Biosynthetic Pathways

It is interesting that both glucose and fructose, which form the disaccharide sucrose, are each incorporated into distinct carbohydrate polymers (i.e. starch and fructan). Sucrose is synthesized in photosynthetically active tissues (source) and transported to tissues where it may metabolized or stored (sink). Sucrose is the most prevalent form of transport sugar in plants and provides a shared starting point for the starch and fructan pathways in sink tissues. Beyond having a common initial substrate, however, the pathways are profoundly dissimilar (Figure 1).

At least two separate enzymes are necessary for conversion of sucrose into polymers containing up to 100 fructose residues in plants [7]. The first step in the pathway involves the enzyme sucrose:sucrose:fructosyltransferase (SST, EC 2.4.1.99), which acts directly on sucrose to produce the trisaccharide kestose. Kestose is the substrate for chain elongation, which is catalyzed by the enzyme fructan:fructan:fructosyltransferase (FFT, EC 2.4.1.100) in Jerusalem artichoke tubers. Variation among fructan-producing species results in polymers of altered size or branching pattern. The reaction also creates free glucose, which does not accumulate in cells actively synthesizing polymer [7].

The complexity of starch granule assembly contrasts greatly with fructan biosynthesis. Even an abridged version of the starch biosynthetic pathway demonstrates that it is far more complex (Figure 1). Similar to the fructan pathway in sink tissues, starch synthesis begins with sucrose. Unlike the fructan pathway, the first step in starch synthesis, catalyzed by the enzyme sucrose synthase, results in the production of a nucleotide sugar and free fructose. Additional metabolic steps include the enzymes UDP-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, multiple forms of starch synthase and multiple forms of starch branching enzymes. The native starch granule is believed to be further shaped during assembly by debranching enzymes and dextranases [8]. The enzymatic steps required for covalent addition of phosphate to starch are not yet completely understood and the role of starch phosphorylase in starch synthesis or degradation remains unclear. Perhaps the most interesting difference between the two pathways is that unlike fructan production, starch synthesis proceeds through phosphorylated and nucleotide-primed sugar intermediates.

Dissimilarities in the starch and fructan biosynthetic pathways include catalytic enzymes, metabolic intermediates, subcellular location and the resulting end-products. This might give the impression that the two polymers accumulate in various unrelated plant species growing in vastly different environments. Despite this impression, many plants are known to synthesize both starch and fructan [9]. The two polymers may be created at separate sites in a plant or together in leaves, roots or stems. One of the most intriguing questions regarding plants that produce more than one type of polymer is how they regulate partitioning of available carbohydrate among the two separate pathways. The methods involved in regulating carbohydrate partitioning in plants that produce only starch do not apply to plants that produce both starch and fructan [7], Preferential diversion of sucrose into water-soluble fructan synthesis in the leaves of Lolium temulantum has been demonstrated [10]. Increased sucrose concentration in many temperate grass species also leads to preferential synthesis of fructan [11]. Evidence suggests that the fructan pathway, in plants that also produce starch, serves as a buffer for periods when sucrose supply is in excess of utilization [12-14], Fructan accumulation due to excess sucrose may occur under oxygen stress, but particularly at reduced temperature where the fructan metabolizing enzymes are known to be quite active [11, 15-18]. Reduced starch synthesis in sink tissue may be attributed to the sensitivity of enzymes in the starch biosynthetic pathway to low temperature [19].

Understanding the factors, which regulate division of a carbohydrate among endogenous metabolic pathways is also critical to the success of transforming a starch-storing plant into one that accumulates fructan. It is not at all clear whether sucrose may be diverted preferentially from starch synthesis in a transgenic plant or how much may be used to synthesize a non-native polymer in starch-storing tissue. Many of the genes associated with fructan synthesis in plants have only recently been cloned [20-21], They may be used as a tool for addressing questions regarding sucrose partitioning, but the precise mechanism by which long chain fructan is synthesized in plants is not entirely understood and regulation of the pathway requires additional study.

The less complex enzymology of bacterial fructan synthesis is much more clearly understood [22], Bacterial fructosyltransferases (EC 2.4.1.10) also catalyze the polymerization of fructose, using sucrose as a substrate (Figure 1). The catalytic sites and critical amino acids in bacterial enzymes have been identified and characterized by site-directed mutagenesis [2325], Microbes are also capable of producing very large fructose polymers, which may find use as hydrocolloids in food and industrial applications. More importantly, large bacterial polymers would contribute less to the osmotic value of the cell, compared to smaller plant fructans. Collectively, the relatively simple bacterial pathway, broad pH optimum and production of larger polymers all represent potential advantages over the use of plant enzymes in addressing difficult questions regarding carbon partitioning and polymer synthesis in transgenic plants.

2.1. Targeting a Bacillus fructosyltransferase to vacuoles

The presence of pathways producing both glucose and fructose polymers in specific plant species is not an evidence that superimposing a new pathway in transgenic plants will be successful. Synthesis of fructan is preferred over starch under specific conditions in source tissue, but it is not clear whether the same is true for transgenic sink tissue. Competition for substrate with native pathways is a concern. However, the bacterial enzymes have a clear advantage over plant SST genes with a nearly 10-fold higher affinity for sucrose [18], The vacuole has been proposed as the site of fructan storage in most plants [26], Targeting a bacterial enzyme to the vacuole must also be considered, and is certainly possible due to the availability of well characterized secretory and sorting signals from native vacuole-targeted proteins [27-28].

Addition of a fructan pathway, through expression of a Bacillus amyloliquefaciens SacB gene, to starch-storing tissue in maize has been reported [29]. The mature coding sequence of the SacB gene was fused to the vacuole sorting signal of the sweet potato storage protein (sporamin) or barley lectin and transformed into maize callus [29]. Tissue specific expression in maize endosperm was accomplished through the use of a maize 10 kD zein promoter. The phenotype of seeds from lines containing this endosperm-specific, vacuole-targeted SacB construct was indistinguishable from wild-type kernels. Also, no differences in soluble sugars, mature seed dry weight or germination rate could be detected in transgenic, compared to wildtype controls [29]. High molecular weight fructose polymer was detected by thin layer chromatography (TLC) analysis in transgenic seeds containing the SacB gene. Gel permeation chromatography also demonstrated that high molecular weight fructan accumulated in transgenic seeds, similar to that produced by bacteria in fermentation culture.

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