Fructans and freezing tolerance
Fructans are linear or branched polymers of fructose that occur in about 15% of flowering plant species, including many which are cultivated commercially – they are synthesized from sucrose in the vacuole, where they are stored as reserve nonstructural carbohydrates. However, their role is wider than just storage. For example, fructan metabolism may impact on the tolerance of plants to drought and frost, and may aid in the defense against infection by fructan-producing pathogens. In this issue (pp. 917–932), Van den Ende et al. report exciting new findings pertaining to the physiological role(s) of fructan. These authors employed a diverse experimental approach that led to the cloning and biochemical characterization of two 6-kestose exohydrolase (6-KEH) isozymes from cold-hardened wheat crown tissue. The 6-KEHs are novel among enzymes of fructan metabolism because of their high specificity for a single substrate, 6-kestose, and their localization in the apoplastic fluid of fructan-containing sink tissues. The thorough examination of fructan metabolism by Van den Ende et al. provides perhaps the best evidence to date that fructan degradation in the apoplast functions to stabilize cell membranes exposed to freezing temperature.
‘6-kestose exohydrolase degradation of apoplastic 6-kestose to sucrose and fructose may be a mechanism to protect membranes from freezing’
Synthesis and degradation
Determining the metabolic pathway(s) of fructan synthesis/degradation proved to be a painstaking endeavor owing to the unusual kinetic properties of the enzymes. Nearly 30 years elapsed before the conceptual model for fructan synthesis proposed by Edelman & Jefford (1968) was shown to be correct (reviewed in Vijn & Smeekens, 1999). The enzymes sucrose:sucrose 1-fructosyl transferase (1-SST) and fructan: fructan 1-fructosyl transferase (1-FFT) synthesize inulin, the most basic fructan. Both 1-SST and 1-FFT are nonspecific with regard to substrate utilization, and the activity is essentially nonsaturable and dependent on both enzyme and substrate concentration (Koops & Jonker, 1996). Subsequently, the enzymes that catalyze the formation of more complex fructans have been isolated and have been shown to have similar properties to 1-SST and 1-FFT.
Compared with fructan synthesis, relatively little is known about the degradation and subsequent mobilization of fructans from the vacuole (Vijn & Smeekens, 1999). Fructan exohydolase (FEH) enzymes have been localized in vacuoles and catalyze the removal of terminal fructose residues. In addition to degrading fructans before mobilization out of the vacuole, vacuolar FEHs are thought to function as trimming enzymes during fructan synthesis (Van den Ende et al.).
Vijn & Smeekens (1999) discussed the long-held suspicions of researchers that fructans have physiological roles not directly associated with the role as a storage form of carbohydrate. As predicted by Vijn & Smeekens (1999), the advent of molecular genetics has been a great benefit to researchers concerned with fructan metabolism. Some surprising results have opened avenues of research not previously considered. Livingston & Henson (1998) reported the presence of fructans and FEH activity in the apoplastic fluid of second-phase cold-hardened crown tissue of oat, leading to the suggestion that fructan metabolism may contribute to the mechanism of freezing tolerance. Van den Ende et al. confirm and greatly extend this research. The two 6-kestose exohydrolase enzymes cloned from a cDNA library prepared from cold-hardened wheat crown tissue were rigorously characterized at the molecular and biochemical levels. The absolute specificity of 6-KEH for 6-kestose provides unequivocal evidence that the enzymes are indeed 6-KEHs, enzymes never before detected in plants. The activity of the 6-KEHs was predominantly found in the apoplastic fluid of the crown tissue, consistent with the array of fructan substrates found in the apoplasic fluid. Furthermore, both 6-KEHs have N-terminal sequences consistent with secretion from the cell. Although the evidence for localization of the 6-KEHs in the apoplastic fluid is not totally definitive, when considered with the novel properties of 6-KEH (especially the extreme specificity for a single substrate) and the expression of 6-KEH in sink tissues that must tolerate freezing temperature, it is logical to conclude that 6-KEH functions to degrade apoplastic 6-kestose to sucrose and fructose as a mechanism to protect membranes from freezing. Alternatively, the authors note that 6-KEH may function to inhibit infection by fructan-producing pathogens, a role that has been proposed for FEH enzymes that occur in plants such as sugar beet that do not synthesize fructans (Van den Ende et al., 2003, 2004).
Transport to the apoplast
Although only briefly discussed by Van den Ende et al., there remains a relevant unresolved question pertaining to fructan metabolism. Specifically, how are fructans, which are synthesized and stored in the vacuole, transported to the apoplast? The authors speculate that fructan transport may occur by a vesicle-mediated mechanism (exocytosis) as described by Echeverria (2000). New results of E. Etxeberria (aka E. Echeverria, pers. comm.) indicate that a similar mechanism exists for import of solutes from the apoplast to cellular compartments (endocytosis). Such a mechanism of transport is attractive because multiple solutes of varying sizes could be transported with no requirement for specific membrane carriers. It seems essential that the mechanism(s) of fructan transport from the vacuole be resolved in order obtain a complete picture of the physiological roles of fructan in plants.
In a more general sense, the experimental approach of Van den Ende et al. provides new information at several levels, from the gene to the intact tissue. Indeed, at least two reports could have been derived from the wealth of data reported in this paper, leading to a more impressive resumé but a much less satisfying story for the scientific community. It can be expected that breakthroughs, such as the discovery of 6-KEH reported in this issue, will soon be forthcoming, leading to new avenues of research. Advances in understanding fructan metabolism may provide new strategies to bioengineer stress tolerance in commercially cultivated plant species and may also impact exploitation of the commercial utility of fructans that has been hampered by the difficulties in obtaining homogeneous complex fructans.