Carbohydrate Metabolism In Developing Tubers

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3.1. Sucrose degradation

There is general consensus that SuSy plays an important role in sucrose mobilization in developing tubers. Morrell and ap Rees (55) correlated the maximum catalytic activities of acid invertase, alkaline invertase and SuSy with the net rate of sucrose degradation in vivo in developing tubers of three potato cultivars and found that only SuSy could sustain the rates observed in all cultivars. SuSy activity markedly increases at the onset of tuber formation, remains high during the phase of tuber filling and declines at maturity (34, 56). Indeed, Sung et al. (57) claimed that SuSy activity could be used as a marker of sink strength when comparing individual growing potato tubers. When potato tubers are detached from the mother plant a rapid decrease in SuSy activity is observed (58, 59, 60) and this is associated with decreased sucrose content and starch synthesizing capacity and increased invertase activity (59).

The role played by SuSy in sucrose mobilization during potato tuber filling was investigated in transgenic potato plants expressing an antisense RNA (61) corresponding to the T-type SuSy isoform. In the transgenic potato plants, a tuber-specific inhibition of SuSy activity was observed even though the expression of the antisense RNA was driven by the constitutive 35S CaMV promoter. This indicates that independent SuSy forms are responsible for SuSy activity in different potato organs. Although no change in tuber yield (fresh weight) was observed in glasshouse-grown plants, a significant reduction (up to 50%) in tuber dry weight as a result of decreased starch and protein accumulation was observed in lines containing less than 30% residual SuSy activity. This suggests that SuSy activity is in excess of the requirement to sustain sucrose mobilization and that the rate of sucrose degradation may not be the main determinant of sink strength in potato tubers as previously proposed (57). Although a marked (40fold) induction of invertase activity (acid and alkaline) was observed in the transgenic tubers, the additional sucrolytic potential only marginally compensates for the loss of sucrose synthase activity and a massive accumulation of glucose and fructose was observed. The possibility exists that the pathway of sucrose utilization may determine the further processing of the products of sucrose breakdown. This was proposed to be the case in cotton fiber cells where

Amor et al. (62) noted that free UDPglucose (UDPGlc) did not serve as an efficient substrate for cellulose biosynthesis compared with UDPGlc channeled directly from a membrane-associated SuSy. Doehlert (63) and, later, Herbers and Sonnewald (64) suggested that channeling of imported carbohydrates into the SuSy pathway is required for starch biosynthesis whilst sucrose hydrolysis via invertase preferentially allocates substrates for glycolysis or oil synthesis (in maize kernels). This hypothesis also provides an explanation for the reduction in sink strength in transgenic potato plants expressing yeast invertase in the tuber cells cytosol (47). In spite of the increased sucrolytic potential in these tubers, the sucrolytic products appear to be preferentially channeled into the glycolytic pathway or into futile cycles rather than being directed into the starch biosynthetic pathway. How this selective substrate channeling is induced remains to be established.

3.2. Fructose metabolism

Given that SuSy catalyses a ready reversible reaction close to equilibrium in vivo (36) the net sucrolytic flux in developing tubers will be modulated by the relative rates of substrate (sucrose) input and product (UDPGlc and fructose) removal. It has been argued that this type of activity modulation is more elastic and responsive to changes in demand by sink tissues and less complicated than for example metabolite regulation of an enzyme catalyzing an irreversible step such as invertase (36, 65). The rate of fructose removal seems particularly important for the maintenance of net sucrose degradation given that fructose acts as a competitive inhibitor of SuSy activity in the sucrolytic direction (66). Potato tubers lack a hexokinase capable of phosphorylating both fructose and glucose. Instead they contain a specific fructokinase (FK) activity and a hexokinase (HK) activity which is less specific and phosphorylates man-nose, glucose and other hexoses but not fructose (32, 67, 68). The pattern of FK activity closely follows that of SuSy during tuber development (31). Potato FK was first purified by Gardner et al. (69) who described the enzyme as a dimer with a native MW of 70 kDa. The authors

Figure 4. Hexose phosphorylation rate in vivo by developing potato tuber discs at increasing exogenous sugar concentration. Also shown are the maximal catalytic activity of HK (glucose substrate), FK and SuSy and the estimated rate of starch synthesis. Note that the highest exogenous glucose concentrations used, the rate of glucose phosphorylation in vivo approximates the maximal rate of glucose phosphorylation in vitro. On the other hand, fructose phosphorylation saturates at less than 5% of the FK maximal catalytic activity and is close to the estimated rate of starch synthesis in the system (adapted from ref.70).

Figure 4. Hexose phosphorylation rate in vivo by developing potato tuber discs at increasing exogenous sugar concentration. Also shown are the maximal catalytic activity of HK (glucose substrate), FK and SuSy and the estimated rate of starch synthesis. Note that the highest exogenous glucose concentrations used, the rate of glucose phosphorylation in vivo approximates the maximal rate of glucose phosphorylation in vitro. On the other hand, fructose phosphorylation saturates at less than 5% of the FK maximal catalytic activity and is close to the estimated rate of starch synthesis in the system (adapted from ref.70).

identified three FK isoforms which all showed a high degree of specificity for fructose and lack of specificity for the nucleoside triphosphate acting as phosphoryl donor. FK activity does not appear to be allosterically regulated but is significantly inhibited by physiological concentrations of fructose (68, 69). Viola (70) showed that the maximal activities of FK extracted from tubers were greatly in excess of unidirectional rates of sucrose degradation in isolated parenchyma. However, when challenged with exogenous sugars, the fructose phosphorylating capacity of the tissue appeared saturated at values close to the estimated rates of sucrose degradation in vivo which represent only a fraction of the maximal activity of fructokinase in vivo. Based on these results, it was hypothesized that the enzyme is strongly regulated in vivo and that it may play a key role in sucrose mobilization (Figure 4).

The bases of FK regulation in vivo have not been elucidated although FK activity appears to be reversibly modulated by reduction and oxidation (R. Viola, manuscript in preparation) in a manner similar to the potato tuber AGPase (71). However, the physiological significance of this observation remains to be established. A cDNA clone of a FK gene was isolated from potato tubers (72). High levels of the corresponding mRNA were found in tubers, particularly during the early stages of development (73). In tomato, two FK cDNA, Frkl and Frk2, have been isolated (74). The Frk2 cDNA encodes a deduced protein with more than 90% identity to the potato FK. In contrast, the Frkl cDNA encodes a deduced protein that shares only 55% amino acid identity with Frk2. The mRNA corresponding to Frk2 accumulates to high levels in young, developing tomato fruit, whereas the Frkl mRNA accumulated to higher levels late in fruit development. The results indicate that fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development. The presence of additional FK genes in potato tubers remains to be established. Antisense repression of FK activity in potato tubers resulted in a proportional decrease in the ability to phosphorylate exogenous ,4C-fructose by the tuber tissue (H.V. Davies, N. Harris, M.A. Taylor, S. Jarvis, H.A. Ross, S. Millam, M. Blundy, M. Burrell and R. Viola, unpublished results). However, tuber yield is affected only in the most extreme lines (FK activity <10% of the wildtype) which also show a minor reduction in tuber dry weight content. At present, it is unclear why a substantial reduction in the apparent fructose phosphorylating capacity in vivo has little impact on dry matter accumulation in the transgenic tubers.

3.3. Metabolism of other sucrolytic products

The combined activities of SuSy and FK yield fructose 6P (Fru6P) and UDPglucose (UDPGlc). Radiolabelling experiments showed rapid interconversion between Fru6P, glucose 6P (Glc6P), glucose IP (GlclP) and UDPGlc in developing tubers (75). The extractable activities of the enzymes involved in the interconversion of these intermediates in developing potato tubers are usually greatly in excess of the estimated rates of sucrose mobilization. No change in tuber carbohydrate metabolism was observed when UDPGlc pyrophosphorylase (UGPase) activity was reduced by over 95% through antisense repression (76). This indicates that 4-5% UGPase activity is sufficient for the enzyme to function in tuber growth and development. To my knowledge, no attempt has been made to manipulate cytosolic phosphogluco-mutase (PGM) or phosphoglucose isomerase (PGI) activity in potato tubers or in other storage organs. However, the absence of cytosolic PGM activity in the endosperm of many maize

Table 1

5'^C-values of carbohydrates isolated from leaves and immature tubers of glasshouse-grown potato plants (cv Russet Burbank). The tissues were collected at 10 am on a sunny day in July. Data are the mean ± SE of 3-9 individual extracts (n in parentheses. N.S., not significant (adapted from ref.85)

Table 1

5'^C-values of carbohydrates isolated from leaves and immature tubers of glasshouse-grown potato plants (cv Russet Burbank). The tissues were collected at 10 am on a sunny day in July. Data are the mean ± SE of 3-9 individual extracts (n in parentheses. N.S., not significant (adapted from ref.85)

Leaves

Tubers

Fisher's P values

Fructose

-27.3 ± 0.7 (9)

-25.8 ± 0.5 (9)

<0.001

Glucose

-25.5 ±0.4 (9)

-24.8 ± 0.5 (9)

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