The circular pathway operating between sucrose and hexose phosphates is well established and has been intensively investigated (Figures 25.1 and 25.2). In the last decade a battery of transgenic plants expressing sense or antisense constructs targeted against most of the genes involved in this pathway (and ectopic expression of proteins specifically directed at many of the metabolic intermediates) have been generated. Moreover, in the last 2 years the genes for plastidial and cytosolic phosphoglucomutase (Tauberger et al., 2000; Fernie et al., 2001b) and sucrose phosphate phosphatase (Lunn et al., 2000) have been cloned suggesting that the jigsaw puzzle of genes is now complete.
Sucrose delivered to the tuber can be cleaved in one of three ways (i) as described above in the apoplast by acid invertase or in the cytosol by either (ii) alkaline invertase or (iii) sucrose synthase (SuSy). As indicated in Figures 25.1 and 25.2 the primary route of sucrose cleavage mirrors the mechanism of unloading with invertase activities being high during the early stages of tuber initiation whilst SuSy dominates in the developing tuber (Appeldorn et al., 1999), whereas the opposite is true for the developing tomato fruit (Damon et al., 1988; Robinson et al., 1988; DemnitzKing et al., 1997). Cereal seeds tend to have high apoplasmic invertase activities to facilitate unloading, however, as mentioned above these are not a prerequisite for unloading hence it is most likely that both pathways have a role to play in sucrose mobilisation within this sink organ (Schmalstig and Hitz, 1987).
The products of sucrose cleavage enter into metabolism by the concerted action of fructokinase and UDPglucose pyrophosphorylase (Zrenner et al., 1993) or fructokinase and hexokinase (Smith et al., 1993; Veramendi et al., 1999, 2002) in the case of the SuSy and invertase pathways, respectively. The hexose phosphates produced by these reactions are equilibrated by the action of cytosolic isoforms of phosphoglucose isomerase and phosphoglucomutase (Fernie et al., 2001b). Sucrose mobilisation has been subject to intense investigation in all crop species, however, perhaps the greatest scrutiny has been applied to the potato.
Experiments of Fu and Park (1995a) (Fu et al., a,b) revealed that potato contains two differentially expressed classes of genes encoding for SuSy in potato which they named SuS3 and SuS4. Both classes were shown to contain 13 introns, including a particularly long leader intron, and their coding regions were found to be 87% identical at the nucleotide level. Using GUS fused to the 5′ flanking sequences they found that SuS3 is strongly expressed in vascular tissues of leaves, stems, roots and tubers—implying a possible role in energy provision in phloem cells: whereas SuS4 genes are strongly expressed in sink tissues such as root tips, basal tissues of the shoot and potato tubers. The SuS4 genes correspond to the cDNA for the T-type isoform first cloned by Salanoubat and Belliard (1987) who proposed that this isoform plays a dominant role in the metabolism of sucrose within the tuber. The T-type isoform was subject to antisense inhibition using the 35S promoter (Franck et al., 1980), and a reduction in SuSy activity was only found in the tubers (Zrenner et al., 1993). In tubers a reduction in activity of up to 95% resulted in a reduction in starch and storage protein content of mature tubers but surprisingly no changes in the level of sucrose. There was, however, a significant increase in the level of hexoses which was in keeping with an observed 40-fold increase in the invertase activity of these lines. The exact reason for this compensatory increase is unknown but it serves to provide further evidence of the flexibility inherent within plant metabolism. Such metabolic flexibility appears to be a central feature of plant metabolism and probably accounts for the absence of major metabolic effects of many transgenic manipulations (e.g., Burrell et al., 1994; Hajirezaei et al., 1994; Fernie et al., 2001c). Thus, when taken together the results of the studies of Zrenner and co-workers strongly indicate that SuSy plays a significant role in determining potato tuber sink strength—at least when activities are reduced. The fact that the enzyme is likely to operate close to equilibrium in vivo (Geigenberger and Stitt, 1993), probably explains the failure to increase sink strength via overexpression strategies (Howard et al., 2001). Further transgenic studies confirmed the pre-eminance of the SuSy route of sucrose cleavage within the developing tuber. Potato plants repressed in the activities of hexokinases (Veramendi et al., 2002, 1999) or of acid invertase (Zrenner et al., 1996) exhibiting very little difference from wild-type tubers implying that these enzymes do not play such a crucial role in metabolism during this stage of the tuber life cycle.
Although the net flux in the tuber is one of sucrose degradation, the rate of sucrose (re)synthesis within this and other tissues is considerable (Wendler et al., 1991; Geigenberger et al., 1997; 1999a; Fernie et al., 2001c; Nguyen-Quoc and Foyer, 2001). This process can also proceed via two different pathways: the reverse of the SuSy degradative pathway or the reactions catalysed by sucrose phosphate synthase and sucrose phosphate phosphatase (Geigenberger and Stitt, 1993). There is clear evidence from feeding experiments with labelled sugars that both pathways contribute to sucrose (re)synthesis within the developing tuber (Geigenberger and Stitt, 1993). It is thought that the combined operation of these pathways with the degradative pathway allows the cell to respond sensitively to both variations in sucrose supply and the cellular demand of carbon for biosynthetic processes (Hatzfeld and Stitt, 1990). The importance of SuSy is demonstrated following antisense repression of this enzyme which resulted in a reduced starch content, coupled to a reduced tuber dry weight and a reduction in storage proteins. Furthermore, when SuSy is bypassed either by introduction of a yeast invertase (Sonnewald et al., 1997; Trethewey et al., 1998) or a bacterial sucrose phosphorylase (Trethewey et al., 2001) there are similar dramatic repercussions on metabolism.
In contrast, sucrose synthesis via sucrose phosphate synthase is relatively well defined (Geigenberger et al., 1997). Although the reaction catalysed by sucrose phosphate synthase is not irreversible, the efficient removal of its product by sucrose phosphate phosphatase means that sucrose phosphate synthase is the first commited reaction in the sucrose synthetic pathway. In photosynthetic tissue sucrose phosphate synthase has been found to be regulated at a variety of levels including allosteric activation by glucose 6-phosphate and inhibition by inorganic phosphate (which allows sucrose synthesis to proceed at times when substrate is plentiful) and deactivated by protein phosphorylation (Reimholz et al., 1994). There is now a growing body of correlative evidence that the potato tuber enzyme is regulated in an analogous manner to the leaf enzyme (Geigenberger et al., 1994, 1999a; Fernie et al., 2001b). The complex regulation of this enzyme suggests that it plays an important metabolic role, a view that is supported by antisense studies which reveal only a minor influence on starch metabolism but a major role in sucrose synthesis in response to water stress (Geigenberger et al., 1997).
In sharp contrast to the lack of changes observed following antisense repression of the majority of enzymes involved in sucrose degradation in the potato are the severe changes seen following repression of the same enzmyes in tomato. With tomato plants altered in invertase (Dickinson et al., 1991; Klann et al., 1996), SuSy (D'Aoust et al., 1999) and hexokinase (Dai et al., 1999) activities display both dramatic phenotypes and marked shifts in metabolism. Transgenic tomatoes expressing a yeast invertase in their apoplast were severely repressed in their growth (Dickinson et al., 1991), however, this transgene was under the control of the tissue-constitutive 35S promoter, and it is most likely that this phenotype results from a block in photoassimilate export. Interestingly, when the expression of one of the tomatoes' six endogenous invertase genes, TIV1, was reduced constitutively by antisense repression of the fruit size was also reduced (Klann et al., 1996) and the precise role of this or any of the other tomato invertases remains to be elucidated. In contrast, the role of SuSy in tomato fruit is well defined and analogous to that in the potato tuber, it seems to be an important determinant of sink strength as indicated by the decreased fruit setting and sucrose unloading capacity of plants exhibiting reduced expression of the enzmye (D'Aoust et al., 1999). Marked phenotypic changes were also observed on the overexpression of an Arabidopsis hexokinase gene in transgenic tomato plants which displayed dramatic growth reduction and early senescence changes which were not seen in the potato (Veramendi et al., 1999). However, it is likely that many of these changes result from the dramatic reduction of leaf photosynthesis in these plants (Dai et al., 1999). In addition to these phenotypic changes, changes in the sugar content and its subsequent mobilisation via metabolism were observed in all the above examples and also in situations wherein the activities of sucrose phosphate synthase (Nguyen-Quoc et al., 1999) were altered transgenically and various invertase activities were altered by conventional plant breeding (Chetelat et al., 1995; Fridmann et al., 2000; Husain et al., 2001). One clear reason for the differences observed on manipulating certain enzyme activities in tomato as opposed to potato is that the tomato fruit goes through large developmental changes whereas the potato tuber does not. This is also reflected in their metabolism. For example starch accumulation in potato proceeds linearly with time (Moorby and Milthorpe, 1975) but is merely transient in tomato fruit (Schaeffer and Petroikova, 1997).
The phenotypes observed in maize seeds on the modification of pathways of sucrose mobilisation are equally severe. We have described the minature1 mutant of maize deficient in cell wall invertase above. Maize further contains two isoforms of SuSy SH1 and SUS1 encoded by the Shrunken1 (Sh1) and Sucrose synthase I (Sus1) loci, respectively. Although both genes are expressed in the developing endosperm, SH1 contributes the majority of the total SuSy enzyme activity. Seeds of the shrunken1 mutant have a mild starch deficiency and degenerate storage cell whereas those of the sus1 mutant shows no phenotypic change (Chourey et al., 1998). Furthermore, distinct phenotypes have been observed on studies of transgenic carrot plants wherein the activities of either invertase or SuSys were altered in the taproots. When invertase activity is severely reduced the carrot plants fail to develop roots suggesting a crucial role for this enzyme in the regulation of carbon portioning (Tang et al., 1999). Reduction of the SuSy activity of carrot roots results in decreased root growth. The transgenic plants also displayed a reduced sucrose utilisation but an increase in starch and cellulose accumulation which was interpreted to suggest that SuSy played an important developmental role within the carrot plant (Tang and Sturm, 1999).
When taken together these examples highlight that plant species vary dramatically with respect to the importance of various routes of both sucrose delivery and utilisation and that successful manipulation at a certain metabolic loci in one species does not imply that such a manipulation will be possible in other species.
It is also important to note that hexose phosphates have a crucial mediatory role in the sucrose to starch transition; they also serve as precursor substrates for glycolysis and oxidative pentose phosphate pathway. However, these pathways have also been the subject of many transgenic studies in heterotrophic tissues and this subject area has been extensively reviewed elsewhere in recent years (Stitt and Sonnewald, 1995; Neuhaus and Emes, 2000; Given, 1999).
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