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- Materials and Methods
Fructans, fructose-based oligo- and polysaccharides, are storage compounds in about 15% of flowering plant species (Hendry, 1993). They are classified into several forms depending on their glycosidic linkages. Inulin-type fructans with linearly β(2→1)-linked fructofuranosyl units occur mainly in dicot species. Levan-type fructans with β(2→6)-linked fructofuranosyl units and mixed-levan type fructans with both β(2→1)- and β(2→6)-linked fructofuranosyl units (graminans) are found in monocot species (Pollock & Cairns, 1991). Inulin biosynthesis involves two enzymes: sucrose : sucrose 1-fructosyl transferase (1-SST, EC 22.214.171.124) and fructan : fructan 1-fructosyl transferase (1-FFT, EC.126.96.36.199) (Van Laere & Van den Ende, 2002). Together with 1-SST and 1-FFT, Sucrose : fructan 6-fructosyl transferase (6-SFT) determines graminan biosynthesis in cereals (Bancal et al., 1992; Sprenger et al., 1995; Kawakami & Yoshida, 2002; Kawakami et al., 2002; Nagaraj et al., 2004). Fructan : fructan 6G-fructosyl transferase (6G-FFT, Shiomi, 1981, 1989; Ritsema et al., 2003) or other 6-FT-like fructosyl transferases (Pavis et al., 2001) are probably implicated in the synthesis of fructan neoseries found in Asparagus officinalis, Allium cepa, Lolium perenne and Avena sativa (Livingston et al., 1993).
Graminan-type fructans are the predominant storage carbohydrates in vegetative tissues of temperate grasses and cereals such as wheat and barley. Wheat fructan levels might be controlled not only by fructan biosynthetic enzymes, but also by fructan exohydrolases (FEHs), releasing terminal fructoses from fructans. These are classified into two types of enzyme: 1-FEHs and 6-FEHs, based on the linkage type [β(2→1) vs β(2→6)] that is preferentially hydrolysed. 1-FEH is coexpressed with fructan biosynthetic enzymes and might act as a β(2→1) trimmer during the period of active fructan biosynthesis (Van den Ende et al., 2003a). Graminans are believed to accumulate mainly in the vacuole, and are subject to degradation by vacuolar FEHs (Wagner & Wiemken, 1986).
Similarly to raffinose oligosaccharides (Tapernoux-Luthi et al., 2004), fructans may be able to protect plants against drought and freezing (Pilon-Smits et al., 1995; Park et al., 1999; Konstantinova et al., 2002; Amiard et al., 2003; Parvanova et al., 2004), probably by stabilizing membranes (Vereyken et al., 2001; Hincha et al., 2002, 2003). Induction of 1-SST and 6-SFT, and subsequent accumulation of fructans in winter cereals hardened above freezing temperatures (first-phase cold hardening, 1-PH), was demonstrated to be essential for winter survival (Olien & Clark, 1993; Yoshida et al., 1998; Gaudet et al., 1999; Kawakami & Yoshida, 2002). In wheat, 1-PH resulted in strongly decreased FEH activities. It was suggested that the observed varietal difference in fructan accumulation under frost is affected largely by the FEH activity levels (Yukawa et al., 1995). Compared with winter wheat, spring wheat was unable to induce 1-SST activity in leaves under cold stress (Savitch et al., 2000).
A significant increase in fructan concentrations in the apoplast of oat crowns was found during second-phase cold hardening (hardening below freezing temperatures, 2-PH), suggesting the presence of two different fructan pools under freezing stress. Compared with total extracts, 2-PH apoplastic fluids showed an increased low degree of polymerization (DP)/high DP fructan and hexose/sucrose ratio, correlating well with the increased FEH and invertase activities in the apoplast (Livingston & Henson, 1998). These results were indicative for the presence of a true apoplastic FEH in cold-hardened oat. FEHs were induced in chicory and orchardgrass by freezing temperatures (Van den Ende & Van Laere, 1996; Yamamoto et al., 1999; Michiels et al., 2004), but also after defoliation (Yamamoto & Mino, 1985; Prud’homme et al., 1992; Morvan-Bertrand et al., 2001; Van den Ende et al., 2001; Asega & de Carvalho, 2004). Gibberellin and ABA might be important hormones for FEH regulation (Morvan et al., 1997; Yang et al., 2004).
So far, plant 1-FEH cDNAs have been cloned from chicory (Van den Ende et al., 2000, 2001) and wheat (Van den Ende et al., 2003a). Plant FEH enzymes are clearly different from invertases (Van Laere & Van den Ende, 2002) as they are unable to degrade sucrose (Suc). Surprisingly, it was found that 6-FEH activities occur in nonfructan plants, and a 6-FEH was recently purified and cloned from sugar beet (Van den Ende et al., 2003b). In order to find new FEH genes putatively related to freezing tolerance, a wheat 1-FEH probe was used to screen a cold-hardened wheat crown cDNA library.