Fructan accumulation and transcription of candidate genes during cold acclimation in three varieties of Poa pratensis

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Abstract

Poa pratensis, a type species for the grass family (Poaceae), is an important cool season grass that accumulates fructans as a polysaccharide reserve. We studied fructan contents and expression of candidate fructan metabolism genes during cold acclimation in three varieties of P. pratensis adapted to different environments: Northern Norway, Denmark, and the Netherlands. Fructan content increased significantly during cold acclimation and varieties showed significant differences in the level of fructan accumulation. cDNA sequences of putative fructosyltransferase (FT), fructan exohydrolase (FEH), and cold acclimation protein (CAP) genes were identified and cloned. In agreement with a function in fructan biosynthesis, transcription of a putative sucrose:fructan 6-fructosyltransferase (Pp6-SFT) gene was induced during cold acclimation and fructan accumulation in all three P. pratensis varieties. Transcription of putative PpFEH and PpCAP genes was also induced by cold acclimation; however, transcription of these two genes was several-fold higher in the variety from Norway compared to the other two varieties. The results presented here suggest that Pp6-SFT is involved in fructan biosynthesis in P. pratensis. FEHs have previously been suggested to be involved in fructan biosynthesis and freezing tolerance, and induced expression of PpFEH during fructan accumulation could also suggest a role in fructan biosynthesis. However, based on the different PpFEH transcription rates among varieties and similar expression of PpFEH and PpCAP, we suggest that PpFEH is more likely to be involved in mediating freezing tolerance, e.g., by regulating the cell osmotic potential through fructan degradation.

Introduction

Fructans – oligo/polymers of fructose – occur in about 15% of the flowering plant species and include many economically important cereals and grasses such as barley (Hordeum vulgare), wheat (Triticum aestivum), oat (Avena sativa), perennial ryegrass (Lolium perenne), timothy (Phleum pratense), fescues and Poa pratensis (Hendry, 1993). Similar to starch, fructans largely serve as storage polysaccharides in plants. In addition, fructans are involved in maintenance of osmotic potentials, membrane stability (Hendry, 1993, Livingston et al., 2009, Valluru and Van den Ende, 2008, Vereyken et al., 2003), cold hardiness and freezing tolerance (Kawakami and Yoshida, 2002). Freezing tolerance in wheat has been shown to be positively correlated with high fructan contents (Yoshida et al., 1998) and perennial ryegrass over-expressing two wheat fructosyltransferase (FT) genes shows increased freezing tolerance (Hisano et al., 2004). In addition, increased contents of fructans in forage grasses are positively correlated to palatability and nutritive value for livestock (Biggs and Hancock, 1998, Longland and Byrd, 2006, Mayland et al., 2000, Zhao et al., 2008). Fructans are also gaining attention in relation to biotechnological and industrial applications such as prebiotics, immunomodulators, nutraceutical formulations, emulsions and cosmetics (Coudray et al., 2003, Vereyken et al., 2003, Vijn and Smeekens, 1999).

The functional properties of fructans depend on their linkage types and degree of polymerization (Vereyken et al., 2003). Most dicots have inulin type (β-2,1 linked) fructans, while grasses mainly have mixed or graminan type (β-2,1 and β-2,6 linked) and neo-series type (fructose is linked to sucrose at the 6th carbon atom of glucose) or levan type (β-2,6 linked) fructans. For example, wheat and perennial ryegrass contain short-chain (up to ∼30 sugar units) graminan and neo-series type fructans, while timothy and P. pratensis contain long-chain (∼200 sugar units) levans (Chatterton and Harrison, 1997, Solhaug, 1991, Spollen and Nelson, 1988, Wei et al., 2002). Long-chain fructans, in particular, are known to increase forage quality and nutraceutical effects (Biggs and Hancock, 1998, Coudray et al., 2003, Wiele et al., 2007).

Four main FTs are involved in fructan biosynthesis (Valluru and Van den Ende, 2008): sucrose:sucrose 1-fructosyltransferase (1-SST) transfers (β-2,1 linkage) the fructose residue from one sucrose molecule to the fructosyl residue of another sucrose molecule, fructan:fructan 1-fructosyltransferase (1-FFT) transfers (β-2,1 linkage) the fructose residue from one fructan molecule to the fructosyl residue of another fructan molecule, fructan:fructan 6G-fructosyltransferase (6G-FFT) transfers the fructose residue from one fructan molecule to the glucosyl residue of another fructan or sucrose molecule and sucrose:fructan 6-fructosyltransferase (6-SFT) mainly transfers (β-2,6 linkage) the fructose residue from a sucrose molecule to the fructosyl residue of a fructan molecule. In addition, fructan 1/6-exohydrolase (1/6-FEH) degrades fructan molecules either with β-2,1 or β-2,6 specificity. Differences in the amount and structure of fructans result from the differential regulation and expression of genes encoding the enzymes involved in fructan metabolism as well as different substrate affinities of the encoded enzymes (Hellwege et al., 1998, Itaya et al., 2007). For example, timothy 6-SFT has high affinity for 6-kestotriose and long-chain β-2,6 linked fructans as substrates and thus produces long-chain levan type fructans, while short chain fructans in wheat and perennial ryegrass are the result of FTs with higher affinity for sucrose or 1-kestotriose (Tamura et al., 2009). Recently, it has been shown that just three amino acid changes can transform a FT's substrate affinity (Lasseur et al., 2009). Recent studies on gene expression in perennial ryegrass during cold acclimation showed increased and coordinated expression of multiple genes encoding enzymes involved in fructan biosynthesis (Hisano et al., 2008). Interestingly, FEHs have been shown to be involved in fructan biosynthesis in wheat and perennial ryegrass, possibly functioning as β-2,1 trimmers by preventing the formation of inulin-type fructans (Lothier et al., 2007, Van den Ende et al., 2003). In addition, FEHs have been suggested to mediate freezing tolerance by degrading fructans, thereby generating a mixture of sugars to regulate the osmotic potential of the cell and stabilize the cell membrane during freezing (Valluru et al., 2008, Van den Ende et al., 2005). Fructans may work alongside cold acclimation proteins (CAP) – cold regulated multi-spanning transmembrane proteins – which are also known to mediate freezing tolerance in the plant cell membrane (Breton et al., 2003, Zhang et al., 2009).

P. pratensis L. (also known as Kentucky bluegrass and in the following denoted as P. pratensis) is an important cool season forage and turf grass that reproduces both sexually, by outcrossing and selfing, and apomictically. The contrasting modes of reproduction facilitate the high polyploidy levels and unusual chromosome numbers (x = 7, 2n = 28–147) observed in P. pratensis (Speckmann and van Dijk, 1972). P. pratensis shows persistence and resilience to cold and seasonal drought (Kanneganti and Kaffka, 1995) and good performance under frequent and close defoliation (Bryan et al., 2000, Durr et al., 2005). Contemporary fructan research is mainly focused on a few plant species including barley, wheat and ryegrass, and despite being a type species for the grass family (Poaceae), little is known about the fructan metabolism in P. pratensis. The content and composition of fructans has been reported for Poa secunda (=Poa ampla) and a putative 6-SFT cDNA sequence is also known for this species (Chatterton and Harrison, 1997, Wei et al., 2002). The aims of the present study were to (1) study fructan accumulation during cold acclimation in three P. pratensis varieties adapted to different environments, (2) identify candidate genes for fructan metabolism and cold acclimation in P. pratensis, and (3) study expression levels of these candidate genes during cold acclimation.

Section snippets

Plant materials and growth conditions

The seeds of three varieties of Poa pratensis (var. Holt, Conni and Evora) were sown in pots (∼480 mL) containing soil mixture. Conni originates from Møn, Denmark (54°58′N, 12°17′E), Evora from Emmeloord, Netherlands (52°42′N, 5°44′E) and Holt from Troms, Norway (69°04′N, 18°09′E). Following germination, plants were grown in a greenhouse (at 9 h photoperiod and ∼25 °C) for 12 weeks from November 2008 to January 2009. Plants (at the pre-elongation stage) were transferred to a growth chamber and

Fructan accumulation in response to low temperatures

Fructan accumulation in leaves of P. pratensis was induced by cold (Fig. 1D). Prior to cold acclimation, mean fructan levels for all varieties were below 0.6% (w/w) and remained at that level for the first 4 days of cold acclimation. Increased fructan contents were observed after 8 days of cold acclimation for Conni, and after 16 days for Holt and Evora. Fructan contents of 1.9%, 3.3% and 5.3% were observed for Evora, Holt and Conni, respectively, after 16 days of cold acclimation. For all

Fructan accumulation is induced by cold acclimation in P. pratensis

P. pratensis has been shown to contain levan type (β-2,6 linked) fructans (Solhaug, 1991), which is similar to P. secunda and timothy (Tamura et al., 2009, Wei et al., 2002), but distinct from perennial ryegrass, which primarily contains graminan or mixed type (β-2,1 and β-2,6 linked) fructans. Many temperate grasses store fructans as reserve polysaccharides in response to increased sugar contents and suboptimal growing conditions under decreasing temperatures. Fructans have been associated

Acknowledgements

The authors thank Graminor AS, Bjørke, Norway for providing P. pratensis (var. Holt) seeds and Betina Hansen for assistance in the greenhouse. This study was made possible by the funding from the Danish Seed Council and Ministry of Food, Agriculture and Fisheries, Danish Food Industry Agency.

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