Fructan structures in Lolium species
The results of the linkage analysis reported here for L. perenne indicate similarities with but differences from, the structures of fructans reported in other members of Lolium. In common with L. rigidum and L. temulentum, 1-kestotriose and 6G-kestotriose were the most abundant trisaccharides in L. perenne. 6-kestotriose was present in much lower amounts (Smouter & Simpson, 1991; Sims et al., 1992).
The DP4 fructan molecules of L. rigidum contained exclusively 2,1-linked fructose residues, 6G,1-kestotetraose and 1 and 6G-kestotetraose being present in higher concentrations than 1,1-kestotetraose (St. John et al., 1997). The two latter DP4 molecules have also been identified in L. perenne (present study) and L. temulentum (Sims et al., 1992). Additionally, these two species contained a DP4 fructan molecule with a 2,6-linked fructose residue attached to the 6G-kestotriose (6G,6-kestotetraose). The 6G,1-kestotetraose has been reported in the three species.
Amongst the DP5 fructan isomers, 1,1 and 6G-kestopentaose and 1 and 6G,1-kestopentaose were common to the three L. species. The 6G,6,6-kestopentaose identified in L. perenne was also found in L. temulentum, but not in L. rigidum. However, about half of the DP5 fructan molecules in L. rigidum were not identified (St. John et al., 1997). These unknown isomers contained almost as much 2,6-linked as 2,1-linked fructose residues. It is possible therefore that the 2,6-linked fructose residues belong to the 6G,6,6-kestopentaose.
Fructans of DP > 8 from L. perenne were similar to the mean size of both fraction C described by Sims et al. (1992) in L. temulentum and the fructan mixture analyzed by Bonnett et al. (1994) in L. rigidum. The majority (76%, 66%, respectively) of high molecular weight fructans from both L. perenne and L. rigidum contained an internal glucose. By contrast, L. temulentum exhibits a progressive increase in the proportion of linear fructans with a terminal glucose residue, as the mean size of the fructans increase. In the fraction containing the highest mean DP (30) fructans, the amount of internal glucose was too small to be quantified. Consequently, high molecular weight fructan molecules in L. temulentum are thought to contain exclusively terminal glucose residues. Marked differences also exist in the relative abundance of 2,6-and 2,1-fructose-fructose linkages among the three Lolium species. There is a higher proportion of 2,6-linked fructose in high molecular weight fructan from L. rigidum (83 : 1, 2,6-linked fructose : 2,1-linked fructose) and L. perenne (72 : 1) than from L. temulentum (40 : 1).
Bonnett et al. (1997 ) proposed that branch points could be used as taxonomic markers to distinguish the Triticodae from the Poodae, since they appeared to be characteristic of the fructan from the Triticodae but absent from or rare in fructan from the Poodae to which Lolium species belong. Indeed, in L. rigidum and L. temulentum , 2,1,6-linked fructose residues were found in very low abundance (2% of all the residues), indicating less than one branch point per molecule. In L. perenne , however, branch points represented up to 3.45% of total residues, which correspond to three to four branch points per molecule, assuming that fructan isomers of DP > 8 all contained the same amount of 2,1,6-linked fructose residues. In Triticum , a member of the Supertribe Triticodae, fructans with a mean DP of nine contained an average of three branch points per molecule ( Carpita et al., 1989 ; Praznik et al., 1992 ). Therefore, the relative abundance of branch points might not be a useful taxonomic marker to distinguish the Triticodae ( Triticum ) and the Poodae ( Lolium ). The 1 and 6-kestotetraose (bifurcose), a branched isomer of DP4, is common to the members of the Triticodae studied so far: ( Triticum ( Carpita et al., 1989 ); Hordeum ( Simmen et al., 1993 ); and Bromus ( Chatterton et al., 1993b )). However, it has never been identified in Lolium species, including L. perenne . Unfortunately, this particular fructan cannot be used either as a taxonomic marker to separate the two Supertribes since it has been reported in Dactylis , a member of the Poodae ( Chatterton et al., 1993c ).
It is unclear whether differences in fructan structures are a result of species differences or of the conditions under which plant material are grown. For example, Bancal et al. (1992) obtained a very different proportion of each fructan isomer from excised wheat leaves induced to accumulate fructan than from field-grown stems and sheaths. Sims et al. (1992) used excised leaves of L. temulentum induced to synthesize fructans by continuous illumination for 24 h whereas Bonnett et al. (1994) extracted fructans from 10-d-old seedlings of L. rigidum induced to accumulate fructan by cooling the meristems and exposing the shoots to continuous high light for 4 d. Interestingly, these fructan-inducible conditions applied to 8-wk-old plants of L. perenne led to a complement of high molecular weight fructan similar to the one reported for L. rigidum. These results suggest then that differences in fructan composition reported in L. species may be related to the conditions under which fructans were accumulated. Recently, Chatterton & Harrison (1997) showed that fructan metabolism in leaves of Poa ampla was altered by a 5°C change in the day/night growth temperature. Poa ampla plants grown in the colder environment contained polymers with 2,6 linkages but no. 2,1-linked fructans. Considerable variation in the abundance of fructan isomers may also exist among different tissues of the same plant. The outer leaf sheath of tall fescue (Festuca arundinacea) contains significantly more fructan DP > 6 and significantly less fructan DP3–6 than the expanding leaves (Housley & Volenec, 1988). Similar results are reported here for L. perenne. Mature leaf sheaths were characterized by high DP fructans whereas elongating leaf bases contained more low DP fructans. These differences could reflect differences between tissues, that is in the nature of the enzymes present or in their regulation. Differences could also be the consequence of the length of time during which fructan have accumulated. Clearly, fructan in bases of elongating leaves have accumulated for only a few days (Morvan-Bertrand et al., 1999), a time presumably insufficient to allow the synthesis of significant amounts of high DP fructan. In contrast, fructan in leaf sheaths that have accumulated over a long period were essentially of high molecular weight. An argument in favor of this hypothesis is that in tall fescue, 3-d-old leaf sheaths contained fructans with a similar average DP as leaf bases (Volenec, 1986).
Fructan synthesis pathway in Lolium
Lolium species produce a complex of fructan structures that belong to three different series: the inulin series, containing exclusively 2,1-linked fructose residues attached to the fructose of the initial sucrose; the inulin neoseries, with an internal glucose residue and β(2–1) linked fructose residues; and the levan neoseries, with an internal glucose residue and β(2–6) linked fructose residues. Grass fructans are synthesized de novo from sucrose, with sucrose as the sole substrate ( Cairns et al., 1999 ) but the enzymatic mechanism is still a matter of debate because all the enzymes or genes of the pathway have not yet been purified. However, based on the current knowledge obtained for different species and assuming that in grasses multiple fructosyl transferases operate ( Vijn & Smeekens, 1999 ), a set of four enzymes would be necessary to account for the synthesis of the three fructan types found in Lolium . Both 1-SST and 1-FFT activities have been measured and the corresponding enzymes have been partially purified from Lolium ( St. John et al., 1997 ) and from other grasses ( Jeong & Housley, 1992 ; Simmen et al., 1993 ). These enzymes could be responsible for the synthesis of the inulin type fructans. 6G-kestotriose is likely the product of the 6G-FT cloned by Vijn et al. (1997 ) in onion. As a majority of fructans from Lolium are based on the 6G-kestotriose, we suggest that most of the flux of C from sucrose to 1-kestotriose is probably mediated by this particular enzyme. Based on the fact that the β(2–6) linkages prevail in grasses, Duchateau et al. (1995 ) suggested that the 6-SFT that catalyzes the transfer of a fructosyl residue from sucrose to a fructan in a β(2–6) linkage, may be the key enzyme for the formation of the (2–6)-linked fructans. Nevertheless, in barley from which 6-SFT has been purified, the main product of the enzyme is bifurcose, a branched DP4 fructan which has not been found in Lolium species. Consequently, if 6-SFT also occurs in Lolium , its affinity for 1-kestotriose must be lower than the affinity of 6G-FT for the same trisaccharide. A lower affinity for 1-kestotriose would result in the synthesis of 6G-kestotriose instead of bifurcose. Furthermore, the affinity of 6-SFT for 6G-kestotriose or for β(2–6)-linked neokestose type fructans must be higher than for 6-kestotriose because no. 6,6-kestotetraose has been found in Lolium . In contrast barley does synthesize 6,6-kestotetraose ( Duchateau et al., 1995 ). Another alternative would imply a 6-FFT (6-fructan-fructanfructosyl transferase) activity which specifically forms β(2–6) fructose linkages without using sucrose as the fructosyl donor.
Fructan hydrolysis in Lolium
Three carbohydrates – (loliose (peak no. 2), 1 and 6G-kestotetraose (peak no. 6), and an unidentified fructan (peak no. 8)) – appear to resist hydrolysis when all others are being mobilized. The reason for this is unknown, but could be attributed either to the specificity of the hydrolytic enzymes involved or to the roles of these carbohydrates in plants: source of C, metabolic intermediate or phloemic carrier of C.
According to the recent studies of Bonnett & Simpson (1993, 1995) and Marx et al. (1997), several isoforms of FEH exist in Lolium species. Some hydrolyzed β(2–1) linked fructans faster than β(2–6) linked fructans, while others exhibited β(2–6) specific activity. The present work showed that in roots and old sheaths of L. perenne, β(2–6) fructans were degraded in the same proportion as β(2–1) fructans after defoliation. Consequently, both FEH isoforms were probably induced by defoliation.
In old sheaths, the amount of high DP fructans decreased without the accumulation of low DP fructans. This result was unexpected, since oligomeric products were released from FEH enzymes purified from oat or barley after one catalytic cleavage (Henson & Livingston, 1996, 1998). This result has been used as evidence for a multichain rather than a single-chain mechanism of hydrolysis. Our results suggest that catalysis in L. perenne occurs either by single-chain attack and/or that the affinity of FEH enzymes is greatest for small fructans. This is, in fact, in accordance with the results obtained by Bonnett & Simpson (1993) on L. rigidum. The higher affinity of FEH enzymes for small molecules may preclude the transient accumulation of smaller partial hydrolysis products.