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Keywords:

  • carbohydrate;
  • cold tolerance;
  • fructan synthesis;
  • fructosyltransferase;
  • Lolium perenne (perennial ryegrass);
  • Pichia pastoris

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • • 
    Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described.
  • • 
    Six cDNAs (prft1–prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed.
  • • 
    One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment.
  • • 
    Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fructans are linear or branched forms of fructose polymers, which are derived from sucrose. Fructans are present in 15% of the angiosperm flora, and are particularly widespread in grasses (Chatterton et al., 1989; Hendry, 1993). Fructans accumulate in plant cells as a carbohydrate reserve in addition to or instead of starch (Hendry, 1993), and are also thought to be involved in the maintenance of osmotic potentials (Pavis et al., 2001a). Fructans in plants are classified into five classes based on the degree of polymerization and types of linkage (β(2-1) or β(2-6)) between glucose and fructose, as well as between adjacent fructoses: inulin series, levan series, mixed levan, inulin neoseries and levan neoseries (reviewed by Vijn & Smeekens, 1999; Chalmers et al., 2005).

Fructan is synthesized by a combination of multiple fructosyltransferases (FTs). Four kinds of FT have been identified in plants: sucrose-sucrose 1-fructosyltransferase (1-SST), fructan-fructan 1-fructosyltransferase (1-FFT), sucrose-fructan 6-fructosyltransferase (6-SFT), and fructan-fructan 6G-fructosyltransferase (6G-FFT) (reviewed by Vijn & Smeekens, 1999; for fructan biosynthesis pathways see Fig. 8). 1-SST catalyses the transfer of a fructose unit from one sucrose molecule to the fructosyl residue of another sucrose molecule via a β(2-1) linkage and produces 1-kestose. 1-FFT catalyses the elongation of fructose units on 1-kestose via a β(2-1) linkage, which generally results in inulin series fructans. It also attaches fructose units on fructan with a β(2-6) linkage via a β(2-1) linkage in plants that accumulate a branched type of fructan (Kawakami & Yoshida, 2005). 6-SFT catalyses the transfer of a fructose unit from sucrose to a wide variety of acceptors, including sucrose, 1-kestose and 6-kestose, and produces fructans having fructose units bound to each other via a β(2-6) linkage, such as levan and the branched types of fructan. 6G-FFT catalyses the transfer of a fructose unit from a fructan (e.g. 1-kestose) to C6 of the glucose unit of another fructan or sucrose to produce precursors of inulin neoseries fructans or levan neoseries fructans (Vijn & Smeekens, 1999; Chalmers et al., 2005). Following isolation of the 6-SFT gene in barley (Sprenger et al., 1995), several FT genes have now been isolated from various plant species mainly belonging to the Gramineae, Liliaceae and Asteraceae (reviewed by Ritsema & Smeekens, 2003; Chalmers et al., 2005).

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Figure 8. Possible pathways of fructan biosynthesis. Possible reactions catalysed by 1-SST, 6G-FFT, 6-SFT, 1-FFT and 6-FFT are shown. The functional analysis of proteins expressed in Pichia pastoris in this study revealed that the prft4 gene encodes 1-SST, and the prft3 and prft5 both encode 6G-FFT. The 6G-FFT protein also exhibited 1-FFT activity. The deduced protein sequences of the other three genes (prfts 1, 2 and 6), the function of which was not determined by the analysis, showed similarity with the sequence of 6-SFT.

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Accumulation of fructans in plants has been found to be associated with tolerance of cold and drought, particularly in the development of freezing tolerance (Olien, 1984; Pontis, 1989; Tognetti et al., 1990; Livingston & Henson, 1998; Yoshida et al., 1998; Kawakami & Yoshida, 2002). Winter cereals and grasses enhance their freezing tolerance through acclimation to low temperatures from autumn to winter. In wheat, this hardening process is associated with an increase in the cellular contents of not only mono- and disaccharides such as sucrose, but also fructan. Levels of fructan in wheat varieties are positively correlated with freezing tolerance (Yoshida et al., 1998), and accumulation of fructan in wheat during hardening is accompanied by an increase in FT gene mRNA (Kawakami & Yoshida, 2002). A seasonal increase in the level of FT gene mRNA towards winter has also been detected in chicory (Van Laere & Van den Ende, 2002). Similarly, an increase in the mRNA level of a putative 6-SFT gene in response to cold treatment of plants has been detected in grasses (Wei & Chatterton, 2001; Wei et al., 2002). Further understanding of relationships between fructan levels and cold tolerance of plants has been obtained in experiments using transgenic plants. Transgenic tobacco plants expressing the Bacillus subtilis fructan polymerase gene SacB (Konstantinova et al., 2002) and transgenic perennial ryegrass plants expressing wheat 6-SFT or 1-SST genes (Hisano et al., 2004) exhibit an increased level of fructan accumulation as well as an increased level of tolerance of freezing. These plants thus provide additional direct evidence of an association between fructan accumulation and freezing tolerance (Konstantinova et al., 2002; Hisano et al., 2004). These results also indicate that an increased level of FT gene transcription is linked to an increase in fructan content. However, little is known about the regulatory mechanisms affecting fructan biosynthesis in cold conditions with regard to the expression of multiple FT genes.

Like many other plants growing in global temperate regions, the agronomically important pasture grass species perennial ryegrass (Lolium perenne) can acquire freezing tolerance through cold acclimation. Changes in gene expression at low temperatures have been detected for a large number of genes in plants, although limited information is available on the relationship between changes in gene expression and tolerance to low temperature (for a review see Pearce, 1999). Nevertheless, an increase in the level of freezing tolerance as a consequence of an increase in fructan content through overexpression of a single FT gene has been observed in perennial ryegrass (Hisano et al., 2004). This suggests that the accumulation of fructan is of critical importance in the acquisition of freezing tolerance for plant species such as perennial ryegrass, in which fructans are the main storage carbohydrates (reviewed by Chalmers et al., 2005). Two FT genes have been identified in perennial ryegrass: 1-SST (Chalmers et al., 2003) and 6G-FFT (Lasseur et al., 2006), but little is known about the expression of these genes in a cold environment.

In the present study, we describe the isolation, functional characterization and genetic mapping of cDNAs from multiple FT genes in perennial ryegrass. We also describe associated changes in carbohydrate levels and mRNA levels of FT genes during cold treatment. This is the first report of the transcription of multiple FT genes being induced by low temperature in a coordinated way, which is associated with the accumulation of fructans.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials

Lolium perenne L. (perennial ryegrass) is an outcrossing temperate grass species, and cultivars are generally bred as synthetic varieties based on a number of heterozygous parents. Although a high degree of heterozygosity is retained within varieties, individual genotypes can be maintained through clonal replication. Genotypes of L. perenne cv. Aberystwyth S23 were used for the isolation of FT genes. For analyses of gene copy number, gene expression and carbohydrate content, genotypes from cv. Riikka were used. In order to position the FT genes on the reference genetic linkage map for perennial ryegrass, 190 plants were genotyped from a true F2 mapping population comprising selfed progeny from a single F1 plant produced by a cross between inbred lines derived from the perennial ryegrass cultivars Perma and Aurora (Armstead et al., 2004).

Construction of the cDNA library and the isolation of FT genes

To construct the perennial ryegrass cDNA library, total RNA was isolated from cold-treated plants. Following 30 d growth in 16 h light at 22°C and 8 h dark at 18°C in a plant growth room, plants were subjected to a cold treatment in which they were kept under light for 8 h at 6°C and then in the dark for 16 h at 2°C, each day in a cold acclimation room for 1, 4, 7, 14 or 30 d. The isolation of total RNA, synthesis of cDNA, construction of cDNA library and screening of the cDNA library were performed as described previously (Shinozuka et al., 2006). Fragments of FT cDNAs were amplified by PCR using primers (Fructan-pF1 and Fructan-pR1; Table S1 in Supplementary material) designed to anneal sequences conserved between the wheat FT genes wft1 and wft2 (Kawakami & Yoshida, 2002). The amplified fragment was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and was used as a probe to screen the cDNA library. The screened phage clones were excised as pBK-CMV phagemid vectors using the Ex-assist helper phage and cloned into the Escherichia coli XLOLR strain according to the manufacturer's instructions (Stratagene, La Jolla, CA, USA).

DNA sequence analysis

DNA sequences were produced using a DNA sequencer (ABI377; Applied Biosystems, Foster City, CA, USA) and kit according to the manufacturer's instructions. The alignment of DNA sequences was carried out using the ClustalW Multiple Sequence Alignment Program ver. 1.8 (http://clustalw.ddbj.nig.ac.jp/top-e.html) (Thompson et al., 1994). The aligned sequences were displayed using bioedit (Hall, 1999), and further aligned by hand to minimize sequence mismatches. A phylogenetic tree was constructed using the neighbour-joining method (http://clustalw.ddbj.nig.ac.jp/top-e.html) (Saitou & Nei, 1987) based on protein sequences deduced from the nucleotide sequences of FT genes. Estimates of evolutionary distance were obtained using Kimura's two-parameter method (Kimura, 1980) and bootstrap values were calculated with 1000 replicates.

Localization of FT genes on the linkage map

Total DNA was extracted as described by Doyle & Doyle (1987) from leaves of genotypes belonging to the Perma × Aurora mapping family and from inbred Perma and Aurora parental lines. Fragments of the FT genes were amplified by PCR from genomic DNA using primers that were designed from cDNA sequences as listed in Table S1. PCR products were separated by electrophoresis on a 2% (w/v) agarose gel in TBE buffer, and polymorphisms were identified by allele-specific PCR amplification or as cleaved amplified polymorphisms. In order to map the prft3 and prft4 genes, the PCR products were treated with StuI and SacII, respectively, before gel electrophoresis. Segregation data for genotypes from the F2 mapping population were analysed using JoinMap ver. 3.0 software (Van Ooijen & Voorrips, 2001).

Southern blot analysis

Total DNA was extracted from a single plant of perennial ryegrass (cv. Riikka) as described above. Southern blot analysis was performed as described previously (Shinozuka et al., 2006). The 3′-untranslated regions (UTRs) of FT genes were amplified by PCR using specific primers (Table S1) and were used as probes.

Functional characterization of the recombinant FT proteins using the Pichia pastoris expression system

The methylotrophic yeast Pichia pastoris expression system (EasySelect Pichia Expression Kit; Invitrogen, Carlsbad, CA, USA) with pPICZαA was used to investigate the expression of recombinant FT proteins according to the method of Kawakami & Yoshida (2002). The entire regions of the coding sequence of putative FT genes without the termination codon were amplified by PCR (Table S1) and were inserted behind the α-factor signal sequence of pPICZαA. Transformation and culturing of P. pastoris were carried out according to the kit instruction manual. The P. pastoris X-33 strain was transformed using plasmid DNA linearized by digestion with DraI and electroporation. Culture of transgenic P. pastoris and purification of recombinant proteins were performed as described by Kawakami & Yoshida (2002), except that we cultured P. pastoris for 72 h at 28°C and purified the recombinant proteins by Amicon Ultra 10 000 MWCO (Millipore, Bedford, MA USA) with 20 mm citrate-phosphate buffer (pH 5.0). The purified recombinant FT proteins were mixed with the same volume of 100 mm sucrose, 1-kestose or 6-kestose to give a final concentration of 50 mm for the substrate. The reaction mixtures were incubated at 25°C or 4°C for 20 h and the products were analysed by high-performance anion exchange chromatography and a pulsed amperometric detector (HPAEC-PAD) using a DX500 chromatograph with a Carbo Pack PA1 column (Dionex, Sunnyvale, CA, USA). System I of Shiomi's method (Shiomi, 1993) was used for the eluent program. Peak identification was performed using standard fructooligosaccharides (Fructooligosaccharides Standard Set for HPLC; Wako Chemicals, Osaka, Japan).

Gene expression analysis

Following 30 d growth in standard conditions, seedlings of perennial ryegrass (cv. Riikka) were cold-treated in a cold acclimation room for 1, 4, 7, 14 and 30 d, as described above. Tissues were harvested from cold-treated plants 4 h after the start of the light period each day. Total RNA was extracted from the leaf blade and crown tissues of cold-treated plants as described by Chomczynski & Sacchi (1987). One microgram of the total RNA was used as the template for cDNA synthesis. The cDNA synthesis reaction mixture was prepared by mixing 4 µl 5× reaction buffer (250 mm Tris-HCl (pH 8.3), 375 mm KCl, 15 mm MgCl2), 2 µl 0.1 m DTT, 0.5 µl RNaseOUT inhibitor (Toyobo, Osaka, Japan), 1 µl 100 µm oligo(dT)20 primer, 4 µl 2.5 mm dNTPs, the total RNA solution, and water to a final volume of 19 µl. The mixture was heated at 65°C for 5 min and cooled rapidly on ice. After the addition of 1 µl reverse transcriptase (M-MLV, Invitrogen), the cDNA synthesis was performed at 42°C for 1 h. The reverse transcriptase was inactivated by heating the sample at 99°C for 1 min. Reverse transcription-mediated real-time PCR (real-time RT–PCR) was carried out using a 1-µl aliquot of the reaction mixture and DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland) with a DNA Engine Opticon 2 System (MJ Research, Waltham, MA, USA) as described previously (Koseki et al., 2005). The PCR cycle used to analyse transcripts of the prft1 and prft2 genes was 95°C for 10 s, 64°C for 20 s, 72°C for 20 s, and 78°C for 2 s. The annealing temperature was reduced to 60°C to analyse transcripts of the prft3–prft5 genes and the α-tubulin gene. This cycle was repeated 40 times. Fluorescence quantification was carried out before and after the incubation at 78°C to monitor the formation of primer-dimers. A reaction mixture without reverse transcriptase was used as a control to confirm that no amplification occurred from genomic DNA contaminants in the RNA sample. Primers for PCR are listed in Table S1. In all PCR experiments, amplification of a single DNA species was confirmed by both melting curve analysis of real-time PCR and gel electrophoresis of PCR products. The analysis was confined to the prft1–prft5 genes because we were not able to find a PCR condition that ensured prft6-specific amplification of transcripts. The mRNA level of the α-tubulin gene was used as a control for the analysis because expression of the gene is unaffected by low temperatures (Wei & Chatterton, 2001; Wei et al., 2002; Zhu et al., 2005).

Analysis of carbohydrates

Total water-soluble sugars were extracted using finely chopped leaf blade or crown tissues from cold-treated perennial ryegrass plants. Cold treatment and harvesting of tissues from cold-treated plants were performed as described above. Tissues were finely chopped and boiled in deionized water for 1 h. The extracts were passed through a filter with a pore size of 0.45 µm. Total sugars were analysed by HPLC with a combination of Shodex columns KS-802 and KS-803 (Shodex, Tokyo, Japan), with a refractive index detector as described by Yoshida et al. (1998).

Statistical analysis

Pearson's correlation coefficients were calculated between the levels of total fructan and sucrose in both leaf and crown tissues, based on average contents at each time point in the cold treatment. Correlations were also calculated between changes in levels of total fructan content and mRNA levels of FT genes, based on average levels of total fructan content and FT mRNA level (ΔCT) relative to theα-tubulin mRNA level obtained by real-time RT–PCR analysis at each time point in the cold treatment.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation and sequence analyses of FT cDNA clones from perennial ryegrass

In order to obtain cDNAs for FT genes from perennial ryegrass, a cDNA library was constructed using RNA isolated from crown tissues of cold-treated plants and screened with a suitable probe (for details see Materials and Methods). Thirty-eight clones containing sequences highly homologous to FT genes from other plants were produced. Six of these clones contained open reading frames (ORFs) ranging between 1.8 and 2.0 kb, while the remaining clones contained only portions of the ORFs. The six clones were designated as prft1prft6 (after perennial ryegrass FT genes). The deduced amino acid sequence of the prft1 protein was 96% (599/624 amino acids) and 97% (602/623 amino acids) identical, respectively, to the prft2 and prft6 amino acid sequences. Similarly, the sequences of the prft3 and pfrt5 proteins were 90% (583/651 amino acids) identical to each other. Thus the six cDNAs formed three groups: one comprising the prft1, prft2 and prft6 genes; one comprising the prft3 and prft5 genes; and one just containing the pfrt4 gene. Comparisons of the amino acid sequences revealed the presence of highly conserved amino acid residues from all over the protein sequences based on the prft1–prft6 genes and cognate proteins identified previously (Fig. 1). FTs belong to the glycosyl hydrolase 32 family, and contain three conserved sequence motifs, NDPNG, FRDP and WECXD (Verhaest et al., 2005). The crucial catalytic acids in these motifs were conserved in the prft1, prft3 and prft4 proteins and in the FTs from other plants (Fig. 1), as well as in the prft2, prft5 and prft6 proteins (data not shown). Two FT cDNAs have been functionally characterized in perennial ryegrass (Chalmers et al., 2003; Lasseur et al., 2006). Comparisons of the prft1–prft6 protein sequences with these perennial ryegrass FTs indicated that the prft4 protein is almost completely (651/653 amino acids) identical to the 1-SST protein (Chalmers et al., 2003), and the prft3 and prft5 proteins are 99% (641/645 amino acids) and 90% (585/651 amino acids) identical, respectively, to the 6G-FFT protein (Lasseur et al., 2006).

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Figure 1. Comparisons of amino acid sequences deduced from the cDNAs of the prft1, prft3 and prft4 genes in perennial ryegrass (Lolium perenne) and related genes in other plants. The database accession numbers of sequences are as follows: prft1, AB186920; Hordeum vulgare 6-SFT, X83233; Triticum aestivum 6-SFT (Wft1), AB029887; prft4, AB288056; T. aestivum 1-SST (Wft2), AB029888; prft3, AB125218; Alium cepa 6G-FFT, Y07838. Sequences were aligned using ClustalW. Identical amino acids are highlighted in black. The NDPNG, FRDP and WECXD motifs are boxed. The three crucial catalytic amino acids are indicated by arrows. – indicates lack of amino acid.

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Phylogenetic analysis of protein sequences deduced from the nucleotide sequences of FT genes and invertase genes isolated from various plants was carried out (Fig. 2). Sequences for the prft1–prft6 proteins were grouped with FT proteins from other species in the Gramineae in a phylogenetic tree. The groups comprising the prft1, prft2 and prft6 proteins, the prft3 and prft5 proteins, and the prft4 protein were located on different clades, as expected from the sequence comparisons. Sequences of the prft1, prft2 and prft6 proteins were located within a clade comprising the 6-SFT proteins of Triticum aestivum, Hordeum vulgare, Agropyron cristatum and Poa secunda. The prft4 protein sequence was located within a clade comprising the 1-SST of L. perenne and Festuca arundinacea. The sequences for the prft3 and prft5 proteins were located within a clade comprising 6G-FFT of Lperenne, as expected from the sequence comparison. The branch formation of all these sequences was supported by a high bootstrap value.

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Figure 2. Phylogenetic relationships between fructosyltransferases and invertases in plants. The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) based on protein sequences deduced from the nucleotide sequences of the genes. Protein sequences of cell wall invertases were used as the outgroup to root the tree. Bootstrap values of 1000 replicates are shown. The following sequences were included in the analysis. Ace 1-SST, Allium cepa 1-SST (AJ006066); Ace 6G-FFT, A. cepa 6G-FFT (Y07838); Acr 6-SFT, Agropyron cristatum 6-SFT (AF211253); Ao FT1, Asparagus officinalis 6G-FFT (AB084283); Bv vaINV, Beta vulgaris acid vacuolar invertase (AJ422051); Ci 1-SST, Cichorium intybus 1-SST (U81520); Cs 1-FFT, Cynara scolymus 1-FFT (AJ000481); Cs 1-SST, C. scolymus 1-SST (Y09662); Fa 1-SST, Festuca arundinaceae 1-SST (AJ297369); Ht 1-FFT, Helianthus tuberosus 1-FFT (AJ009756); Ht 1-SST, H. tuberosus 1-SST (AJ009757); Hv 1-SST, Hordeum vulgare 1-SST (AJ567377); Hv 6-SFT, H. vulgare 6-SFT (X83233); Le cwINV, Lycopersicon esculentum cell wall invertase (AF506006); Lp 1-SST, Lolium perenne 1-SST (AF492836); Lp 6G-FFT, L. perenne 6G-FFT (AF492836); Nt vINV, Nicotiana tabacum vacuolar invertase (CAC83577); Os cwINV, Oryza sativa cell wall invertase (AB073749); Os cwINV2, O. sativa cell wall invertase 2 (AY340072); Os cwINV3, O. sativa cell wall invertase 3 (AY342320); Os vaINV, O. sativa vacuolar acid invertase (AF276704); Os vaINV2, O. sativa vacuolar acid invertase 2 (AF276703); Ps 6-SFT, Poa secunda 6-SFT (AF192394); Ta 6-SFT Wft1, Triticum aestivum 6-SFT (AB029887); Ta 1-SST Wft2, T. aestivum 1-SST (AB029888); Ta 1-FFTa Wft3, T. aestivum 1-FFT-A (AB088409); Ta1-FFTb Wft4, T. aestivum 1-FFT-B (AB088410); Ta vINV1, T. aestivum vacuolar invertase 1 (AY575717); To 1-SST, Taraxacum officinale 1-SST (AJ250634); Zm cwINV2, Zea mays cell wall invertase 2 (AF165179).

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Genetic mapping of FT gene loci and estimation of copy number of the FT genes

In order to locate FT genes on the perennial ryegrass linkage map, a PCR-based segregation analysis of genomic DNA from the Perma/Aurora F2 mapping population (Armstead et al., 2004) was carried out. We were able to detect polymorphisms between the parental lines from Perma P1 and Aurora P2 for genomic DNA regions based on the pfrt1, prft3 and prft4 genes (Fig. 3a,b,d).

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Figure 3. Localization of the prft1, prft3 and prft4 genes on the perennial ryegrass (Lolium perenne) linkage map. (a) Polymorphisms of PCR-amplified fragment of the prft1 gene between parental plants and segregation in the F2 mapping population. (b) Polymorphisms of SacII-digested PCR-amplified fragment of the prft4 gene between parental plants and segregation in the F2 mapping population. (c) Location of the prft1 and prft4 genes on linkage group (LG) 7. (d) Polymorphisms of StuI-digested PCR-amplified fragment of the prft3 gene between parental plants and segregation in the F2 mapping population. (e) Location of the prft3 gene on LG3. Positions of other molecular markers on LG7 and LG3 (Armstead et al., 2004) are indicated by vertical lines. Map distances between the FT genes and the two flanking molecular markers are shown in cM. P1, Perma; P2, Aurora. Polymorphic fragments derived from P1 and P2 are indicated by filled and open arrows, respectively (a,b,d). ?X174 DNA digested with HaeIII was loaded as a size marker (M).

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The prft1 sequences of P1 and P2 were distinguished from each other by amplification of a 282-bp fragment that was specifically amplified in P2, but not in P1 (Fig. 3a), using primers of which one annealed to a short DNA segment present only in an intron of the gene in P2. The primers also amplified another nonpolymorphic fragment from both P1 and P2 (Fig. 3a), which could be distinguished from the prft1 polymorphism by gel electrophoresis and could come from amplification within a cognate gene copy.

Nucleotide sequences for the prft3 and the prft4 genes were distinguished between parental lines by the presence or absence of restriction sites within PCR-amplified fragments. A portion of the prft3 gene amplified from P1 contained a StuI site, while the corresponding DNA fragment from P2 did not (Fig. 3d). Similarly, a portion of the prft4 gene amplified from P2 contained a SacII site, while the corresponding DNA fragment from P1 did not (Fig. 3b).

Using these polymorphisms, segregation of the genes in the F2 population was determined (Fig. 3a,b,d) and the loci were mapped. The prft1 and prft4 genes were both located within the interval between markers INV1:2 and R2896 in the distal part of the linkage group (LG) 7 (Fig. 3c). The prft3 gene was located within the interval between markers CHO71673.F/R and 3MS12 in the distal part of the LG3 (Fig. 3e).

Copy number of the prft1, prft3 and prft4 genes in the perennial ryegrass genome was estimated by a gel-blot analysis of total DNA isolated from a single individual plant of cv. Riikka using the 3′-UTRs of these genes as probes (Fig. 4). No fragment that cross-hybridizes with different probes was detected, which indicates the high specificity of these probes. Two or three hybridization signals, excluding weakly hybridized ones, were detected per lane. This suggests that copy numbers of each of the prft1, prft3 and prft4 genes are one or two, or possibly two or three, depending on the level of heterozygosity in the genotype used.

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Figure 4. Gel-blot analysis of perennial ryegrass (Lolium perenne) genomic DNA using fructosyltransferase (FT) genes as probes. Total DNA isolated from cv. Riikka was digested with SacI (S), DraI (D), HindIII (H), EcoRI (E) or BamHI (B), and was hybridized with probes specific to the 3′-UTR of prft1 (a), prft3 (b) and prft4 (c) genes. Lambda phage DNA digested with HindIII was loaded as a size marker (M).

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Functional characterization of the FT cDNAs

The prft1–prft6 cDNAs were expressed in P. pastoris, and the products were used for an in vitro functional analysis using sucrose, 1-kestose, or 6-kestose as a substrate. The recombinant protein from the prft4 gene produced 1-kestose when sucrose was provided in the reaction mixture, which indicates that prft4 encodes 1-SST (Fig. 5g; for the synthetic pathways see Fig. 8).

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Figure 5. Anion-exchange HPLC analysis of oligofructans produced by incubation of sucrose or 1-kestose with prft1–prft6 recombinant proteins expressed in Pichia pastoris cells. The reactions were conducted with 50 mm sucrose (a,c,e,g,i,k) or 50 mm 1-kestose (b,d,f,h,j,l) at 25°C for 24 h. Recombinant proteins of the prft1 (a,b), prft2 (c,d), prft3 (e,f), prft4 (g,h), prft5 (i,j), and prft6 (k,l) genes were used for the analysis. Compounds were detected by pulsed amperometric detection and identified using external standards, sucrose, fructose, glucose, 1-kestose and 1-nystose. Neo-kestose, 1&6G-kestotetraose and 1,6G-kestotetraose were identified by the retention times of reaction products of asparagus 6G-FFT, AoFT1 (Ueno et al., 2005). Suc, sucrose; Fru, fructose; Glc, glucose; 1-k, 1-kestose; Nys, 1-nystose; 6G, neo-kestose; 1&6G, 1&6G-kestotetraose; 1,6G, 1,6G-kestotetraose.

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The recombinant proteins from the prft3 and prft5 genes both produced 1&6G-kestotetraose (labelled ‘1&6G’ in Fig. 5f,j), and neo-kestose (‘6G’) when 1-kestose was provided in the reaction mixture. Sucrose and fructose were also released, indicating the presence of 1-kestosidase-like activity in the protein extracts (Fig. 5f,j). The production of 1&6G-kestotetraose and neo-kestose typically indicates that the prft3 and prft5 both encode the 6G-FFT (Fig. 8). 1-Nystose (‘Nys’) was also produced by incubating prft3 or prft5 proteins with 1-kestose (Fig. 5f,j), which indicates that these proteins also have 1-FFT activity. This was consistent with the production of 1,6G-kestotetraose (‘1,6G’) by the prft5 protein (Fig. 5j). The presence of 1-FFT activity in the 6G-FFT protein, as well as the production of these oligosaccharides, has also been detected in similar experiments using recombinant proteins from perennial ryegrass (Lasseur et al., 2006), asparagus (Ueno et al., 2005) or onion (Ritsema et al., 2003).

No prominent peaks indicative of reaction products from FT activity were detected when recombinant proteins from the prft1, prft2 and prft6 genes were incubated with the substrates, except for a minor peak corresponding to 1-nystose when 1-kestose was provided in the reaction mixture (Fig. 5b,d,l). No product was synthesized by incubating 6-kestose with any of the proteins (data not shown). Identical chromatography patterns were detected between reactions performed at 25 and 4°C for all the recombinant proteins, except that a 10–20% reduction in peak reaction products was detected at 4°C for recombinant proteins from the prft3–prft5 genes.

Changes in levels of carbohydrate in response to low temperature

Changes in fructan and sucrose contents and total glucose and fructose contents in response to low temperature were analysed. Plants were subjected to cold treatment using a day temperature of 6°C for 8 h and a night temperature of 2°C for 16 h. The fructan content in both leaf and crown tissues increased dramatically with prolonged cold treatment for at least up to 30 d (Fig. 6a,b; for total fructan content see Fig. S1). In crown tissues, the fructan level increased in plants that were cold-treated for 4 d. Total fructan content increased 13-fold and 29-fold in leaf and crown tissues, respectively, and was threefold higher in crown tissues than in leaf tissues, after 30 d of the cold treatment (Fig. S1).

image

Figure 6. Changes in fructan and sugar contents during cold treatment of perennial ryegrass (Lolium perenne) plants. Fructan contents in (a) leaf tissue; (b) crown tissue. The contents of fructans comprising three sugar units (trisaccharides, open bars), fructans comprising four to six sugar units (oligofructans, grey bars), and fructans comprising seven or more sugar units (long-chain fructans, black bars) are shown. Sucrose (hatched bars) and total glucose plus fructose contents (stippled bars) in (c) leaf tissue; (d) crown tissue. Tissues were sampled after cold-treatment for 0, 1, 4, 7, 14 and 30 d, and were subjected to quantification by HPLC. Data represent mean ± SE obtained from three replicates of the analysis.

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In addition to the total fructan content, the content of fructans comprising three sugar units (trisaccharides), fructans comprising four to six sugar units (oligofructans), and fructans comprising seven or more sugar units (long-chain fructans) were quantified separately (Fig. 6a,b). An increase in trisaccharides was detected during cold treatment in both leaf and crown tissues, indicating de novo synthesis of fructans by the treatment. Although all three classes of fructan increased as a consequence of cold treatment in both tissues, there was a marked difference in the relative amount of fructans of different size classes between leaf and crown tissues. In leaf tissues, trisaccharide and long-chain fructan levels were similar to each other, while the oligofructan level was lower than the levels of these fructans at most time points during the cold treatment for up to 14 d. After 30 d of cold treatment there was no significant difference between trisaccharide and oligofructan levels, which were only slightly lower than the level of long-chain fructans (Fig. 6a). By contrast, in crown tissues there was a pronounced increase in the proportion of long-chain fructans with an increase in the period of cold treatment (Fig. 6b). This also revealed that the observed higher level of total fructan in crown tissues after 30 d cold treatment was attributed mostly to the increase in the level of long-chain fructan molecules. Collectively, these data indicate that small fructan molecules are synthesized de novo from sucrose and are sequentially converted into longer-chain fructan molecules during cold treatment. As a consequence of these reactions, fructans are mostly accumulated as long molecules, especially in crown tissues after a long period of cold treatment.

Sucrose content decreased during the first 4 d of cold treatment and then increased continuously during longer cold treatment for up to 30 d (Fig. 6c,d). There was a highly significant positive correlation between changes in the levels of fructan and sucrose in both leaf and crown tissues (correlation coefficients 0.97 and 0.85, respectively). No significant changes were detected for total glucose and fructose contents (Fig. 6c,d).

Changes in the mRNA level of FT genes in response to low temperature

The mRNA levels for the prft1 and prft2 genes increased gradually through the period of cold treatment in both leaf and crown tissues (Fig. 7a–d). A pronounced increase was initiated after approx. 7 d of cold treatment, which continued for at least 30 d. The pattern of changes in mRNA levels for the prft3, prft4 and prft5 genes was different from that for the prft1 and prft2 genes. In particular, the mRNA level for the prft3 and prft4 genes increased within the first 24 h of cold treatment in leaf tissues. The level decreased after the initial increase, and then increased again during longer cold treatment (Fig. 7e–j). Thus, among the genes we identified, there are at least two different patterns of change in mRNA levels in response to low temperature.

image

Figure 7. Changes in mRNA levels of prft1–prft5 genes during cold treatment of perennial ryegrass (Lolium perenne) plants. Real-time RT–PCR analysis of RNA extracted from leaf (a,c,e,g,i) or crown (b,d,f,h,j) tissues of plants cold-treated for 0, 1, 4, 7, 14 and 30 d (left to right in each panel). The mRNA levels of prft1 (a,b), prft2 (c,d), prft3 (e,f), prft4 (g,h), and prft5 (i,j) were assessed relative to theα-tubulin mRNA level. Data represent mean ± SE obtained from three replicates of the analyses.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification and mapping of FT genes in perennial ryegrass

We isolated six cDNAs encoding FTs in perennial ryegrass. Expression of recombinant protein in P. pastoris demonstrated that the prft4 gene encodes 1-SST, and the prft3 and the prft5 genes both encode 6G-FFT. High sequence similarity with perennial ryegrass cDNAs that have been functionally characterized (Chalmers et al., 2003; Lasseur et al., 2006) supports these results. For unknown reason(s), no prominent reaction occurred when we used recombinant protein obtained by expressing the prft1, prft2 or prft6 cDNAs in our system. The location of the prft1, prft2 and prft6 protein sequences within a clade comprising 6-SFT sequences from various plants in the phylogenetic analysis indicates that the prft1, prft2 and prft6 genes might encode 6-SFT.

The linkage map position of the prft1, prft3 and prft4 genes was established using a perennial ryegrass mapping population derived from a cross between inbred lines derived from the Perma and Aurora cultivars. Both prft1 and prft4 genes mapped on the distal region of LG7 in the proximity of the locus of an invertase gene (Armstead et al., 2004; Fig. 3c). Three FT genes have previously been mapped to perennial ryegrass LGs using a different mapping population derived from a pair-cross between North African6 (NA6) and Aurora6 (AU6) plants (Chalmers et al., 2005). The 1-SST gene (Lp1-SST; marker xlp1-sst) has been mapped to a distal region of LG7 of NA6. Although the alignment between these different genetic maps has not been fully established, it is very likely that the position of the prft4 gene corresponds to that for xlp1-sst, taking into account that the prft4 gene encodes 1-SST. In NA6, an FT gene marker (xlpft1), the function of which has not been characterized, has also been mapped adjacent to xlp1-sst (Chalmers et al., 2005). It is possible that the prft1 gene corresponds to xlpft1. Alternatively, it is possible that the prft1 gene is not identical to xlpft1 and that multiple FT genes are located in this chromosomal region. The 6-SFT gene in barley has been mapped to the distal region of the short arm of chromosome 1 (7H) corresponding to LG7 of perennial ryegrass (Wei et al., 2000), suggesting that the prft1 gene might encode 6-SFT. In the distal part of LG7, the prft1 and prft4 genes were closely linked with INV1:2, a gene for soluble invertase (Gallagher et al., 2004; Fig. 3c). Considering the possibility that FT genes originated from an invertase gene in plants (Sprenger et al., 1995; Vijn & Smeekens, 1999; Francki et al., 2006), it is possible that colocalization of the FT genes with the invertase gene in this chromosomal region arises from a previous duplication of the invertase gene.

The prft3 gene was mapped to the distal region of perennial ryegrass LG3. An FT gene marker (xlpft4), the function of which has not been characterized, has also been mapped to a distal region of LG3 of AU6 (Chalmers et al., 2005). Likewise, xlpft4 might be identical to the prft3 gene and encode 6G-FFT.

Very high sequence similarity between the prft1 or prft2 and prft6 genes, or between the prft3 and prft5 genes, suggests that some of these genes may be allelic. Similarly, the prft4 gene and the reported 1-SST gene (Chalmers et al., 2003), or the prft3 or prft5 genes and the reported 6G-FFT gene (Lasseur et al., 2006), may also be allelic. These possibilities remain to be examined by a more extensive analysis of the perennial ryegrass genome.

Changes in fructan content as an adaptive response to freezing temperatures and transcriptional regulation of FT genes in response to low temperature

Significant accumulation of fructans was detected in both leaf and crown tissues during cold treatment. There was little increase in plant size during cold treatment. We have measured fructan content in plants that were not cold-treated and were kept for the same period at a normal temperature (18–22°C). We found that fructan levels in plants kept at normal temperature for 14 d corresponded to 15.4 and 18.7% of those of cold-treated plants in leaf and crown tissues, respectively. Although this experiment should not be regarded as a control for the cold treatment, because plants grow considerably faster at a normal temperature, a larger increase in fructan content in cold-treated plants than in plants grown at normal temperature indicates that the observed accumulation of fructan during cold treatment involves cold-specific effects that are considerably larger than developmental effects.

Previous tests of freezing tolerance using crown tissues have shown that a similar cold treatment confers significant freezing tolerance in various perennial ryegrass accessions. For instance, the survival rate of cv. Riikka under freezing conditions at –6°C for 16 h increased from 0 to 62% following treatment (Yamashita et al., 1993). In general, the freezing tolerance of plants increases in proportion to the period of cold acclimation. More than 7 d cold treatment is required to achieve the maximum level of freezing tolerance in perennial ryegrass (Lorenzetti et al., 1971). As a consequence, changes in the fructan content during cold treatment parallel the acquisition of freezing tolerance through cold acclimation, suggesting that fructan may be closely associated with the transition to enhanced freezing tolerance.

Previous expression analyses of FT genes have shown that transcript profiles are generally consistent with both enzymatic activity measurements and levels of fructan accumulation (reviewed by Chalmers et al., 2005), suggesting that the cellular level of fructan is primarily regulated at the level of transcription of FT genes. Analysis of FT gene expression, however, has been carried out mainly during changes in the development of plants (Lüscher et al., 2000; Koroleva et al., 2001; Lidgett et al., 2002; Chalmers et al., 2003; Johnson et al., 2003; Gallagher et al., 2004; Lasseur et al., 2006), and very few analyses of expression of FT genes under cold treatment are known (see below). Moreover, there have been only a few reports that describe relationships between the expression of different FT genes (Wang et al., 2000; Lasseur et al., 2006). Our present data demonstrate for the first time in plants that the transcription of multiple FT genes is induced in a coordinated way by low temperature, and occurs in parallel with the accumulation of fructans.

Two distinct patterns of change in mRNA levels during cold treatment were detected. One pattern involves an abrupt increase in mRNA within the first 24 h of cold treatment, which is followed by a decrease after several days of cold treatment and then an increase during further cold treatment over at least 30 d. This pattern was observed for the prft3 (6G-FFT) and prft4 (1-SST) genes in leaf tissues. The induction of this gene expression in the first 24 h of cold treatment in both leaf and crown tissues probably reflects the initiation of fructan synthesis using sucrose as a substrate, and may also account for the increased fructan content of crown tissues in plants cold-treated for 4 d. This is consistent with the roles of 6G-FFT and 1-SST in the initiation of fructan synthesis (Fig. 8).

Such a two-step change in mRNA level resembles changes in the levels of carbohydrate detected in plants that undergo cold treatment (reviewed by Gaudet et al., 1999). Our electron microscope analyses have also revealed a decrease in mesophyll cell size subsequent to the initial increase during the early period of cold treatment, before it increases again during a longer period of cold treatment in perennial ryegrass (Tominaga et al., 2004). These phenomena probably reflect a response to an initial shock of cold treatment followed by a longer period of adaptation, which can be widely observed in cold-tolerant plants.

Changes in the mRNA level of putative 6-SFT genes during cold treatment in similar controlled conditions have been analysed in wheatgrass (Wei & Chatterton, 2001) and big bluegrass (Wei et al., 2002). An increase followed by a decrease in mRNA level during 15 d of cold treatment was obtained, which corresponds to the pattern found for the prft3 and prft4 genes in the present study. The wheatgrass results also indicated that the level of low molecular-weight fructan increases during the early period of cold treatment. However, because wheatgrass analyses were confined to changes in the mRNA levels of FT genes only up to 15 d of cold treatment, increases in mRNA levels during longer periods of cold treatment, as detected in the present study, were not investigated. We consider that a seasonal increase in the mRNA levels of FT genes observed in field-grown plants towards winter (Kawakami & Yoshida, 2002; Van Laere & Van den Ende, 2002) may be similar to the changes detected in the plants undergoing long-term cold treatment in the present study.

The other pattern is a gradual increase in the mRNA level over a long period of the cold treatment, which was observed for the prft1 and prft2 genes. Although we have not been able to determine the function of the prft1 and prft2 proteins by recombinant protein analysis, the expression profiles found during cold treatment suggest that the prft1 and prft2 proteins are mainly involved in long-term fructan accumulation. The gradual increase in mRNA level may be associated with the acquisition of freezing tolerance and/or storage of carbohydrates as an adaptive response to the onset of winter, as reported in wheat (Kawakami & Yoshida, 2002). Plants that accumulate fructans can store fructose units while keeping osmotic potentials within a limited range. Thus proteins encoded by the prft1 and prft2 genes are likely to be involved in the production of fructans with a large number of linear or branched polymers of fructose units. The results of the phylogenetic analysis (Fig. 2) suggest that 6-SFT may be a likely candidate for the protein encoded by the prft1 and prft2 genes as well as the homologous prft6 gene. Whether 6-SFT activity is present in Lolium species remains to be confirmed, because bifurcose, which is indicative of 6-SFT activity, has not been detected (Pavis et al., 2001b; Chalmers et al., 2005). Fructan-fructan 6-fructosyltransferase (6-FFT) may also be a likely candidate because it may also synthesize large fructan molecules, although the presence of this enzyme has only been postulated (Pavis et al., 2001b).

As a gradual increase was observed in the mRNA levels of the prft3 and prft5 (6G-FFT) genes and the prft4 (1-SST) gene following cold treatment for longer than 7 d, the prft3, prft4 and prft5 genes may also be involved in adaptation to cold conditions, possibly through supplying smaller fructan molecules, which can be used as substrates for polymerization of fructose units.

There is a high positive correlation between the levels of fructan and FT mRNA in response to cold treatment (Table S2), which is more pronounced than those reported previously for developmental changes (Gallagher et al., 2004; Lasseur et al., 2006). This suggests that relationships between fructan accumulation during cold treatment and transcriptional control of FT genes are much stronger than relationships between developmental controls of fructan level and the transcription of FT genes. It should also be noted that a marked difference in the relative amount of fructans of different size classes between leaf and crown tissues (Fig. 6) may reflect a difference in fructan synthesis and/or metabolism between source and sink organs. Because the patterns of change in the mRNA levels of the prft1–prft5 genes in leaf and crown tissues are very similar to each other (Fig. 7), a plausible interpretation of the observed differences in fructan size classes between these tissues is that fructans accumulate mainly in crown tissues as long-chain molecules suitable for carbohydrate storage, whereas in leaf tissues fructans are metabolized while being synthesized, which restricts the preferential accumulation of long-chain fructans.

Overall, these results indicate that there are at least two patterns of expression for FT genes in response to low temperature. One involves induction of FT gene expression in response to both cold shock and a long period of cold conditions. The other pattern involves the induction of FT gene expression in response to a long period of cold only. These different patterns of expression have been assigned to FT genes of different functions. This coincidence between gene expression and function of FTs suggests that the genes have acquired diversity during evolution in both function and gene expression in response to cold conditions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Yoshiya Shimamoto, Jun Abe, Tetsuya Yamada and Sohei Kobayashi for valuable suggestions on genetic studies in perennial ryegrass, Atsuo Kimura, Masayuki Okuyama, Keiji Ueno and Akira Kawakami for helpful advice on functional analysis of recombinant proteins, Yoko Tominaga for valuable information regarding cold-inducible genes, and Azusa Kameyama for technical assistance in genotyping FT genes. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Changes in total fructan content during cold treatment of plants: fructan content in (a) leaf tissue; (b) crown tissue. Tissues were sampled after cold-treatment for 0, 1, 4, 7, 14, and 30 d, and were subjected to quantification by HPLC. Data represent the mean and SE obtained from three replicates of the analysis.

Table S1 List of PCR primers used in the present study

Table S2 Correlation coefficients between changes in fructan content and mRNA levels of FT genes during cold treatment

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FilenameFormatSizeDescription
NPH_2409_sm_FigS1.ppt166KSupporting info item
NPH_2409_sm_TableS1.xls26KSupporting info item
NPH_2409_sm_TableS2.xls19KSupporting info item