucrose is the major substrate of fructan metabolism. Sucrose content in sheaths increased in parallel with fructan accumulation, in agreement with previous studies that demonstrated that sucrose induces fructan synthesis (Pontis 1970, Housley and Pollock 1985, Gallagher et al. 2004). The transcript levels in blade tissues were clearly lower than in sink tissues. In contrast, fructan levels in the middle section of the blade increased >2-fold after 4 d of treatment (Fig. 7B). This increase might be produced by other FTs or by fructan transport from sheaths to blade tissues (Wang and Nobel 1998). The transcriptional pattern under control and low temperature treatment studied here suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions. This is in contrast to observations in other species, such as wheat, wheatgrass and bluegrass, where fructan accumulation and 6-SFT expression is low, but is induced by low temperatures during cold hardening (Tognetti et al. 1990, Wei and Chatterton 2001, Kawakami and Yoshida 2002, Wei et al. 2002). This particular gene regulation pattern, distinct from previously reported 6-SFT genes in other species (Wei and Chatterton 2001, Kawakami and Yoshida 2002, Wei at al. 2002) without the need for a previous cold treatment to induce high gene expression, could play an important role in this forage species, contributing to a high level of fructan accumulation in the basal parts of the plant, leading to a species well adapted to continuous grazing. The accumulation of storage compounds close to ground level has been reported as a strategy for many forage species to sustain regrowth immediately after defoliation (Smith et al. 1998, Johnson et al. 2003). The significant decrease in 6-SFT transcript levels in basal leaf sections immediately following the initiation of cold treatment (1 d) and the recovery of gene function with



a final drop after 7 d of treatment at low temperatures indicates a particular gene regulation pattern probably involving different response elements in this gene. Future studies, including the isolation and concerted gene expression analyses of different genes of B. pictus fructan metabolism, such as 1-SST and FEHs under different stress and physiological conditions and their expression in heterologous systems such as Pichia pastoris or transgenic plants to confirm functionality, will not only contribute to the general knowledge of fructan metabolism but will also improve the use of this species as forage and as a possible source of marker genes for assisted breeding or biotechnological applications.

Materials and Methods Plant material Seeds of B. pictus were kindly provided by Dr. A. Soriano and Dr. G. Schrauff from the Faculty of Agronomy, Buenos Aires University. Seeds came from Río Mayo, Chubut Province, Argentina, located at 45°S latitude. Bromus pictus plants were grown in greenhouse conditions in 8 liter pots, at 23°C. Plants were watered three times a week and fertilized weekly with Hakaphos (NPK 18–18–18, Compo, France). Adult plants were moved to growth chambers and grown under the same temperature and irrigation conditions with a 16 h light photoperiod [photosynthetically active radiation (PAR) 108 µmol m–2 s–1].

Isolation of the full-length fructosyltransferase cDNA RNA was extracted from B. pictus mature sheaths using Trizol solution according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A 1 µg aliquot of RNA was used to perform RT–PCR with SuperScript III Reverse Transcriptase (Invitrogen) using random primers and a specific protocol for GC-rich transcripts according to 5′ RACE Kit instructions (Invitrogen). PCR was performed using oligonucleotides with a design based on conserved regions of plant FTs (U2 and L2). Oligonucleotide sequences are shown in Supplementary Table S1. Fragments obtained by RT–PCR were ligated in pGEMT-Easy vector (Promega, Madison, WI, USA) and the constructions were used to transform competent Escherichia coli cells. Several positive clones were sequenced with a genetic analyzer ABI3130XL (Applied Biosytems, Foster City, CA, USA). Based on the first 760 bp fragment sequence, oligonucleotides were designed to perform RACE experiments (A and B, Supplementary Table S1). The 3′ end of the cDNA was amplified by performing 3′ RACE (Invitrogen) with primer B. A 1,100 bp fragment containing a stop codon at position 247 and the complete 3′untranslated region (UTR), including the polyadenylation sequence, was obtained. The 5′ end was completed by a twostep strategy. First, a 156 bp fragment was obtained by

RT–PCR using reverse primer L57 and a forward degenerate primer (DEG2) (Supplementary Table S1 and Fig. 1). In a second step, the 5′ end fragment, including the 5′UTR (61 bp), was isolated by 5′ RACE using a reverse primer complementary to the 5′ end of the previous fragment and the L260 primer. Sequence analysis of all the cloned fragments confirmed that they belong to the same cDNA. In order to amplify the entire cDNA fragment, primers UTR2 and L19 were used (Supplementary Table S1). PCR amplification included 6% dimethylsulfoxide (DMSO) and Expand Long Range Enzyme Mix (Roche, Germany). PCR conditions were as follows: 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 59°C for 1 min and 72°C for 2.5 min. Final extension was at 72°C for 10 min. The 2,151 bp fragment obtained was cloned and sequenced as mentioned above. RT–PCR with Pfx Taq polymerase (Invitrogen) was performed to analyze possible allele variability. For these amplifications, 6% DMSO was also included together with the PCRx Enhancer Solution (Invitrogen). The sequence was deposited in GenBank: accession number FJ424612. This cDNA, named Bp6-SFT, was compared with other cloned 6-SFTs using ClustalW (Thompson et al. 1994) and analyzed with the bioinformatics tools SignalP 3.0 (Bendtsen et al. 2004) and Signal Peptide Prediction (SIG-Pred: http://bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html) to predict the putative presence of a signal peptide, molecular weight and pI. Phylogenetic analyses were performed using protein sequences deduced from the nucleotide sequences of cloned and previously characterized FTs, FEHs, and vacuolar and cell wall invertases. Multiple alignments of protein sequences of Bp6-SFT with other 6-SFTs were carried out with ClustalW (Thompson et al. 1994) and corroborated with MAFFT (Katoh et al. 2005). To determine which evolution model best fits the data, we ran Prot-Test (Phylemon integrated web tool, http://phylemon.bioinfo.cipf.es). The model with the highest score was WAG. A phylogenetic tree was constructed using this model with the maximum likelihood method implemented in ProML from Phylip software package (Felsenstein 2005) and via the Phylemon integrated web tool. Bootstrap values were calculated using 100 replicates.

Genomic sequence of Bp6-SFT To determine the genomic sequence of Bp6-SFT, genomic PCR was performed testing different primers, polymerases and amplification cycles. As no amplification of the complete genomic fragment was obtained using the flanking primers UTR2 and L19, a partial fragment amplification strategy was approached. Partial fragments were achieved using the Expand Long Template Enzyme Mix (Roche, Germany), under the following conditions: 94°C for 3 min, followed by 38 cycles at 94°C for 20 s, 60°C for 1 min and 68°C for 2 min. Final extension was performed at 68°C for 10 min. Fragments were cloned and sequenced as described above.


499

F. del Viso et al.

The primer combination UTR2–L8 (Supplementary Table S1) yielded a 1,200 bp fragment containing the first two introns and a 9 bp exon corresponding to the mini-exon of the Bp6-SFT gene. Using primers U2 and L2, a 1,000 bp fragment was amplified that contained the third intron of the Bp6-SFT gene.

Southern blot analyses Genomic DNA was extracted from frozen B. pictus mature blades using Nucleon Phytopure Reagent (Amersham Biosciences, Piscataway, NJ, USA). A 24 µg aliquot of DNA was digested with the restriction enzymes BglI, DraI, EcoRI, EcoRV and PvuII (NEB, Ipswich, MA, USA). Digested DNA was precipitated overnight with sodium acetate and ethanol, washed with 70% ethanol and run overnight on a 0.8% agarose gel. The gel was capillary blotted and UV-fixed to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Buckinghanshire, UK). Membranes were pre-hybridized overnight with ULTRAhyb Hybridization Buffer (Ambion, Austin, TX, USA) at 42°C and hybridized overnight with a 32P-labeled 700 bp probe amplified from Bp6-SFT exon 3 (Fig. 1C). Membranes were washed twice in 2× SSC/0.1% SDS for 10 min at 42°C, and then in 0.2× SSC/0.1% SDS for 5 min at room temperature. Membranes were exposed overnight to radioactive sensitive screens and scanned with a Typhoon Trio scanner (Amersham Bioscences).

Northern blot analyses Bromus pictus mature leaves were dissected into five segments: three 2 cm length segments from the sheaths (sections 1, 2 and 3) and two 4 cm length segments from the blade (a middle section and the tip sections, labeled as 4 and 5, respectively) (Fig. 5A). Sections from different leaves were pooled to total 0.5 g of tissue for each extraction. Sampling was done in triplicate for each section of the leaf, and tissue was harvested 4 h after the light period started. Tissue was frozen and stored at –80°C. RNA was extracted from these five sections using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Two independent extractions from different individuals were performed. A 10 µg aliquot of RNA of each section was run in a 1% 1× MOPS (Sigma, St Louis, MO, USA) agarose gel. The gel was capillary blotted to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech) overnight and UV-fixed. The same buffers and conditions of pre-hybridization, hybridization and washes used for Southern blot were applied. Membranes were exposed for 4 h to overnight to radioactive sensitive screens, and scanned and detected as described above. In the cold treatment experiment, the intensity of each band was quantified by using a Typhoon Trio scanner (Amersham Bioscences). The RNA loaded was quantified by using Typhoon Trio on the gel stained with ethidium bromide. Data from these analyses were used to normalize the inten-

500

sity of radioactivity of each band based on rRNA loaded in each well. The value for the control tissue was set at 1.0 and other data were calculated relative to this value.

Protein extraction and enzyme assays Samples of different leaf sections were powdered in liquid nitrogen using a mortar and pestle. Sampling was performed as for Northern blot assays. Homogenates were prepared by extracting the powder in a buffer containing 100 mM sodium acetate, pH 5.6, 10 mM β-mercaptoethanol and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Extracts were centrifuged at 20,000×g for 30 min. The clarified supernatant was filtered through Sephadex G-50 (Sigma) columns and used to assay FT activity. FT activity was assayed by incubating the extracts for 4 h with 200 mM sucrose at pH 5.0. Fructosylsucrose was determined after degradation of the unreacted sucrose by a commercial sucrase (Megazyme, Bozeman, MN, USA) by the TBA method according to Percheron (1962) and Puebla et al. (1999). Protein content in the extracts was determined by the method of Bradford (1976). The results presented are the mean and standard deviations of at least three independent experiments.

Cold treatment assay Adult plants were transferred to a cold chamber at 4°C with a 16 h light photoperiod (PAR 108 µmol m–2 s–1). Sampling was done in triplicate for each section of the leaf. Samples were harvested after 1, 2, 4 and 7 d of cold treatment 4 h after the start of the light period each day, frozen in liquid nitrogen and kept at –80°C. Control samples were harvested at the same time from plants that remained at 23°C in growth chambers with the same photoperiod, irradiance and irrigation conditions.

Carbohydrate analysis WSCs were extracted from 0.5 g of each leaf section of control and cold-treated plants. Tissues were boiled in ammoniacal water (pH 8.0) three times for 5 min. The extracts were filtered through a 0.22 µm pore size membrane and lyophilyzed overnight. Dried sugars were resuspended in deionized water. Sucrose, fructose and glucose content were quantified using enzymatic assays according to Spackman and Cobb (2001). To determine fructan amounts, sucrose was first removed by digestion with sucrase (Megazyme). Free fructose and glucose were removed by adding sodium hydroxide and heating for 10 min. The fructan content was determined by the TBA method (Percheron 1962). The results presented are the mean and standard deviations of three independent samples. Sugar extracts were desalted with a Dowex 50 (H+)Amberlite IRA 400 (AcO–) ion exchange column and analyzed by high-pH anion-exchange chromatography in a DX-300 Dionex BioLC system with pulsed amperometric



detection (HPAEC-PAD; Dionex Corp., Sunnyvale, CA, USA). Separation of carbohydrates was carried out on a CarboPac PA-100 column (25×0.4 cm) with a PA-100 pre-column and a 20 µl injection loop. The following condition was employed: 50 mM sodium hydroxide–50 mM sodium acetate followed by a 50–300 mM sodium acetate elution gradient during 40 min. The flow rate was 1.0 ml min–1.

UV MALDI-TOF MS analysis Matrices and calibrating chemicals were purchased from Sigma-Aldrich. Measurements were performed using an Ultraflex II TOF/TOF mass spectrometer equipped with a high-performance solid-state laser (λ = 355 nm) and a reflector. The system is operated by the Flexcontrol 2.4 software package (Bruker Daltonics GmbsH, Germany). Samples were irradiated with a laser power of 60% and measured in the reflectron positive-ion mode. The samples were loaded onto a ground steel sample plate (MTP 384 ground steel; BrukerDaltonics GmbsH) as mixtures with the matrix 2,5-dihydroxybenzoic acid (DHB).

Supplementary data Supplementary data are available at PCP online.

Funding The Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PICT 6248); the Instituto Nacional de Tecnología Agropecuaria (INTA) (PE 3454); Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and INTA (PhD student fellowship to F.d.V.); INTA (PhD student fellowship to C.M.F.); ANPCyT [PME 125 grant for the Ultraflex II (Bruker) TOF/TOF mass spectrometer]. Dr. HGP is a career member of CONICET and Emeritus Professor of the Facultad de Ciencias Exactas y Naturales, University of Mar del Plata, Argentina. Dr. RAH and Dr. ASC are career members of CONICET. Dr. HEH is a career member of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC) and Professor at the Facultad de Ciencias Exactas y Naturales, University of Buenos Aires (UBA).

Acknowledgments The authors thank Dr. V. Lia for assistance in phylogenetic analysis, Dr M. del Vas and Dr. A. J. Distefano for assistance in molecular cloning strategies and A. Montenegro for support in the greenhouses. We also thank Dr. A. Soriano and Dr. G. Schrauff for kindly providing B. pictus seeds.

References Altenbach, D., Nuesch, E., Ritsema, T., Boller, T. and Wiemken, A. (2005) Mutational analysis of the active center of plant fructosyltransferases: festuca 1-SST and barley 6-SFT. FEBS Lett. 579: 4647–4653.

Bancal, P., Carpita, N.C. and Gaudillere, J.P. (1992) Differences in fructan accumulated in induced and field-grown wheat plants: an elongation trimming pathway for their synthesis. New Phytol. 120: 313–321. Bendtsen, J.D., Nielsen, H., von Heijne, G. and Brunak, S. (2004) Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783–795. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72: 248–254. Cairns, A.J., Turner, L.B. and Gallagher, J.A. (2008) Ryegrass leaf fructan synthesis is oxygen dependent and abolished by endomembrane inhibitors. New Phytol. 180: 832–840. Chalmers, J., Johnson, X., Lidgett, A. and Spangenberg, G. (2003) Isolation and characterisation of a sucrose:sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne). J. Plant Physiol. 160: 1385–1391. Chalmers, J., Lidgett, A., Cummings, N., Cao, Y., Forster, J. and Spangenberg, G. (2005) Molecular genetics of fructan metabolism in perennial ryegrass. Plant Biotech. J. 3: 459–474. De Roover, J., Vandenbranden, Van Laere, A. and Van den Ende, W. (2000) Drought induces fructan synthesis and 1-SST (sucrose:sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.). Planta 210: 808–814. Felsenstein, J. (2005) PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. Francki, M.G., Walker, E., Forster, J.W., Spangenberg, G. and Appels, R. (2006) Fructosyltransferase and invertase genes evolved by gene duplication and rearrangements: rice, perennial ryegrass, and wheat gene families. Genome 49: 1081–1091. Gallagher, J.A., Cairns, A.J. and Pollock, C.J. (2004) Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L. J. Exp. Bot. 55: 557–569. Hendry, G.A.F. (1993) Evolutionary origins and natural functions of fructans—a climatological, biogeographic and mechanistic appraisal. New Phytol. 123: 3–14. Hendry, G.A.F., Wallace, R.K., (1993) The origin, distribution, and evolutionary significance of fructans. In Science and Technology of Fructans. Edited by Suzuki, M.C. Chatterton, N.J. pp.119–139. CRC Press, Boca RatonFL . Hincha, D.K., Zuther, E., Hellwege, E.M. and Heyer, A.G. (2002) Specific effects of fructo- and gluco-oligosaccharides in the preservation of liposomes during drying. Glycobiology 12: 103–110. Hisano, H., Kanazawa, A., Kawakami, A., Yoshida, M., Shimamoto, Y. and Yamada, T. (2004) Transgenic perennial ryegrass plants expressing wheat fructosyltransferase genes accumulate increased amounts of fructan and acquire increased tolerance on a cellular level to freezing. Plant Sci. 167: 861–868. Hisano, H., Kanazawa, A., Yoshida, M., Humphreys, M.O., Iizuka, M., Kitamura, K., et al. (2008) Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass. New Phytol. 178: 766–780. Housley, T.L. and Pollock, C. (1985) Photosynthesis and carbohydrate metabolism in detached leaves of Lolium temulentum L. New Phytol. 99: 499–507. Ji, X., Van den Ende, W., Schroeven, L., Clerens, S., Geuten, K., Cheng, S., et al. (2007) The rice genome encodes two vacuolar invertases with


501

F. del Viso et al.

fructan exohydrolase activity but lacks the related fructan biosynthesis genes of the Pooideae. New Phytol. 173: 50–62. Johnson, X., Lidgett, A., Chalmers, J., Guthridge, K., Jones, E., Cummings, N., et al. (2003) Isolation and characterisation of an invertase cDNA from perennial ryegrass (Lolium perenne). J. Plant Physiol. 160: 903–911. Katoh, K., Kuma, K., Toh, H. and Miyata, T. (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33: 511–518. Kawakami, A., Sato, Y. and Yoshida, M. (2008) Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance. J. Exp. Bot. 59: 793–802. Kawakami, A. and Yoshida, M. (2002) Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening. Biosci. Biotechnol. Biochem. 66: 2297–2305. Konstantinova, T., Parvanova, D., Atanassov, A. and Djilianov, D. (2002) Freezing tolerant tobacco transformed to accumulate osmoprotectants. Plant Sci. 163: 157–164. Lasseur, B., Lothier, J., Djoumad, A., De Coninck, B., Smeekens, S., Van Laere, A., et al. (2006) Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/ fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.). J. Exp. Bot. 57: 3961. Lasseur, B., Schroeven, L., Lammens, W., Le Roy, K., Spangenberg, G., Manduzio, H., et al. (2009) Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass (Lolium perenne) into a sucrose:sucrose 1-fructosyltransferase. Plant Physiol. 149: 327–339. Li, H.J., Yang, A.F., Zhang, X.C., Gao, F. and Zhang, J.R. (2007) Improving freezing tolerance of transgenic tobacco expressing sucrose:sucrose 1-fructosyltransferase gene from Lactuca sativa. Plant Cell Tissue Organ Cult. 89: 37–48. Li, Y., Su, X., Zhang, B. and Zhang, Z. (2008) Molecular detection and drought resistance analysis of SacB-transgenic poplars (Populus alba×P. glandulosa). Front. Forest China 3: 226–231. Livingston, D.P.I., Chatterton, N.J. and Harrison, P.A. (1993) Structure and quantity of fructan oligomers in oat (Avena spp.). New Phytol. 123: 725–734. Lothier, J., Lasseur, B., Le Roy, K., Van Laere, A., Prud’homme, M.P., Barre, P., et al. (2007) Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization. J. Exp. Bot. 58: 1969–1983. Luscher, M., Hochstrasser, U., Vogel, G., Aeschbacher, R., Galati, V., Nelson, C.J., et al. (2000) Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue. Plant Physiol. 124: 1217–1228. Luscher, M. and Nelson, C.J. (1995) Fructosyltransferase activities in the leaf growth zone of tall fescue. Plant Physiol. 107: 1419–1425. Maleux, K. and Van den Ende, W. (2007) Levans in excised leaves of Dactylis glomerata: effects of temperature, light, sugars and senescence. J. Plant Biol. 50: 671–680. Martinez-Fleites, C., Ortiz-Lombardia, M., Pons, T., Tarbouriech, N., Taylor, E.J., Arrieta, J.G., et al. (2005) Crystal structure of levansucrase

502

from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J. 390: 19–27. Morvan-Bertrand, A., Boucaud, J., Le Saos, J. and Prud’homme, M.P. (2001) Roles of the fructans from leaf sheaths and from the elongating leaf bases in the regrowth following defoliation of Lolium perenne L. Planta 213: 109–120. Nagaraj, V., Altenbach, D., Galati, V., Luscher, M., Meyer, A.D., Boller, T., et al. (2004) Distinct regulation of sucrose:sucrose-1 fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT), the key enzymes of fructan synthesis in barley leaves: 1-SST as the pacemaker. New Phytol. 161: 735–748. Naranjo, C.A., Arias, F.H., Gil, F.E. and Soriano, A. (1990) Bromus pictus of the B. setifolius complex (section Pnigma): numerical taxonomy and chromosome evidence for species rank. Can. J. Bot. 68: 2493–2500. Percheron, F. (1962) Colorimetric determination of fructose and fructofuranosides by the thiobarbituric acid reaction. C. R. Acad Sci., Paris 255: 2521–2522. Pilon-Smits, E., Ebskamp, M., Paul, M.J., Jeuken, M., Weisbeek, P.J. and Smeekens, S. (1995) Improved performance of transgenic fructanaccumulating tobacco under drought stress. Plant Physiol. 107: 125–130. Pontis, H.G. (1970) The role of sucrose and fructosylsucrose in fructan metabolism. Physiol. Plant. 23: 1084–1100. Pontis, H.G. (1988) Fructan and cold stress. J. Plant Physiol. 134: 148–150. Puebla, A.F. (1999) Estudios bioquímicos y moleculares del metabolismo de fructanos en Gramíneas nativas en relación con estreses ambientales. Buenos Aires University, Argentina. Puebla, A.F., Battaglia, M.E., Salerno, G.L. and Pontis, H.G. (1999) Sucrose–sucrose fructosyl transferase activity: a direct and rapid colorimetric procedure for the assay in plant extracts. Plant Physiol. Biochem. 37: 699–702. Puebla, A.F., Salerno, G.L. and Pontis, H. (1997) Fructan metabolism in two species of Bromus subjected to chilling and water stress. New Phytol. 136: 123–129. Ritsema, T., Hernandez, L., Verhaar, A., Altenbach, D., Boller, T., Wiemken, A., et al. (2006) Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box. Plant J. 48: 228–237. Ritsema, T., Verhaar, A., Vijn, I. and Smeekens, S. (2005) Using natural variation to investigate the function of individual amino acids in the sucrose-binding box of fructan:fructan 6G-fructosyltransferase (6G-FFT) in product formation. Plant Mol. Biol. 58: 597–607. Roberfroid, M.B. (2007) Inulin-type fructans: functional food ingredients. J. Nutr. 137: 2493S–2502S. Roth, A., Luscher, M., Sprenger, N., Boller, T. and Wiemken, A. (1997) Fructan and fructan-metabolizing enzymes in the growth zone of barley leaves. New Phytol. 163: 73–79. Santoiani, C.S., Tognetti, J.A., Pontis, H.G. and Salerno, G.L. (1993) Sucrose and fructan metabolism in wheat roots at chilling temperatures. Physiol. Plant. 87: 84–88. Smith, K., Simpson, R.J., Oram, R.N., Lowe, K.F., Kelly, K.B., Evans, P.M., et al. (1998) Seasonal variation in the herbage yield and nutritive value of perennial ryegrass cultivars with high or normal herbage water soluble carbohydrate concentrations grown in three contrasting Australian dairy environments. Aust. J. Exp. Agric. 38: 821–830.



Spackman, V.M.T. and Cobb, A.H. (2001) An enzyme-based method for the rapid determination of sucrose, glucose and fructose in sugar beet roots and the effects of impact damage and postharvest storage in clamps. J. Sci. Food Agric. 82: 80–86. Sprenger, N., Bortlik, K., Brandt, A., Boller, T. and Wiemken, A. (1995) Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley. Proc. Natl Acad. Sci. USA 92: 11652–11656. Sturm, A. (1999) Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. Plant Physiol. 121: 1–8. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. Tognetti, J.A., Salerno, G.L., Crespi, M.D. and Pontis, H.G. (1990) Sucrose and fructan metabolism of different wheat cultivars at chilling temperatures. Physiol. Plant. 78: 554–559. Valluru, R. and Van den Ende, W. (2008) Plant fructans in stress environments: emerging concepts and future prospects. J. Exp. Bot. 59: 2905–2916. Van den Ende, W., and Van Laere, A., (2007) Fructans in dicotyledonous plants: occurrence and metabolism. In Recent Advances in Fructooligosaccharides Research. Edited by Shiomi, N.,Benkeblia, N. and Onodera, S. pp. 1–14. Research Signpost, Kerala, India. van Hijum, S.A., van der Maarel, M.J. and Dijkhuizen, L. (2003) Kinetic properties of an inulosucrase from Lactobacillus reuteri 121. FEBS Lett. 534: 207–210.

Vereyken, I.J., Chupin, V., Islamov, A., Kuklin, A., Hincha, D.K. and de Kruijff, B. (2003) The effect of fructan on the phospholipid organization in the dry state. Biophys. J. 85: 3058–3065. Wagner, W., Keller, F. and Wiemken, A. (1983) Fructan metabolism in cereals: induction in leaves and compartmentation in protoplasts and vacuoles. Z. Pflanzenphysiol. 112: 359–372. Wang, N. and Nobel, P.S. (1998) Phloem transport of fructans in the crassulacean acid metabolism species Agave deserti. Plant Physiol. 116: 709–714. Wei, J.Z. and Chatterton, N.J. (2001) Fructan biosynthesis and fructosyltranferase evolution: the expression of the 6-SFT (sucrose:fructan 6-fructosyltransferase) gene in crested wheatgrass (Agropyrum cristatum). J. Plant Physiol. 158: 1203–1213. Wei, J.Z., Chatterton, N.J., Harrison, P.A., Wang, R.R.C. and Larson, S.R. (2002) Characterization of fructan biosynthesis in big bluegrass (Poa secunda). J. Plant Physiol. 159: 705–715. Yoshida, M., Abe, J., Moriyama, M. and Kuwabara, T. (1998) Carbohydrate levels among winter wheat cultivars varying in freezing tolerance and snow mold resistance during autumn and winter. Physiol. Plant. 103: 8–16. Yoshida, M., Kawakami, A. and Van den Ende, W. (2007) Graminan metabolism in cereals: wheat as a model system. In Recent Advances in Fructooligosaccharides Research. Edited by Shiomi, N., Benkeblia, N. and Onodera, S. pp. 201–212. Research Signpost, Kerala, India.

(Received November 12, 2008; Accepted January 5, 2009)


503