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Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance Abstract Fructans are water-soluble fructose oligomers and polymers that are based on sucrose, and have been implicated in protecting plants against water stress. Rice (Oryza sativa L.) is highly sensitive to chilling temperatures, and is not able to synthesize fructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase, encoded by wft2, or sucrose:fructan 6-fructosyltransferase, encoded by wft1, were introduced into rice plants, and rice transformants that accumulate fructans were successfully obtained. The mature leaf blades of transgenic rice lines with wft2 or wft1 accumulated 16.2 mg g−1 FW of oligo- and polysaccharides mainly composed of inulin oligomers of more than DP7, and 3.7 mg g−1 FW of oligo- and polysaccharides, mainly composed of phlein oligomers of more than DP15, respectively. The transgenic rice seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than non-transgenic rice seedlings, and exhibited enhanced chilling tolerance. The oligo- and polysaccharide concentrations of seedlings expressing wft1 were obviously lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage. This is the first report owing that fructan accumulation enhanced tolerance to non-freezing low temperatures. Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant Introduction Rice (Oryza sativa L.) originated in tropical and subtropical climates and is very sensitive to low temperatures (Graham and Patterson, 1982). Expansion of rice cultivation into regions that experience periodic or sustained low temperatures has increased the risk of crop loss through chilling injury. Tolerance to low temperatures during seed emergence and the early growth stages is an important characteristic for rice seedling establishment, particularly where direct sowing cultivation is practised in colder regions, such as Hokkaido, the northernmost large island of Japan. The first visible symptom of rice seedling damage caused by chilling is wilting, and prolonged low temperatures result in complete dehydration of the leaves. It is thought that transpiration exceeds the rate of water absorption through the root system under low temperature conditions, causing a loss of turgor in leaf tissues (Kabaki and Tajima, 1981). One of the primary plant responses for adaptation to water deficit stress is the accumulation of solutes such as amino acids (e.g. proline), quaternary ammonium compounds (e.g. glycinebetaine), polyols, and sugars (e.g. mannitol, trehalose, sucrose, and fructans) that act as osmoprotectants (Bohnert et al., 1995; Hare et al., 1998; Nuccio et al., 1999). Fructans, a class of water-soluble fructose polymers based on sucrose, accumulate in many bacterial and plant species, in which they serve as an important storage carbohydrate (Pollock and Cairns, 1991) and are implicated in protecting plants against water deficit caused by low matric potential, salinity, or low temperatures (Pontis, 1989; Hendry, 1993; Spollen and Nelson, 1994). Bacterial fructans (levans) are produced extracellularly by the enzymes levansucrase or fructosyltransferase, which produce levans directly from sucrose (Vijn and Smeekens, 1999). In plants, fructans are synthesized in vacuoles from sucrose by the action of two or more different fructosyltransferases, including sucrose:sucrose 1-fructosyltransferase (1-SST), sucrose:fructan 6-fructosyltransferase (6-SFT), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan:fructan 6G-fructosyltransferase (6G-FFT) (Vijn and Smeekens, 1999). Following the initial cloning of Hordeum vulgare (barley) 6-SFT (Hv6-SFT; Sprenger et al., 1995), several genes encoding enzymes associated with fructan biosynthesis have been cloned from various plants, including 1-SST and 1-FFT from Cichorium intybus (chicory; de Halleux and Van Cutsem, 1997; Goblet et al., 1999), Cynara scolymus (globe artichoke; Hellwege et al., 1997, 1998), and Helianthus tuberosus (Jerusalem artichoke; van der Meer et al., 1998); 1-SST and 6G-FFT from Allium cepa (onion; Vijn et al., 1997, 1998); and 1-SST and/or 6-SFT from temperate grasses such as Festuca arundinacea (tall fescue; Lüscher et al., 2000), Agropyron cistatum (crested wheatgrass; Wei and Chatterton, 2001), Poa secunda (big bluegrass; Wei et al., 2002), Lolium perenne (perennial ryegrass; Lidgett et al., 2002; Chalmers et al., 2003), and H. vulgare (Nagaraj et al., 2004). 1-SST and 6-SFT, designated wft2 and wft1, from Triticum aestivum (wheat) were also isolated, and it was found that those two genes are involved in fructan accumulation during cold hardening (Kawakami and Yoshida, 2002). Metabolic engineering for the biosynthesis of fructans has been the focus of considerable attention as a potential strategy for increasing water stress tolerance. Fructan-synthesizing genes have been introduced into non-fructan-accumulating plants such as Nicotiana tabacum (tobacco; Ebskamp et al., 1994; Pilon-Smits et al., 1995; Caimi et al, 1997), Solanum tuberosum (potato; van der Meer et al., 1994; Röber et al., 1996; Pilon-Smits et al., 1996; Caimi et al., 1997), Beta vulgaris (sugar beet; Pilon-Smits et al., 1999), Trifolium repens (white clover; Jenkins et al., 2002), and Zea mays (maize; Caimi et al., 1996) by ectopically expressing levansucrase or fructosyltransferase genes isolated from Bacillus spp., Erwinia amylovora, and Streptococcus spp. Some of those transgenic plants exhibited high fructan accumulation and water stress tolerance. For example, tobacco and sugar beet plants expressing bacterial levansucrase exhibited increased drought tolerance (Pilon-Smits et al., 1995, 1999), freezing tolerance (Konstantinova et al., 2002), or osmotic tolerance (Park et al., 1999). In some cases, aberrant growth phenotypes such as leaf bleaching, necrosis, and stunting were observed in transformants expressing bacterial fructan synthesis genes (Cairns, 2003). Transgenic plants carrying genes encoding plant fructosyltransferases have also been produced and mainly used for functional identification of introduced genes and analysis of fructan accumulation in non-fructan-synthesizing plants. For instance, Hv6-SFT was introduced into tobacco and chicory (Sprenger et al. 1997), the Jerusalem artichoke 1-SST gene and/or 1-FFT gene was introduced into sugar beet (Sévenier et al., 1998) and petunia (van der Meer et al., 1998), and the globe artichoke 1-SST-and/or 1-FFT-encoding genes were introduced into potato (Hellwege et al., 2000). By contrast to levansucrase genes from bacteria, no phenotypic aberrations have been observed in transformants expressing plant fructosyltrannsferase genes (Cairns, 2003). Hisano et al. (2004) reported that transgenic perennial ryegrass expressing wheat 1-SST or 6-SFT genes accumulated more fructans and acquired higher tolerance for freezing at the cellular level compared with non-transgenic plants, but there have been no other reports on increasing chilling stress tolerance (i.e. non-freezing, low temperatures) in transgenic plants carrying fructosyltransferase genes. Rice plants do not accumulate fructans, and seedlings are susceptible to injury at temperatures between freezing and 10 °C. Cairns (2003) indicated that non-fructan plants do not accumulate large amounts of fructans with the introduction of plant-derived fructosyltransferase genes, but because of the potential benefit to rice agronomy in marginal climatic conditions, it would be interesting to know if transgenic rice lines expressing wheat-derived fructosyltransferases accumulate sufficient fructan concentrations in mature leaf blades to affect chilling tolerance in rice seedlings. Two fructosyltransferase genes, encoding 1-SST and 6-SFT, could prove to be useful tools for fructan production in non-fructan plants because both enzymes produce fructans from sucrose as the sole substrate. In this study, transgenic rice plants that accumulate fructans by overexpressing wft1 and wft2 (T. aestivum 6-SFT and 1-SST, respectively) were generated and their mature leaf blade carbohydrate concentrations, fructan oligomer composition, and the correlation between fructan accumulation and seedling chilling tolerance were analysed. Materials and methods Transformation and screening of transgenic plants Full-length wft1 (accession no. AB029887) or wft2 (accession no. AB029888) (Kawakami and Yoshida, 2002) were inserted into pMLH7133, a Ti-based vector, downstream of the first intron of the phaseolin gene under control of the cauliflower mosaic virus (CaMV) 35S promoter (Mitsuhara et al., 1996). The resulting plasmids, pMLH1733-wft1 and pMLH1733-wft2, were introduced into calli of the Japonicum rice cultivar Yukihikari by Agrobacterium-mediated transformation, according to a previously reported protocol (Tanaka et al., 2001). Transformed calli were selected for hygromycin resistance, and transgenic plants were regenerated as previously described (Tanaka et al., 2001). Presence of the transgene in T0 generation plants was examined by PCR amplification. T1 generation lines were screened by HPLC for accumulation of fructans. In the T2 generation, lines homozygous for a transgene were selected by PCR, and the transgene copy number was determined by genomic Southern hybridization. T3 transgenic rice lines were used for further analysis. Transgene transcription Total RNA was isolated from rice leaf tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Northern hybridization was done according to a standard protocol (Sambrook et al., 1989). Total RNA (15 μg) was separated by electrophoresis on 1.5% (w/v) agarose gels, blotted onto hybond N+ membranes (GE Healthcare, Little Chalfont, Bucks, UK), and hybridized with a α-32P-labelled cDNA fragment corresponding to Wft1 or Wft2. The blot was washed twice with 0.1× SSC and 0.1% SDS for 20 min at 65 °C and exposed to X-ray film at –80 °C. Carbohydrate extraction and analysis For analysis of water-soluble carbohydrates in mature leaf blades and shoots from seedlings of T3 transgenic lines and non-transgenic rice plants, the eighth or ninth leaf blades from plants grown for 2 months in a greenhouse at 25 °C/18 °C (12 h/12 h), and shoot and root tissues of seedlings grown for 10 d in a growth chamber at 26 °C/19 °C with a 16 h light/8 h dark cycle were harvested and stored at –80 °C. Total water-soluble carbohydrates were extracted from finely chopped tissues in boiling deionized water for 1 h. Total carbohydrates were analysed by HPLC with a combination of Shodex KS-802 and KS-803 columns (Shodex, Tokyo, Japan) using the refractive index detector as described by Yoshida et al. (1998). Glucose, fructose, sucrose, oligosaccharide, and polysaccharide concentrations were determined. For analysis of fructan oligomers with different glycosidic linkages, high-performance anion exchange chromatography (HPAEC) was performed on a DX 500 chromatograph (Dionex, Sunnyvale, CA, USA) with a Carbo Pac PA-1 anion exchange column and a pulsed amperometric detector (PAD) as described by Shiomi et al. (1997). Peaks for glucose, fructose, sucrose, 1-kestose, 1,1-kestotetraose, 1,1,1-kestopentaose (Wako, Osaka, Japan), and 6-kestose (Iizuka et al., 1993) were identified by comparison with authentic standards. Phlein oligomers based on 6-kestose and bifurcose were putatively identified by comparison of HPAEC retention times with fructan oligomers extracted from wheat tissues 8 d after anthesis, and products generated by recombinant wheat 6-SFT (Wft1) incubated with 100 mM sucrose at 10 °C for 120 h (Kawakami and Yoshida, 2005). Chilling tolerance assay Rice seeds were immersed in distilled water for 3 d at 27 °C, sown on soil in a plastic container, and then transferred to a growth chamber at 26 °C/19 °C with a 16 h light/8 h dark cycle. Ten-day-old seedlings were exposed to 5 °C with continuous light for 11 d. After the chilling treatment, seedlings were transferred to a greenhouse at 26 °C/19 °C with a 16 h light/8 h dark cycle and grown for a further 7 d. The percentage of surviving seedlings was determined with active growth as the main criterion for survivors, and wilting and non-growth as the criteria for scoring as non-survivors. Results Production of transgenic rice plants expressing wft1 or wft2 After regeneration of transformants carrying either wft2 or wft1, several hygromycin-resistant rice plants were self-pollinated and homozygous transgenic lines containing wft2 (I16, I22, I24, and I29) or wft1 (S33, S50, and S51) were obtained in the T3 generation. No apparent phenotypic abnormalities or fertility problems were observed in transgenic lines expressing wft1 or wft2 (Fig. 1). Fig. 1. Open in new tabDownload slide Phenotypes of transgenic rice lines expressing wft1 (6-SFT) or wft2 (1-SST). (A) Transgenic rice line expressing wft2 (I22), non-transgenic rice plant (Cont), and transgenic rice line expressing wft1 (S51) grown in an environmental control room for about 2 months. (B) Spikes of transgenic rice lines expressing wft2 (I16 and I22), wft1 (S50 and S51), and non-transgenic rice plants (Cont). Southern blot analysis revealed that the transgenic lines had one or two copies of the wft1 or wft2 gene in their genomes (data not shown). To verify transgene expression, wft1 and wft2 transcript accumulation was measured in non-transgenic rice plant controls and transgenic rice lines by northern hybridization. The levels of wft2 mRNA were almost the same among transgenic rice lines expressing wft2, but varied widely in wft1 transformants. The S33 line had a relatively low level of wft1 expression compared with S50 and S51 (Fig. 2). Fig. 2. Open in new tabDownload slide Expression of wft1 and wft2 in mature leaf blades of transgenic rice lines and non-transgenic rice plants. Total RNA samples (20 μg) extracted from non-transgenic rice plants (Cont), transgenic rice lines I16, I22, I24, I29 (wft2), S33, S50, and S51 (wft1) were fractioned by electrophoresis on a 1.2% formamide-containing agarose gel. Hybridization was carried out with α-32P-labelled probe for detecting wft1 or wft2. The corresponding ethidium bromide gel image of rRNA shows the relative amounts of total RNA loaded for each sample. Carbohydrate contents and composition of fructan oligomers in mature leaf blades of transgenic rice lines expressing wft1 or wft2 Oligo- and polysaccharide concentrations, and total carbohydrate concentrations in mature leaf blades of transgenic rice lines expressing wft2 were significantly higher than in non-transgenic rice plants (Table 1). Sucrose contents in transgenic and non-transgenic plants were not significantly different. The major HPAEC peaks of oligo- and polysaccharides were mainly fructans and inulin oligomers from 1-kestotriose (DP3) to 1,1,1,1,1-kestoheptaose (DP7), and small amounts of unknown oligosaccharides (Fig. 3). Table 1. Carbohydrate contents of mature leaf blades of non-transgenic and transgenic rice lines expressing wft1 (wheat 6-SFT) or wft2 (wheat 1-SST) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) The values are means ±standard deviation (n=3). Data for the transgenic plants were analysed for significant differences from the data for non-transgenic plants using Student's t-test. An asterisk indicates significance at P < 0.05. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Table 1. Carbohydrate contents of mature leaf blades of non-transgenic and transgenic rice lines expressing wft1 (wheat 6-SFT) or wft2 (wheat 1-SST) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) The values are means ±standard deviation (n=3). Data for the transgenic plants were analysed for significant differences from the data for non-transgenic plants using Student's t-test. An asterisk indicates significance at P < 0.05. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Fig. 3. Open in new tabDownload slide Anion exchange HPLC analysis of soluble carbohydrates in extracts from mature leaf blades of non-transgenic rice plants (a) and transgenic rice lines expressing wft1 (d) or wft2 (b). Inulin extracted from chicory roots was used as a standard for inulin oligomers, and the numbers (3–7) on each peak indicate the corresponding degree of polymerization (DP). Chromatogram (c) indicates peaks of authentic standards: G, glucose; F, fructose; S, sucrose; 1K, 1-kestotriose; 6K, 6-kestotriose; N, 1,1-kestotetraose; fn, 1,1,1-kestopentaose. The amounts of oligo- and polysaccharides that transgenic rice lines expressing wft1 accumulated in mature leaf blades were significantly higher than non-transgenic rice plants, but lower than transgenic rice lines expressing wft2 (Table 1). Oligo- and polysaccharide accumulation in S33 was also lower than in S50 and S51, and the total carbohydrate concentrations of transgenic rice lines expressing wft1 did not differ significantly from non-transgenic rice plants. The major oligo- and polysaccharide HPAEC peaks from mature leaf blades expressing wft1 were fructans, but in small amounts than and with different compositions from those of wft2 lines (Fig. 3). The fructans accumulated in S50 were similar to those found in wheat stems 8 d after anthesis and products generated by recombinant wft1 incubated with 100 mM sucrose (Fig. 4). Fig. 4. Open in new tabDownload slide Anion exchange HPLC analysis of carbohydrates in extracts from mature leaf blades of transgenic rice lines expressing wft1 (c) and wheat stem tissues 8 d after anthesis (d), and oligo-fructans generated by recombinant Wft1 with 100 mM sucrose at 10 °C for 120 h (b). Chromatogram (c) is scaled up longitudinally ×6. Chromatogram (a) indicates the peaks of authentic standards: G, glucose; F, fructose; S, sucrose; 1K, 1-kestotriose; 6K, 6-kestotriose; N, 1,1-kestotetraose; fn, 1,1,1-kestopentaose. Carbohydrate contents and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 Seedlings from wft2-expressing lines (I16, I22, I24, I29) and wft1-expressing lines (S33, S50, S51) were analysed for carbohydrate contents in root and shoot tissues just before chilling treatment. The carbohydrate contents in root tissues were not different among non-transformant rice plants and transgenic rice lines expressing wft1 or wft2 (data not shown). Among the shoot tissues of the control and transformant lines, only S50 had significantly elevated levels of mono- and disaccharides (Fig. 5). Oligo- and polysaccharide concentrations of I16, I22, I24, and I29 were significantly higher than in non-transgenic rice plants, and were notably different from those in transgenic rice lines expressing wft1. The wft1-expressing line S51 accumulated significantly more oligo- and polysaccharides than the non-transformant controls, but far less than the wft2-expressing lines. Lines I16, I22, I29, and S50 had significantly increased total carbohydrate concentrations. I24 also had more total carbohydrate than non-transgenic controls, but the increase was not statistically significant. Fig. 5. Open in new tabDownload slide Carbohydrate concentrations of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants. Data for the transgenic rice lines were analysed for significant differences from the data for non-transgenic rice plants using Student's t-test. An asterisk indicates significant difference from the controls at a P < 0.05 confidence interval. Vertical bars represent the standard deviation of three individual experiments. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Transgenic seedlings from wft2 rice lines I16, I22, I24, and I29, and wft1 lines S33, S50, and S51 were assessed for chilling tolerance at 5 °C for 11 d. Fewer than 10% of non-transgenic control seedlings continued to grow after chilling treatment, but all of the wft2 lines and one wft1 line (S50) had improved survival rates, ranging from greater than 40% (S50) to almost 90% (I29) (Fig. 6). However, the resumption of growth by S50 after chilling was slower than that of the wft2 lines. wft1 lines S33 and S51 responded to cold about the same as the non-transgenic controls (Fig. 6). A correlation analysis of chilling tolerance and carbohydrate contents showed that glucose, oligo- and polysaccharides, and total water-soluble carbohydrates were positively correlated with tolerance (Table 2). Table 2. Correlation coefficients between carbohydrate concentrations and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* *, ** Significance at P ≤0.05 or P ≤0.01 confidence intervals, respectively. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Table 2. Correlation coefficients between carbohydrate concentrations and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* *, ** Significance at P ≤0.05 or P ≤0.01 confidence intervals, respectively. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Fig. 6. Open in new tabDownload slide Chilling tolerance of transgenic rice seedlings expressing wft1 or wft2. Rice seedlings of non-transgenic and transgenic lines expressing wft1 (S33, S50, and S51) or wft2 (I16, I22, I24, and I29) grown at 26 °C/19 °C for 10 d were chilled at 5 °C for 11 d and transferred to a growth chamber at 26 °C/19 °C for 7 d. Chilling tolerance was assessed by percentage survival of the plants after return to normal growth temperatures. Vertical bars represent the standard deviations of two individual experiments. Discussion Improving chilling tolerance in summer crops has been a long-standing goal of agronomists. The advent of the practical application of agricultural biotechnology presents a number of possibilities for reaching this goal, including manipulation of organic solute contents. As one possible approach, genes which encode either 6-SFT or 1-SST were introduced into non-fructan-accumulating rice to produce plants that accumulate large amounts of fructans. Catalytic ability of Wft2 (1-SST) and Wft1 (6-SFT) in vivo The mature leaf blades of transgenic rice lines expressing wft2 grown in a greenhouse for about 2 months accumulated large amounts of inulin oligomers from 1-kestotriose (DP3) to at least 1,1,1,1,1-kestoheptose (DP7) (Table 1, Fig. 3). The primary function of 1-SST is thought to be the transfer of a fructosyl unit from one sucrose to another, resulting in the formation of the trisaccharide 1-kestose (Edelman and Jefford, 1968). Some studies, however, have shown that purified 1-SST mediates the synthesis of 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) during prolonged incubation with sucrose (Koops and Jonker, 1996; Lüscher et al., 1996; Van den Ende and Van Laere, 1996a). Van den Ende and Van Laere (1996b) reported that the synthesis of inulin oligomers exceeding DP3 proceeded during incubation conditions with high 1-SST concentrations as well as long reaction times. Incubation of recombinant 1-SST produced in Pichia pastoris with 100 mM 1-kestose resulted in the formation of 1,1-kestotetraose (Kawakami and Yoshida, 2005), suggesting that Wft2 possesses an additional activity for transferring a fructosyl unit from a fructan or sucrose to other fructans by β-(2,1)-linkage. Thus, transgenic rice lines that express high levels of Wft2 accumulate inulin oligomers exceeding DP3 during long-term growth. Production of inulin oligomers more than DP7 by 1-SST in vitro or in vivo experiments has not been reported. Mature leaf blades of transgenic rice lines expressing wft1 accumulated fructan oligomers, but the amounts were significantly lower than those accumulated by rice lines expressing wft2 (Table 1). 6-SFT predominantly catalyses the transfer of a fructosyl unit from one sucrose to a fructan or another sucrose in a β-(2,6)-linkage, but exhibits much higher levels of invertase activity than fructosyltransferase activity when incubated with sucrose as the sole substrate (Duchateau et al., 1995; Kawakami and Yoshida, 2002). Since wild-type rice plants do not accumulate fructans, the transgenic rice lines expressing wft1 would not be able to exert sufficient fructosyltransferase activity and could therefore not accumulate large amounts of fructans. The fructans in wheat stem tissues were mainly composed of phleins based on 6-kestose and 1,6-kestotetraose without branches of β-(2,1)-linked fructosyl units (Bancal et al., 1992). The products generated by recombinant Wft1 incubated with 100 mM sucrose were also phleins of a similar composition to those of fructans in wheat stem tissues (Fig. 4). By contrast to the fructans of wheat stem tissues, and products generated by recombinant Wft1, transgenic rice lines expressing wft1 mainly accumulated phlein oligomers enriched in β-(2,6)-linked fructosyl units (Fig. 4). The leaves of transgenic tobacco plants expressing barley 6-SFT accumulated small amounts of a series of high oligomers, up to DP12 (Sprenger et al., 1997). If major peaks [marked by asterisks in chromatogram (c) of Fig. 4] indicate different lengths of phlein oligomers based on 6-kestose, the S50 line expressing wft1 would contain phlein oligomers higher than DP15. Content of fructans in mature leaf blades of transgenic rice expressing wft1 or wft2 A previous review (Cairns, 2003) showed that transgenic plants (sugar beets, potato, tobacco, petunia, and chicory) expressing 1-SST or 6-SFT accumulated fructans at concentrations of <0.6 mg g−1 FW in leaf tissues, which was much lower than transgenic plants expressing bacterial fructosyltransferase genes. In this study, oligo- and polysaccharide concentrations in mature leaf blades of transgenic rice lines expressing wft1 and wft2 were 3.7 and 16.2 mg g−1 FW on average, respectively (Table 1), which is comparable with transgenic potato, tobacco, and sugar beets expressing bacterial fructosyltransferases (Cairns, 2003). Tissues containing high concentrations of sucrose, such as sugar beet roots and potato tubers, accumulate large amounts of fructans compared with leaf tissues in transformants expressing fructosyltransferase (Sprenger et al., 1997; Sévenier et al., 1998; Hellwege et al., 2000). Actually, mean sucrose concentrations in mature rice leaf tissues were 24 mg g−1 FW (Table 1), which is higher than in other plant species such as tobacco (Sprenger et al., 1997), potato (Hellwege et al., 2000), or petunia (van den Meer et al., 1998), indicating that sucrose substrate concentrations have an effect on fructan accumulation, and that plants with high sucrose concentrations have the potential to accumulate large amounts of fructans with the introduction of plant fructosyltransferases. Some transgenic plants transformed by bacterial fructosyltransferases exhibit aberrant phenotypes such as stunting, leaf bleaching, necrosis, and inhibition of development (Röber et al., 1996; Caimi et al., 1997; Turk et al., 1997; Jenkins et al., 2002). However, these aberrations have not been reported in transformants carrying plant-derived fructosyltransferases. Cairns (2003) suggested that the aberrant phenotypes were caused by extreme accumulation of fructans in the tissues of transgenic plants expressing bacterial fructosyltransferases. However, transgenic rice lines expressing wft1 or wft2, which accumulated amounts of fructans comparable with transformants expressing bacterial fructosyltransferases, apparently grew normally. The phenotypic aberrations are therefore not likely to have been caused by high concentrations of fructans in plant tissues per se. The conformation, degree of polymerization, or subcellular location of accumulated fructans may be the more important influences on growth aberrations, but the reasons are, for the present, a matter of speculation. Clearly, however, interference with water status and transport, and the concomitant effects on phytohormone regulation of tissue growth and differentiation would be obvious targets of future research. Recently, it has been reported that a rice vacuolar invertase possesses high fructan exohydrolase activity (Ji et al., 2007), indicating that the transgenic rice lines with wft1 or wft2 might not only synthesize fructans but would also be able to degrade them, and that the released fructose could then be used as an energy source. That is, fructans may function as additional temporary storage carbohydrates rather than as harmful heterogeneous substances in rice plants expressing wheat fructosyltransferases. Chilling tolerance of fructan-accumulating rice seedlings Chilling stress causes water deficits by an imbalance between water transport from root tissues and transpiration from leaf tissues (Tajima et al., 1983), production of reactive oxygen species (Hodgers et al., 1997), and damage of cell membranes (Crowe et al., 1992). Osmolytes such as amino acids, quanternary ammonium compounds, polyols, and soluble sugars that accumulate in plant tissues reduce the degree of damage caused by chilling injury (Bohnert et al., 1995; Couée et al., 2006). Ten-day-old seedlings of transgenic rice lines expressing wft2 (I16, I22, I24, and I29) contained low levels of oligo- and polysaccharides compared with mature leaf blades, but significantly higher amounts than non-transgenic rice seedlings, which correlated well with their enhanced chilling tolerance (Figs 5, 6). It is also apparent that oligo- and polysaccharide concentrations are more highly correlated with chilling tolerance than other carbohydrate contents (Table 2). The significant correlation between glucose contents and chilling tolerance could be caused by increasing released glucose by fructosyltransferase activity from one sucrose to another, or to a fructan. The oligo- and polysaccharide concentrations of wft1 transformant seedlings were obviously lower than those of transgenic rice lines expressing wft2 (Fig. 5). Among the three lines expressing wft1, only S50 had elevated amounts of mono- and disaccharides and exhibited increased tolerance compared with non-transgenic rice plants (Figs 5, 6), but there was no correlation between increasing concentrations of oligo- and polysaccharides and tolerance to chilling injury. Significant increases in mono- and disaccharides induced by some factor other than Wft1 might contribute to the increased chilling tolerance of S50. The positional effects of transformation would have to be addressed, particularly for wft1 transformants, before a definitive mechanism can be ascribed to these differences. Determination of the temporal changes in water-soluble carbohydrates during and after chilling treatments is necessary for a more detailed analysis of the mechanisms by which fructan accumulations affect chilling tolerance. The present results indicate that fructans primarily increase chilling tolerance of rice. Fructans are highly hydrophilic substances and might play an essential role as osmolytes. Moreover, fructans stabilize cellular membrane structures under low temperature and drought conditions (Demel et al., 1998; Hincha et al., 2000, 2002). It has been reported that the function of proton pumping across the tonoplast membrane of rice was damaged by low temperature (Kasamo et al., 2000). Therefore, fructans that have accumulated in transgenic rice lines expressing wft2 would protect protein or membrane structures in those tissues. On the other hand, fructan contents in wft2-expressing rice lines are sufficient to increase osmotic pressure in the vacuole. The change in osmotic pressure should cause the translocation of water molecules from the cytosol to tonoplast and induce osmotic signalling or drought signalling in a cell. It is well known that the cold signalling is cross-linked by drought signalling and osmotic signalling (Yamaguchi-Shinozaki and Shinozaki, 2006). The signal cascade induces expression of multiple genes associated with abiotic and biotic stresses (Zhu et al., 2007). Therefore, there is a possibility that fructan accumulations in the transgenic rice lines activate the signal cascade, induce expression of multiple genes such as cold-responsive genes, and indirectly increase chilling tolerance. Further analysis of the expression pattern of cold-responsive genes and related genes in fructan-accumulating rice lines is needed to elucidate the mechanism by which fructan accumulations increase chilling tolerance of plants. Conclusion Transgenic rice lines were successfully developed that accumulate fructans at concentrations comparable with those of transgenic plants expressing bacterial fructosyltransferases, and that have significantly increased tolerance to chilling injury. This is the first report that fructan accumulation enhances tolerance to non-freezing low temperatures in plants. The results of this study suggest that fructans produced by plant-derived fructosyltransferases can be agronomically useful for manipulating chilling tolerance, and that plant species containing large amounts of sucrose, such as rice, are suitable for transgenic conversion to plants that accumulate large amounts of fructans. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2008 The Author(s). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

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Abstract Fructans are water-soluble fructose oligomers and polymers that are based on sucrose, and have been implicated in protecting plants against water stress. Rice (Oryza sativa L.) is highly sensitive to chilling temperatures, and is not able to synthesize fructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase, encoded by wft2, or sucrose:fructan 6-fructosyltransferase, encoded by wft1, were introduced into rice plants, and rice transformants that accumulate fructans were successfully obtained. The mature leaf blades of transgenic rice lines with wft2 or wft1 accumulated 16.2 mg g−1 FW of oligo- and polysaccharides mainly composed of inulin oligomers of more than DP7, and 3.7 mg g−1 FW of oligo- and polysaccharides, mainly composed of phlein oligomers of more than DP15, respectively. The transgenic rice seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than non-transgenic rice seedlings, and exhibited enhanced chilling tolerance. The oligo- and polysaccharide concentrations of seedlings expressing wft1 were obviously lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage. This is the first report owing that fructan accumulation enhanced tolerance to non-freezing low temperatures. Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant Introduction Rice (Oryza sativa L.) originated in tropical and subtropical climates and is very sensitive to low temperatures (Graham and Patterson, 1982). Expansion of rice cultivation into regions that experience periodic or sustained low temperatures has increased the risk of crop loss through chilling injury. Tolerance to low temperatures during seed emergence and the early growth stages is an important characteristic for rice seedling establishment, particularly where direct sowing cultivation is practised in colder regions, such as Hokkaido, the northernmost large island of Japan. The first visible symptom of rice seedling damage caused by chilling is wilting, and prolonged low temperatures result in complete dehydration of the leaves. It is thought that transpiration exceeds the rate of water absorption through the root system under low temperature conditions, causing a loss of turgor in leaf tissues (Kabaki and Tajima, 1981). One of the primary plant responses for adaptation to water deficit stress is the accumulation of solutes such as amino acids (e.g. proline), quaternary ammonium compounds (e.g. glycinebetaine), polyols, and sugars (e.g. mannitol, trehalose, sucrose, and fructans) that act as osmoprotectants (Bohnert et al., 1995; Hare et al., 1998; Nuccio et al., 1999). Fructans, a class of water-soluble fructose polymers based on sucrose, accumulate in many bacterial and plant species, in which they serve as an important storage carbohydrate (Pollock and Cairns, 1991) and are implicated in protecting plants against water deficit caused by low matric potential, salinity, or low temperatures (Pontis, 1989; Hendry, 1993; Spollen and Nelson, 1994). Bacterial fructans (levans) are produced extracellularly by the enzymes levansucrase or fructosyltransferase, which produce levans directly from sucrose (Vijn and Smeekens, 1999). In plants, fructans are synthesized in vacuoles from sucrose by the action of two or more different fructosyltransferases, including sucrose:sucrose 1-fructosyltransferase (1-SST), sucrose:fructan 6-fructosyltransferase (6-SFT), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan:fructan 6G-fructosyltransferase (6G-FFT) (Vijn and Smeekens, 1999). Following the initial cloning of Hordeum vulgare (barley) 6-SFT (Hv6-SFT; Sprenger et al., 1995), several genes encoding enzymes associated with fructan biosynthesis have been cloned from various plants, including 1-SST and 1-FFT from Cichorium intybus (chicory; de Halleux and Van Cutsem, 1997; Goblet et al., 1999), Cynara scolymus (globe artichoke; Hellwege et al., 1997, 1998), and Helianthus tuberosus (Jerusalem artichoke; van der Meer et al., 1998); 1-SST and 6G-FFT from Allium cepa (onion; Vijn et al., 1997, 1998); and 1-SST and/or 6-SFT from temperate grasses such as Festuca arundinacea (tall fescue; Lüscher et al., 2000), Agropyron cistatum (crested wheatgrass; Wei and Chatterton, 2001), Poa secunda (big bluegrass; Wei et al., 2002), Lolium perenne (perennial ryegrass; Lidgett et al., 2002; Chalmers et al., 2003), and H. vulgare (Nagaraj et al., 2004). 1-SST and 6-SFT, designated wft2 and wft1, from Triticum aestivum (wheat) were also isolated, and it was found that those two genes are involved in fructan accumulation during cold hardening (Kawakami and Yoshida, 2002). Metabolic engineering for the biosynthesis of fructans has been the focus of considerable attention as a potential strategy for increasing water stress tolerance. Fructan-synthesizing genes have been introduced into non-fructan-accumulating plants such as Nicotiana tabacum (tobacco; Ebskamp et al., 1994; Pilon-Smits et al., 1995; Caimi et al, 1997), Solanum tuberosum (potato; van der Meer et al., 1994; Röber et al., 1996; Pilon-Smits et al., 1996; Caimi et al., 1997), Beta vulgaris (sugar beet; Pilon-Smits et al., 1999), Trifolium repens (white clover; Jenkins et al., 2002), and Zea mays (maize; Caimi et al., 1996) by ectopically expressing levansucrase or fructosyltransferase genes isolated from Bacillus spp., Erwinia amylovora, and Streptococcus spp. Some of those transgenic plants exhibited high fructan accumulation and water stress tolerance. For example, tobacco and sugar beet plants expressing bacterial levansucrase exhibited increased drought tolerance (Pilon-Smits et al., 1995, 1999), freezing tolerance (Konstantinova et al., 2002), or osmotic tolerance (Park et al., 1999). In some cases, aberrant growth phenotypes such as leaf bleaching, necrosis, and stunting were observed in transformants expressing bacterial fructan synthesis genes (Cairns, 2003). Transgenic plants carrying genes encoding plant fructosyltransferases have also been produced and mainly used for functional identification of introduced genes and analysis of fructan accumulation in non-fructan-synthesizing plants. For instance, Hv6-SFT was introduced into tobacco and chicory (Sprenger et al. 1997), the Jerusalem artichoke 1-SST gene and/or 1-FFT gene was introduced into sugar beet (Sévenier et al., 1998) and petunia (van der Meer et al., 1998), and the globe artichoke 1-SST-and/or 1-FFT-encoding genes were introduced into potato (Hellwege et al., 2000). By contrast to levansucrase genes from bacteria, no phenotypic aberrations have been observed in transformants expressing plant fructosyltrannsferase genes (Cairns, 2003). Hisano et al. (2004) reported that transgenic perennial ryegrass expressing wheat 1-SST or 6-SFT genes accumulated more fructans and acquired higher tolerance for freezing at the cellular level compared with non-transgenic plants, but there have been no other reports on increasing chilling stress tolerance (i.e. non-freezing, low temperatures) in transgenic plants carrying fructosyltransferase genes. Rice plants do not accumulate fructans, and seedlings are susceptible to injury at temperatures between freezing and 10 °C. Cairns (2003) indicated that non-fructan plants do not accumulate large amounts of fructans with the introduction of plant-derived fructosyltransferase genes, but because of the potential benefit to rice agronomy in marginal climatic conditions, it would be interesting to know if transgenic rice lines expressing wheat-derived fructosyltransferases accumulate sufficient fructan concentrations in mature leaf blades to affect chilling tolerance in rice seedlings. Two fructosyltransferase genes, encoding 1-SST and 6-SFT, could prove to be useful tools for fructan production in non-fructan plants because both enzymes produce fructans from sucrose as the sole substrate. In this study, transgenic rice plants that accumulate fructans by overexpressing wft1 and wft2 (T. aestivum 6-SFT and 1-SST, respectively) were generated and their mature leaf blade carbohydrate concentrations, fructan oligomer composition, and the correlation between fructan accumulation and seedling chilling tolerance were analysed. Materials and methods Transformation and screening of transgenic plants Full-length wft1 (accession no. AB029887) or wft2 (accession no. AB029888) (Kawakami and Yoshida, 2002) were inserted into pMLH7133, a Ti-based vector, downstream of the first intron of the phaseolin gene under control of the cauliflower mosaic virus (CaMV) 35S promoter (Mitsuhara et al., 1996). The resulting plasmids, pMLH1733-wft1 and pMLH1733-wft2, were introduced into calli of the Japonicum rice cultivar Yukihikari by Agrobacterium-mediated transformation, according to a previously reported protocol (Tanaka et al., 2001). Transformed calli were selected for hygromycin resistance, and transgenic plants were regenerated as previously described (Tanaka et al., 2001). Presence of the transgene in T0 generation plants was examined by PCR amplification. T1 generation lines were screened by HPLC for accumulation of fructans. In the T2 generation, lines homozygous for a transgene were selected by PCR, and the transgene copy number was determined by genomic Southern hybridization. T3 transgenic rice lines were used for further analysis. Transgene transcription Total RNA was isolated from rice leaf tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Northern hybridization was done according to a standard protocol (Sambrook et al., 1989). Total RNA (15 μg) was separated by electrophoresis on 1.5% (w/v) agarose gels, blotted onto hybond N+ membranes (GE Healthcare, Little Chalfont, Bucks, UK), and hybridized with a α-32P-labelled cDNA fragment corresponding to Wft1 or Wft2. The blot was washed twice with 0.1× SSC and 0.1% SDS for 20 min at 65 °C and exposed to X-ray film at –80 °C. Carbohydrate extraction and analysis For analysis of water-soluble carbohydrates in mature leaf blades and shoots from seedlings of T3 transgenic lines and non-transgenic rice plants, the eighth or ninth leaf blades from plants grown for 2 months in a greenhouse at 25 °C/18 °C (12 h/12 h), and shoot and root tissues of seedlings grown for 10 d in a growth chamber at 26 °C/19 °C with a 16 h light/8 h dark cycle were harvested and stored at –80 °C. Total water-soluble carbohydrates were extracted from finely chopped tissues in boiling deionized water for 1 h. Total carbohydrates were analysed by HPLC with a combination of Shodex KS-802 and KS-803 columns (Shodex, Tokyo, Japan) using the refractive index detector as described by Yoshida et al. (1998). Glucose, fructose, sucrose, oligosaccharide, and polysaccharide concentrations were determined. For analysis of fructan oligomers with different glycosidic linkages, high-performance anion exchange chromatography (HPAEC) was performed on a DX 500 chromatograph (Dionex, Sunnyvale, CA, USA) with a Carbo Pac PA-1 anion exchange column and a pulsed amperometric detector (PAD) as described by Shiomi et al. (1997). Peaks for glucose, fructose, sucrose, 1-kestose, 1,1-kestotetraose, 1,1,1-kestopentaose (Wako, Osaka, Japan), and 6-kestose (Iizuka et al., 1993) were identified by comparison with authentic standards. Phlein oligomers based on 6-kestose and bifurcose were putatively identified by comparison of HPAEC retention times with fructan oligomers extracted from wheat tissues 8 d after anthesis, and products generated by recombinant wheat 6-SFT (Wft1) incubated with 100 mM sucrose at 10 °C for 120 h (Kawakami and Yoshida, 2005). Chilling tolerance assay Rice seeds were immersed in distilled water for 3 d at 27 °C, sown on soil in a plastic container, and then transferred to a growth chamber at 26 °C/19 °C with a 16 h light/8 h dark cycle. Ten-day-old seedlings were exposed to 5 °C with continuous light for 11 d. After the chilling treatment, seedlings were transferred to a greenhouse at 26 °C/19 °C with a 16 h light/8 h dark cycle and grown for a further 7 d. The percentage of surviving seedlings was determined with active growth as the main criterion for survivors, and wilting and non-growth as the criteria for scoring as non-survivors. Results Production of transgenic rice plants expressing wft1 or wft2 After regeneration of transformants carrying either wft2 or wft1, several hygromycin-resistant rice plants were self-pollinated and homozygous transgenic lines containing wft2 (I16, I22, I24, and I29) or wft1 (S33, S50, and S51) were obtained in the T3 generation. No apparent phenotypic abnormalities or fertility problems were observed in transgenic lines expressing wft1 or wft2 (Fig. 1). Fig. 1. Open in new tabDownload slide Phenotypes of transgenic rice lines expressing wft1 (6-SFT) or wft2 (1-SST). (A) Transgenic rice line expressing wft2 (I22), non-transgenic rice plant (Cont), and transgenic rice line expressing wft1 (S51) grown in an environmental control room for about 2 months. (B) Spikes of transgenic rice lines expressing wft2 (I16 and I22), wft1 (S50 and S51), and non-transgenic rice plants (Cont). Southern blot analysis revealed that the transgenic lines had one or two copies of the wft1 or wft2 gene in their genomes (data not shown). To verify transgene expression, wft1 and wft2 transcript accumulation was measured in non-transgenic rice plant controls and transgenic rice lines by northern hybridization. The levels of wft2 mRNA were almost the same among transgenic rice lines expressing wft2, but varied widely in wft1 transformants. The S33 line had a relatively low level of wft1 expression compared with S50 and S51 (Fig. 2). Fig. 2. Open in new tabDownload slide Expression of wft1 and wft2 in mature leaf blades of transgenic rice lines and non-transgenic rice plants. Total RNA samples (20 μg) extracted from non-transgenic rice plants (Cont), transgenic rice lines I16, I22, I24, I29 (wft2), S33, S50, and S51 (wft1) were fractioned by electrophoresis on a 1.2% formamide-containing agarose gel. Hybridization was carried out with α-32P-labelled probe for detecting wft1 or wft2. The corresponding ethidium bromide gel image of rRNA shows the relative amounts of total RNA loaded for each sample. Carbohydrate contents and composition of fructan oligomers in mature leaf blades of transgenic rice lines expressing wft1 or wft2 Oligo- and polysaccharide concentrations, and total carbohydrate concentrations in mature leaf blades of transgenic rice lines expressing wft2 were significantly higher than in non-transgenic rice plants (Table 1). Sucrose contents in transgenic and non-transgenic plants were not significantly different. The major HPAEC peaks of oligo- and polysaccharides were mainly fructans and inulin oligomers from 1-kestotriose (DP3) to 1,1,1,1,1-kestoheptaose (DP7), and small amounts of unknown oligosaccharides (Fig. 3). Table 1. Carbohydrate contents of mature leaf blades of non-transgenic and transgenic rice lines expressing wft1 (wheat 6-SFT) or wft2 (wheat 1-SST) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) The values are means ±standard deviation (n=3). Data for the transgenic plants were analysed for significant differences from the data for non-transgenic plants using Student's t-test. An asterisk indicates significance at P < 0.05. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Table 1. Carbohydrate contents of mature leaf blades of non-transgenic and transgenic rice lines expressing wft1 (wheat 6-SFT) or wft2 (wheat 1-SST) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) Carbohydrate contents (mg g−1 FW) F G S Mono+Di Oligo+Poly Total Transgenic rice lines with wft2 I16 2.7 (±0.6)* 2.4 (±0.8)* 26.9 (±6.6) 32.4 (±7.8) 17.3 (±6.1)* 49.4 (±9.6)* I22 2.3 (±0.4)* 1.5 (±0.3)* 20.9 (±3.7) 25.6 (±4.3) 15.5 (±3.7)* 40.2 (±7.4)* I24 4.7 (±1.0)* 3.3 (±1.1)* 25.4 (±4.2) 36.5 (±5.5) 18.0 (±4.0)* 55.7 (±7.5)* I29 2.2 (±0.5)* 1.6 (±0.4)* 24.5 (±3.8) 29.6 (±4.7) 13.9 (±3.5)* 44.4 (±3.7)* Average 3.0 (±1.2)* 2.2 (±1.0)* 24.4 (±4.8) 31.0 (±6.2) 16.2 (±4.3)* 48.3 (±9.2)* Transgenic rice lines with wft1 S33 1.3 (±0.1)* 0.7 (±0.1) 13.5 (±2.2)* 16.5 (±2.5)* 2.4 (±0.2)* 18.9 (±2.7)* S50 1.3 (±0.2) 0.9 (±0.2) 16.7 (±1.9) 19.5 (±2.3) 3.7 (±0.3)* 23.7 (±2.2) S51 1.9 (±0.5) 1.1 (±0.3) 27.4 (±2.5) 31.8 (±3.2) 4.2 (±1.1)* 36.2 (±3.2) Average 1.5 (±0.5) 1.0 (±0.3) 20.0 (±6.8) 23.6 (±7.6) 3.7 (±1.1)* 27.3 (±8.3) Non-transgenic rice plants Average 1.2 (±0.1) 1.0 (±0.1) 24.0 (±5.8) 26.9 (±6.4) 0.1 (±0.1) 26.9 (±6.3) The values are means ±standard deviation (n=3). Data for the transgenic plants were analysed for significant differences from the data for non-transgenic plants using Student's t-test. An asterisk indicates significance at P < 0.05. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Fig. 3. Open in new tabDownload slide Anion exchange HPLC analysis of soluble carbohydrates in extracts from mature leaf blades of non-transgenic rice plants (a) and transgenic rice lines expressing wft1 (d) or wft2 (b). Inulin extracted from chicory roots was used as a standard for inulin oligomers, and the numbers (3–7) on each peak indicate the corresponding degree of polymerization (DP). Chromatogram (c) indicates peaks of authentic standards: G, glucose; F, fructose; S, sucrose; 1K, 1-kestotriose; 6K, 6-kestotriose; N, 1,1-kestotetraose; fn, 1,1,1-kestopentaose. The amounts of oligo- and polysaccharides that transgenic rice lines expressing wft1 accumulated in mature leaf blades were significantly higher than non-transgenic rice plants, but lower than transgenic rice lines expressing wft2 (Table 1). Oligo- and polysaccharide accumulation in S33 was also lower than in S50 and S51, and the total carbohydrate concentrations of transgenic rice lines expressing wft1 did not differ significantly from non-transgenic rice plants. The major oligo- and polysaccharide HPAEC peaks from mature leaf blades expressing wft1 were fructans, but in small amounts than and with different compositions from those of wft2 lines (Fig. 3). The fructans accumulated in S50 were similar to those found in wheat stems 8 d after anthesis and products generated by recombinant wft1 incubated with 100 mM sucrose (Fig. 4). Fig. 4. Open in new tabDownload slide Anion exchange HPLC analysis of carbohydrates in extracts from mature leaf blades of transgenic rice lines expressing wft1 (c) and wheat stem tissues 8 d after anthesis (d), and oligo-fructans generated by recombinant Wft1 with 100 mM sucrose at 10 °C for 120 h (b). Chromatogram (c) is scaled up longitudinally ×6. Chromatogram (a) indicates the peaks of authentic standards: G, glucose; F, fructose; S, sucrose; 1K, 1-kestotriose; 6K, 6-kestotriose; N, 1,1-kestotetraose; fn, 1,1,1-kestopentaose. Carbohydrate contents and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 Seedlings from wft2-expressing lines (I16, I22, I24, I29) and wft1-expressing lines (S33, S50, S51) were analysed for carbohydrate contents in root and shoot tissues just before chilling treatment. The carbohydrate contents in root tissues were not different among non-transformant rice plants and transgenic rice lines expressing wft1 or wft2 (data not shown). Among the shoot tissues of the control and transformant lines, only S50 had significantly elevated levels of mono- and disaccharides (Fig. 5). Oligo- and polysaccharide concentrations of I16, I22, I24, and I29 were significantly higher than in non-transgenic rice plants, and were notably different from those in transgenic rice lines expressing wft1. The wft1-expressing line S51 accumulated significantly more oligo- and polysaccharides than the non-transformant controls, but far less than the wft2-expressing lines. Lines I16, I22, I29, and S50 had significantly increased total carbohydrate concentrations. I24 also had more total carbohydrate than non-transgenic controls, but the increase was not statistically significant. Fig. 5. Open in new tabDownload slide Carbohydrate concentrations of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants. Data for the transgenic rice lines were analysed for significant differences from the data for non-transgenic rice plants using Student's t-test. An asterisk indicates significant difference from the controls at a P < 0.05 confidence interval. Vertical bars represent the standard deviation of three individual experiments. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Transgenic seedlings from wft2 rice lines I16, I22, I24, and I29, and wft1 lines S33, S50, and S51 were assessed for chilling tolerance at 5 °C for 11 d. Fewer than 10% of non-transgenic control seedlings continued to grow after chilling treatment, but all of the wft2 lines and one wft1 line (S50) had improved survival rates, ranging from greater than 40% (S50) to almost 90% (I29) (Fig. 6). However, the resumption of growth by S50 after chilling was slower than that of the wft2 lines. wft1 lines S33 and S51 responded to cold about the same as the non-transgenic controls (Fig. 6). A correlation analysis of chilling tolerance and carbohydrate contents showed that glucose, oligo- and polysaccharides, and total water-soluble carbohydrates were positively correlated with tolerance (Table 2). Table 2. Correlation coefficients between carbohydrate concentrations and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* *, ** Significance at P ≤0.05 or P ≤0.01 confidence intervals, respectively. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Table 2. Correlation coefficients between carbohydrate concentrations and chilling tolerance of transgenic rice seedlings expressing wft1 or wft2 and non-transgenic rice plants F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* F G S Mono+Di Oligo+Poly Total Correlation coefficients between carbohydrate contents and survival rate (%) after chilling treatments 0.421 0.754* –0.597 –0.233 0.864** 0.813* *, ** Significance at P ≤0.05 or P ≤0.01 confidence intervals, respectively. F, Fructose; G, glucose; S, sucrose; Mono+Di, mono- and disaccharides; Oligo+Poly, oligo- and polysaccharides; Total, total water-soluble carbohydrates. Open in new tab Fig. 6. Open in new tabDownload slide Chilling tolerance of transgenic rice seedlings expressing wft1 or wft2. Rice seedlings of non-transgenic and transgenic lines expressing wft1 (S33, S50, and S51) or wft2 (I16, I22, I24, and I29) grown at 26 °C/19 °C for 10 d were chilled at 5 °C for 11 d and transferred to a growth chamber at 26 °C/19 °C for 7 d. Chilling tolerance was assessed by percentage survival of the plants after return to normal growth temperatures. Vertical bars represent the standard deviations of two individual experiments. Discussion Improving chilling tolerance in summer crops has been a long-standing goal of agronomists. The advent of the practical application of agricultural biotechnology presents a number of possibilities for reaching this goal, including manipulation of organic solute contents. As one possible approach, genes which encode either 6-SFT or 1-SST were introduced into non-fructan-accumulating rice to produce plants that accumulate large amounts of fructans. Catalytic ability of Wft2 (1-SST) and Wft1 (6-SFT) in vivo The mature leaf blades of transgenic rice lines expressing wft2 grown in a greenhouse for about 2 months accumulated large amounts of inulin oligomers from 1-kestotriose (DP3) to at least 1,1,1,1,1-kestoheptose (DP7) (Table 1, Fig. 3). The primary function of 1-SST is thought to be the transfer of a fructosyl unit from one sucrose to another, resulting in the formation of the trisaccharide 1-kestose (Edelman and Jefford, 1968). Some studies, however, have shown that purified 1-SST mediates the synthesis of 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) during prolonged incubation with sucrose (Koops and Jonker, 1996; Lüscher et al., 1996; Van den Ende and Van Laere, 1996a). Van den Ende and Van Laere (1996b) reported that the synthesis of inulin oligomers exceeding DP3 proceeded during incubation conditions with high 1-SST concentrations as well as long reaction times. Incubation of recombinant 1-SST produced in Pichia pastoris with 100 mM 1-kestose resulted in the formation of 1,1-kestotetraose (Kawakami and Yoshida, 2005), suggesting that Wft2 possesses an additional activity for transferring a fructosyl unit from a fructan or sucrose to other fructans by β-(2,1)-linkage. Thus, transgenic rice lines that express high levels of Wft2 accumulate inulin oligomers exceeding DP3 during long-term growth. Production of inulin oligomers more than DP7 by 1-SST in vitro or in vivo experiments has not been reported. Mature leaf blades of transgenic rice lines expressing wft1 accumulated fructan oligomers, but the amounts were significantly lower than those accumulated by rice lines expressing wft2 (Table 1). 6-SFT predominantly catalyses the transfer of a fructosyl unit from one sucrose to a fructan or another sucrose in a β-(2,6)-linkage, but exhibits much higher levels of invertase activity than fructosyltransferase activity when incubated with sucrose as the sole substrate (Duchateau et al., 1995; Kawakami and Yoshida, 2002). Since wild-type rice plants do not accumulate fructans, the transgenic rice lines expressing wft1 would not be able to exert sufficient fructosyltransferase activity and could therefore not accumulate large amounts of fructans. The fructans in wheat stem tissues were mainly composed of phleins based on 6-kestose and 1,6-kestotetraose without branches of β-(2,1)-linked fructosyl units (Bancal et al., 1992). The products generated by recombinant Wft1 incubated with 100 mM sucrose were also phleins of a similar composition to those of fructans in wheat stem tissues (Fig. 4). By contrast to the fructans of wheat stem tissues, and products generated by recombinant Wft1, transgenic rice lines expressing wft1 mainly accumulated phlein oligomers enriched in β-(2,6)-linked fructosyl units (Fig. 4). The leaves of transgenic tobacco plants expressing barley 6-SFT accumulated small amounts of a series of high oligomers, up to DP12 (Sprenger et al., 1997). If major peaks [marked by asterisks in chromatogram (c) of Fig. 4] indicate different lengths of phlein oligomers based on 6-kestose, the S50 line expressing wft1 would contain phlein oligomers higher than DP15. Content of fructans in mature leaf blades of transgenic rice expressing wft1 or wft2 A previous review (Cairns, 2003) showed that transgenic plants (sugar beets, potato, tobacco, petunia, and chicory) expressing 1-SST or 6-SFT accumulated fructans at concentrations of <0.6 mg g−1 FW in leaf tissues, which was much lower than transgenic plants expressing bacterial fructosyltransferase genes. In this study, oligo- and polysaccharide concentrations in mature leaf blades of transgenic rice lines expressing wft1 and wft2 were 3.7 and 16.2 mg g−1 FW on average, respectively (Table 1), which is comparable with transgenic potato, tobacco, and sugar beets expressing bacterial fructosyltransferases (Cairns, 2003). Tissues containing high concentrations of sucrose, such as sugar beet roots and potato tubers, accumulate large amounts of fructans compared with leaf tissues in transformants expressing fructosyltransferase (Sprenger et al., 1997; Sévenier et al., 1998; Hellwege et al., 2000). Actually, mean sucrose concentrations in mature rice leaf tissues were 24 mg g−1 FW (Table 1), which is higher than in other plant species such as tobacco (Sprenger et al., 1997), potato (Hellwege et al., 2000), or petunia (van den Meer et al., 1998), indicating that sucrose substrate concentrations have an effect on fructan accumulation, and that plants with high sucrose concentrations have the potential to accumulate large amounts of fructans with the introduction of plant fructosyltransferases. Some transgenic plants transformed by bacterial fructosyltransferases exhibit aberrant phenotypes such as stunting, leaf bleaching, necrosis, and inhibition of development (Röber et al., 1996; Caimi et al., 1997; Turk et al., 1997; Jenkins et al., 2002). However, these aberrations have not been reported in transformants carrying plant-derived fructosyltransferases. Cairns (2003) suggested that the aberrant phenotypes were caused by extreme accumulation of fructans in the tissues of transgenic plants expressing bacterial fructosyltransferases. However, transgenic rice lines expressing wft1 or wft2, which accumulated amounts of fructans comparable with transformants expressing bacterial fructosyltransferases, apparently grew normally. The phenotypic aberrations are therefore not likely to have been caused by high concentrations of fructans in plant tissues per se. The conformation, degree of polymerization, or subcellular location of accumulated fructans may be the more important influences on growth aberrations, but the reasons are, for the present, a matter of speculation. Clearly, however, interference with water status and transport, and the concomitant effects on phytohormone regulation of tissue growth and differentiation would be obvious targets of future research. Recently, it has been reported that a rice vacuolar invertase possesses high fructan exohydrolase activity (Ji et al., 2007), indicating that the transgenic rice lines with wft1 or wft2 might not only synthesize fructans but would also be able to degrade them, and that the released fructose could then be used as an energy source. That is, fructans may function as additional temporary storage carbohydrates rather than as harmful heterogeneous substances in rice plants expressing wheat fructosyltransferases. Chilling tolerance of fructan-accumulating rice seedlings Chilling stress causes water deficits by an imbalance between water transport from root tissues and transpiration from leaf tissues (Tajima et al., 1983), production of reactive oxygen species (Hodgers et al., 1997), and damage of cell membranes (Crowe et al., 1992). Osmolytes such as amino acids, quanternary ammonium compounds, polyols, and soluble sugars that accumulate in plant tissues reduce the degree of damage caused by chilling injury (Bohnert et al., 1995; Couée et al., 2006). Ten-day-old seedlings of transgenic rice lines expressing wft2 (I16, I22, I24, and I29) contained low levels of oligo- and polysaccharides compared with mature leaf blades, but significantly higher amounts than non-transgenic rice seedlings, which correlated well with their enhanced chilling tolerance (Figs 5, 6). It is also apparent that oligo- and polysaccharide concentrations are more highly correlated with chilling tolerance than other carbohydrate contents (Table 2). The significant correlation between glucose contents and chilling tolerance could be caused by increasing released glucose by fructosyltransferase activity from one sucrose to another, or to a fructan. The oligo- and polysaccharide concentrations of wft1 transformant seedlings were obviously lower than those of transgenic rice lines expressing wft2 (Fig. 5). Among the three lines expressing wft1, only S50 had elevated amounts of mono- and disaccharides and exhibited increased tolerance compared with non-transgenic rice plants (Figs 5, 6), but there was no correlation between increasing concentrations of oligo- and polysaccharides and tolerance to chilling injury. Significant increases in mono- and disaccharides induced by some factor other than Wft1 might contribute to the increased chilling tolerance of S50. The positional effects of transformation would have to be addressed, particularly for wft1 transformants, before a definitive mechanism can be ascribed to these differences. Determination of the temporal changes in water-soluble carbohydrates during and after chilling treatments is necessary for a more detailed analysis of the mechanisms by which fructan accumulations affect chilling tolerance. The present results indicate that fructans primarily increase chilling tolerance of rice. Fructans are highly hydrophilic substances and might play an essential role as osmolytes. Moreover, fructans stabilize cellular membrane structures under low temperature and drought conditions (Demel et al., 1998; Hincha et al., 2000, 2002). It has been reported that the function of proton pumping across the tonoplast membrane of rice was damaged by low temperature (Kasamo et al., 2000). Therefore, fructans that have accumulated in transgenic rice lines expressing wft2 would protect protein or membrane structures in those tissues. On the other hand, fructan contents in wft2-expressing rice lines are sufficient to increase osmotic pressure in the vacuole. The change in osmotic pressure should cause the translocation of water molecules from the cytosol to tonoplast and induce osmotic signalling or drought signalling in a cell. It is well known that the cold signalling is cross-linked by drought signalling and osmotic signalling (Yamaguchi-Shinozaki and Shinozaki, 2006). The signal cascade induces expression of multiple genes associated with abiotic and biotic stresses (Zhu et al., 2007). Therefore, there is a possibility that fructan accumulations in the transgenic rice lines activate the signal cascade, induce expression of multiple genes such as cold-responsive genes, and indirectly increase chilling tolerance. Further analysis of the expression pattern of cold-responsive genes and related genes in fructan-accumulating rice lines is needed to elucidate the mechanism by which fructan accumulations increase chilling tolerance of plants. Conclusion Transgenic rice lines were successfully developed that accumulate fructans at concentrations comparable with those of transgenic plants expressing bacterial fructosyltransferases, and that have significantly increased tolerance to chilling injury. This is the first report that fructan accumulation enhances tolerance to non-freezing low temperatures in plants. The results of this study suggest that fructans produced by plant-derived fructosyltransferases can be agronomically useful for manipulating chilling tolerance, and that plant species containing large amounts of sucrose, such as rice, are suitable for transgenic conversion to plants that accumulate large amounts of fructans. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2008 The Author(s).

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Journal of Experimental Botany – Oxford University Press

Published: Mar 1, 2008

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