Altered Metabolic Fluxes Result from Shifts in Metabolite Levels in Sucrose

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002 25 Original Article

A. R. Fernie et al.Metabolic ﬂuxes in sucrose phosphorylase-expressing tubers

Plant, Cell and Environment (2002) 25, 1219–1232

Altered metabolic ﬂuxes result from shifts in metabolite levels in sucrose phosphorylase-expressing potato tubers

A. R. FERNIE, A. TIESSEN, M. STITT, L. WILLMITZER & P. GEIGENBERGER

Max-Planck-Institut für Molekulare Pﬂanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany

ABSTRACT sucrose synthase is important to maintain a relatively large sucrose pool and to minimize the ATP consumption As reported in a previous paper (Plant, Cell and Environ- required for normal metabolic function in the wild type. ment 24, 357–365, 2001), introduction of sucrose phosphorylase into the cytosol of potato results in increased Key-words: energy; glycolysis; palatinose; potato (Solanum respiration, an inhibition of starch accumulation and tuberosum) starch synthesis; sucrose degradation; sucrose decreased tuber yield. Herein a more detailed investigation phosphorylase; sucrose synthase; tuber. into the effect of sucrose phosphorylase expression on tuber metabolism, in order to understand why storage and growth are impaired is described. (1) Although the activity INTRODUCTION of the introduced sucrose phosphorylase was low and Conversion of sucrose to starch in growing potato tubers is accounted for less than 10% of that of sucrose synthase its well characterized with respect to the pathway and the expression led to a decrease in the activities of enzymes of location of the individual steps (Kruger 1997) but much less starch synthesis relative to enzymes of glycolysis and rela- is known about the regulation of this important pathway. It tive to total amylolytic activity. (2) Incubation of tuber discs has been demonstrated that ADP-glucose pyrophosphory- in [14C]glucose revealed that the transformants display a lase (AGPase), plastidial phosphoglucomutase (PGM) and two-fold increase of the unidirectional rate of sucrose import of ATP into the plastid via the amyloplast adenylate breakdown. However this was largely compensated by a translocator exert influence over the control of starch syn- large stimulation of sucrose re-synthesis and therefore the thesis in potato tubers (Tjaden et al. 1998; Geigenberger, net rate of sucrose breakdown was not greatly affected. Müller-Röber & Stitt 1999a; Fernie et al. 2001b). AGPase Despite this fact major shifts in tuber metabolism, includ- catalyses the first irreversible step that is unique to the ing depletion of sucrose to very low levels, higher rates of pathway of starch synthesis, and is activated by 3-phospho- glycolysis, and larger pools of amino acids were observed glycerate (3PGA) and inhibited by Pi (inorganic phos- in these lines. (3) Expression of sucrose phosphorylase led phate; Sowokinos & Preiss 1982; Hnilo & Okita 1989). to a decrease of the cellular ATP/ADP ratio and energy These allosteric properties allow the rate of starch synthesis charge in intact growing tubers. It was estimated that at to respond to changes in levels of glycolytic metabolites least 30% of the ATP formed during respiration is con- that are generated when the balance between the rate of sumed as a result of the large acceleration of the cycle of sucrose breakdown and flux into respiratory pathways sucrose breakdown and re-synthesis in the transformants. changes (Geigenberger et al. 1997; Geigenberger, Geiger & Although the absolute rate of starch synthesis in short-term Stitt 1998a). However, on their own, they are not sufficient labelling experiments with discs rose, starch synthesis fell to explain other metabolic scenarios in which the rate of relative to other fluxes including respiration, and the overall starch synthesis is altered; in particular when the sucrose starch content of the tubers was lower than in wild-type level is altered. In these conditions, the rate of starch syn- tubers. (4) External supply of amino acids to replace thesis changes reciprocally to the levels of the glycolytic sucrose as an osmoticum led to a feed-back inhibition of intermediates (Geigenberger et al. 1994; Geiger, Stitt & glycolysis, but did not restore allocation to starch. (5) How- Geigenberger 1998). ever, an external supply of the non-metabolizable sucrose Genetic manipulation of cytosolic sucrose mobilization analogue palatinose – but not sucrose itself – stimulated within the tuber by the ectopic expression of yeast invertase flux to starch in the transformants. (6) The results indicate resulted in a massive metabolic shift, involving induction that the impaired performance of sucrose phosphorylase- both of glycolysis and a rapid cycle of sucrose breakdown expressing tubers is attributable to decreased levels of and re-synthesis, whereas starch accumulation was sucrose and increased energy consumption during sucrose decreased (Sonnewald et al. 1997; Trethewey et al. 1998, futile cycling, and imply that sucrose degradation via 1999a, b). Similarly, the expression of sucrose phosphorylase led to an elevation in the activities of several glycolytic Correspondence: Peter Geigenberger. Fax: +49 3315678408; e-mail: enzymes and the levels of glycolytic intermediates, and an [email protected] even more dramatic decrease of tuber starch content (Tre-

1220 A. R. Fernie et al. thewey et al. 2001). In both cases the lower starch content from 9-week-old, daily-watered plants with high activities was intriguing because the tubers contained elevated levels of sucrose synthase, which is taken as an indicator for rap- of hexose-phosphates and 3-PGA, the substrate and acti- idly growing tubers (Merlo et al. 1993), were used for the vator of the AGPase reaction, respectively. experiments. The generation of transgenic plants has been The role of sugars in the regulation of metabolic and described previously (Trethewey et al. 2001). developmental processes in plants has recently received much attention (see Smeekens 2000) and there is a growing Analysis of enzyme activities body of evidence that sugar-mediated regulation contrib- utes to the regulation of metabolism in potato tubers (see Unless described here enzyme analyses were performed as Fernie, Roessner & Geigenberger 2001a). Furthermore, described in Geigenberger et al. (1998b). In the case of several independent studies (Borisjuk et al. 1998; Geiger sucrose synthase, two different assay conditions were used: et al. 1998; Weber et al. 1998; Grimmer, Bachﬁscher & 150 mM sucrose and 4 mM UDP (Vmax), and 15 mM sucrose

Komor 1999) indicate that the sucrose concentration may and 0·4 mM UDP (Vsel). Sucrose phosphorylase activity was be a key determinant in the regulation of carbon partition- determined using the extraction buffer as detailed by Gei- ing in heterotrophic tissues. The invertase and sucrose genberger & Stitt (1993) and an assay modified from phosphorylase lines are both characterized by a dramatic Birkenberg & Brenner (1984) in that the reaction was buff- reduction of sucrose. Sucrose might act as a signal or, alter- ered at pH 7·0 using Hepes instead of phosphate buffer. natively, influence the osmotic relations. Sucrose represents Starch synthases, branching enzymes and the specific activ- a major osmotic component of the cytosol, and decreased ities of the cytosolic and plastidic isoforms of phosphoglu- levels of this metabolite are often accompanied by major comutase were determined as described in Fernie et al. changes in the levels of other osmotically active com- (2001b). pounds, including amino acids (Hare, Cress & Van Staden 1998). Analysis of metabolite levels Intriguingly, the general metabolic phenotype resulting from expression of a sucrose phosphorylase in potato Sucrose, starch and amino acid levels were determined as tubers resembles that following expression of invertase, described in Geigenberger et al. (1996) with the exception even though the introduced activity was far lower in the of proline which was determined as detailed by Bates, Wal- case of sucrose phosphorylase (Trethewey et al. 2001). Fur- dren & Teare (1973). Phospho-esters and nucleotides were ther, the decrease in starch accumulation was larger in the determined as detailed by Geigenberger et al. (1997). sucrose phosphorylase lines than the invertase lines (Tre- thewey et al. 2001). Taken together, these results indicate Tuber disc labelling experiments that the metabolic consequences following expression of heterologous sucrose degrading activities are not due to an Tuber discs (diameter 8 mm, thickness 1–2 mm) were cut increased rate of sucrose degradation per se. To test this directly from growing tubers attached to the fully photo- hypothesis directly we have now measured the rates of synthesizing mother plant, washed three times with 10 mM sucrose degradation and re-synthesis, and the overall activ- 2-(N-morpholino) ethane sulfonic acid (MES) (pH 6·5; ities of sucrose synthase and sucrose-phosphate synthase in KOH) and then incubated (eight discs in a volume of 4 mL sucrose phosphorylase-expressing tubers. To evaluate the in a 100 mL Erlenmeyer flask shaken at 90 r.p.m) for 25 min alternative possibility that the metabolic shifts are due to or 2 h in 10 mM Mes-KOH (pH 6·5) containing 2 mM glu- differences between properties of the endogenous sucrose cose including 1·4 KBq µmol−1 [U-14C]-glucose (Amer- synthase and the introduced sucrose phosphorylase, we sham-Buchler, Freiburg, Germany) supplemented with analysed whether sucrose phosphorylase led to an acceler- asparagine, mannitol, palatinose, proline or sucrose at var- ated energy consumption in tuber tissue, and whether ious concentrations. Then the discs were harvested, washed external feeding of sucrose, amino acids or the sucrose three times in buffer (100 mL per eight discs), and frozen analogue palatinose to discs of transgenic tubers were able in liquid nitrogen. to increase the rate of starch synthesis. Fractionation of 14C labelled components MATERIALS AND METHODS Tuber discs were extracted with 80% (v/v) ethanol at 80 °C Plant material (1 mL per two discs), re-extracted in two subsequent steps with 50% (v/v) ethanol (1 mL per two discs at each step), Transformants and wild-type potato plants (Solanum the combined supernatants dried under an air stream at ° tuberosum L. cv. Desirée, Saatzucht Fritz Lange, Bad 40 C, taken up in 1 mL H2O, and labelled separated by ion- Schwartau, Germany) were grown in soil (3 L pots) supple- exchange chromatography and thin-layer chromatography mented with Hakaphos grün (100 g per 230 L soil; BASF, exactly as described in Geigenberger et al. (1997). The Ludwigshafen, Germany) in a growth chamber insoluble material left after ethanol extraction was analysed (350 µmol photons m−2 s−1 irradiance, 14 h/10 h day/night for label in starch as in Merlo et al. (1993). Specific activities regime, 20 °C, 50% relative humidity). Growing tubers of the hexose phosphate pool were estimated by dividing

Metabolic fluxes in sucrose phosphorylase-expressing tubers 1221 the label retained in the phospho-ester fraction by the phosphorylation (Huber et al. 1996). This contrasts with summed carbon in this fraction as detailed in Geigenberger sucrose-phosphate synthase (SPS), in which the maximal et al. (1997). In the case of the 25 min feeding experiments catalytic activity was not signiﬁcantly changed, but the the speciﬁc activities were divided by two in order to get activity assayed under non-saturating substrate concentra- the average value during incubation. tions (Vsel) was markedly increased in all transgenic lines. Potato tuber SPS is regulated by reversible protein phosphorylation which changes the kinetic properties of the Statistical analysis enzyme rather than the maximal catalytic activity, and can

Where differences are described in the text as signiﬁcant, a be tracked as a change in the Vsel/Vmax activity ratio (see t-test was performed using the algorithm incorporated into Trethewey et al. 1999b). There was an up to three-fold

Microsoft Excel 7·0 (Microsoft Corp., Seattle, WA, USA) increase in the Vsel/Vmax ratio in all transgenic lines, in com- that yielded a value below 5% (P < 0·05). parison with the wild type, indicating that the activation state of SPS was increased. The expression of sucrose phosphorylase was accompa- RESULTS nied by an up to a 50% decrease in the activity of AGPase Determination of key enzyme activities in lines SP-29 and SP-12, by a reduction in the activity of mediating the sucrose–starch transition soluble starch synthase in line SP-29, and by a decrease in starch phosphorylase activity in lines SP-2, SP-29 and SP- Developing tubers of the transformants contained sucrose 12 (Table 1). There were no clear changes of the activities phosphorylase at an overall activity that was less than 10% of other enzymes of starch metabolism. No significant of that of sucrose synthase (Table 1). The transformants changes were observed in the activities of hexokinases, fruc- contained significantly increased activity of sucrose syn- tokinases and invertases compared to the wild type, whereas thase, when the latter was assayed using saturating sub- glycolytic activities were increased (data not shown, see also strates. However, under non-saturating assay conditions Trethewey et al. 2001). The activity of sucrose phosphory- which are closer to the in-vivo situation, sucrose synthase lase was also accompanied by a significant decrease in activity remained unchanged compared with the wild type, sucrose and starch levels (with the exception of the starch indicating that the increase in overall activity was largely level in line SP-11; Table 1) similar to that previously compensated in planta, possibly via reversible protein observed (Trethewey et al. 2001; Roessner et al. 2001).

Table 1. Sucrose levels, starch levels, and enzyme activities in growing tubers of sucrose phosphorylase-expressing transgenic plants. Sucrose synthase (SuSy) activity was assayed in the presence of 150 mM sucrose and 4 mM UDP (termed Vmax), or in the presence of 15 mM sucrose and 0·4 mM UDP (termed Vsel). Sucrose phosphate synthase (SPS) activity was assayed in the presence of 12 mM Fru-6-P, 36 mM Glc-6-P and 6 mM UDPGlc (termed Vmax), or in the presence of 2 mM Fru-6-P, 6 mM Glc-6-P, 6 mM UDPGlc and 5 mM Pi (termed Vsel). The activation state is calculated as Vsel/Vmax ratio and given as a percentage

Parameter Wild type SP-11 SP-2 SP-29 SP-12

Sucrose (µmol g FW−1) 8·88 ± 0·77 2·34 ± 0·31a 3·48 ± 0·95a 4·44 ± 0·32a 3·39 ± 0·88a Starch (µmol Glc g FW−1) 703 ± 62 592 ± 57 529 ± 46a 489 ± 29a 472 ± 57a Enzyme activities (nmol g FW−1 min−1): Sucrose phosphorylase n.d. 123 ± 7a 162 ± 27a 161 ± 9a 171 ± 32a ± ± ± ± ± SPS Vmax 797 66 638 44 684 64 651 60 784 51 ± ± a ± a ± a ± a SPS Vsel 53 1 125 13 147 25 153 12 190 29 SPS activation state (%) 6·8 ± 0·5 19·8 ± 2·0a 21·3 ± 2·3a 23·7 ± 2·0a 24·8 ± 4·7a ± ± a ± a ± a ± a SuSy Vmax 1088 84 1560 119 2022 185 1724 64 1727 223 ± ± a ± ± ± SuSy Vsel 241 33 157 6 159 25 241 50 201 7 SuSy activation state (%) 22 ± 1 10 ± 1a 8 ± 1a 14 ± 2a 12 ± 1a UGPase 8100 ± 763 n.m. 8274 ± 921 7723 ± 581 7821 ± 682 Cytosolic PGM 1842 ± 129 n.m. 1576 ± 131 1778 ± 133 1815 ± 234 Plastidial PGM 1542 ± 204 n.m. 1842 ± 230 1724 ± 185 1482 ± 134 AGPase 777 ± 54 651 ± 53 643 ± 48 446 ± 52a 432 ± 59a Soluble starch synthase 142 ± 11 n.m. 119 ± 16 98 ± 13a 108 ± 12 GB starch synthase 29 ± 5 n.m. 22 ± 4 28 ± 4 24 ± 3 Branching enzyme Φ 145 ± 15 n.m. 137 ± 23 129 ± 9 141 ± 18 Starch phosphorylase 486 ± 8 n.m. 312 ± 10a 301 ± 14a 328 ± 8a Total amylase 478 ± 31 n.m. 531 ± 29 517 ± 32 596 ± 50

The data presented are the mean ± SE (n = 4–5 tubers from different plants). GB, granule bound. aValues that were determined by the t- test to be signiﬁcantly different from the wild type (P < 0·05). φ, branching enzyme is expressed as fold-stimulation of glycogen phosphorylase; n.m.; not measured; n.d., not detectable.

Table 2. Redistribution of radiolabel within tuber discs excised from wild-type and sucrose phosphorylase-expressing lines following incubation in 2 mM [U-14C]glucose and different sugar supplements. Discs were cut from developing tubers of 10-week-old plants, washed three times in buffer, and then pre-incubated for 20 min in 10 mM MES-KOH (pH 6·5) supplemented as detailed. Incubations then received [U-14C]glucose to a ﬁnal speciﬁc activity of 5625 dpm nmol−1. The discs were then incubated for a further 25 min, washed three times, extracted and analysed for radiolabel in insolubles, sucrose, P-ester, organic acids and amino acids

Distribution of radiolabel, % of total metabolized recovered in: Genotype/ Uptake treatment (dpm g FW−1 25 min−1) Insolubles Sucrose P-esters Org acids Amino acids

Wild type 25 mM Man 172232 ± 3425 25·1 ± 2·1 34·8 ± 1·1 26·9 ± 2·0 5·2 ± 0·5 8·0 ± 0·8 25 mM Suc 180616 ± 7829 28·9 ± 2·0 32·6 ± 2·0 22·9 ± 0·9 6·4 ± 0·2 9·2 ± 0·3 100 mM Suc 197856 ± 4717 38·6 ± 2·9b 30·0 ± 0·3 17·9 ± 2·1b 5·4 ± 0·6 8·1 ± 0·6 SP-11 25 mM Man 162608 ± 7243 9·6 ± 1·3a 61·9 ± 1·3a 19·2 ± 0·5a 4·2 ± 0·6 5·1 ± 0·2a 25 mM Suc 164070 ± 5674 8·9 ± 0·8a 61·9 ± 2·4a 19·6 ± 0·3a 4·3 ± 0·8a 5·4 ± 0·7a 100 mM Suc 174452 ± 3113a 9·4 ± 0·5a 63·7 ± 0·8a 18·4 ± 0·4 3·0 ± 0·2a 5·5 ± 0·2a SP-2 25 mM Man 189238 ± 9621 12·1 ± 1·1a 59·5 ± 2·7a 18·9 ± 1·2a 3·9 ± 0·5 5·7 ± 0·5a 25 mM Suc 200577 ± 7414 11·5 ± 1·2a 58·5 ± 2·7a 19·8 ± 0·8a 4·4 ± 0·6a 5·8 ± 0·4a 100 mM Suc 203695 ± 6923 13·7 ± 1·3a 62·4 ± 2·3a 15·4 ± 0·5 3·6 ± 0·5a 5·0 ± 0·4a SP-29 25 mM Man 186322 ± 8772 11·3 ± 2·0a 65·1 ± 2·0a 16·9 ± 0·9a 2·8 ± 0·2a 3·9 ± 0·2a 25 mM Suc 178165 ± 10800 8·0 ± 0·9a 69·1 ± 0·8a 15·9 ± 0·5a 2·7 ± 0·2a 4·2 ± 0·2a 100 mM Suc 229617 ± 5676a 9·5 ± 1·7a 71·3 ± 2·6a 12·9 ± 0·7ab 2·8 ± 0·3a 3·5 ± 0·1a

Data presented are the mean ± SE, n = 4. aValues that were determined by the t-test to be signiﬁcantly different from wild type (P < 0·05). bValues that were determined by the t-test to be signiﬁcantly different in response to sugar supplied (P < 0·05).

Metabolism of labelled glucose in tuber discs of sucrose in the incubation medium significantly stimulated the transgenic lines label incorporation into insoluble compounds (starch) and inhibited label incorporation into sucrose and phospho- In order to analyse the rate of sucrose degradation and its esters in wild-type discs, there was no marked or consistent re-synthesis, freshly cut slices of growing potato tubers effect in discs from the transformants were incubated with 2 mM [U-14C]-glucose in the presence of 25 mM mannitol (Table 2). Labelled glucose was pre- ferred to sucrose due to the large changes in the internal Estimation of rates of sucrose synthesis and sucrose levels observed in the transformants. Furthermore, breakdown, glycolysis and starch synthesis this choice of radio-labelled substrate also provided the opportunity to perform experiments under a range of In short-term labelling experiments the relative distribu- sucrose concentrations and to measure the rate of sucrose tion of label does not always reflect the actual metabolic re-synthesis under these conditions. In both wild-type and fluxes due to isotopic dilution (for details see Geigenberger transgenic tissue, 14C glucose uptake rose slightly when 25 et al. 1997). We divided label retention in the phospho-ester or 100 mM sucrose was included in the medium. The metab- fraction after 25 min (Table 2) by the summed carbon in the olism of label differed markedly between wild-type discs hexose phosphate pool (data not shown), and then divided and discs from sucrose phosphorylase-expressing tubers. A again by two to estimate the average specific activity of the markedly and significantly smaller proportion of the label hexose phosphate pool during the 25 min incubation. The was incorporated into insolubles (of which between 90 and endogenous pools of hexose-phosphates (approximately 93% of the label was consistently recovered in starch 150–500 nmol g FW−1, data not shown) and sugars (i.e. regardless of the genotype or incubation conditions) in the sucrose 2000–10000 nmol g FW−1, see Table 1) were much transformants. Label incorporation into the intermediates larger than the amount of external (labelled) glucose that and products of glycolysis (phospho-esters, organic and is taken up in 25 min (approximately 30–40 nmol g FW−1, amino acids) also decreased in the transformants, but less calculated from Table 2). As a result the specific activity of markedly than label incorporation in starch. Moreover a the phospho-ester pool will rise gradually during incuba- markedly and significantly larger proportion of the label tion, and the value obtained after the end of the 25 min was recovered in sucrose in the transformants than in wild- incubation period will overestimate the average value dur- type discs (approximately 60% compared with 30%), ing incubation by a factor of two. reflecting the increased activation state of SPS (see above). Incubation of discs in 100 mM sucrose significantly Intriguingly, whereas inclusion of high concentrations of decreased the proportion of radiolabel in the phospho-

Table 3. Absolute rates of starch synthesis, glycolysis and sucrose re-synthesis within tuber discs excised from wild-type and sucrose phosphorylase overexpressors following incubation as in Table 2. Absolute rates of starch synthesis, glycolysis and sucrose re-synthesis were calculated from the label incorporation data using the specific activity of the hexose phosphate pool in order to account for isotopic dilution factors. The specific activity of the hexose phosphate pool was estimated by dividing the label released by the action of acid phosphatase by the sum of carbon in phosphorylated sugars measured in the same samples at the end of the incubation. Values were corrected by dividing by two, to get the mean specific activity during the course of the 25 min incubation

Metabolic ﬂux (nmol hexose equivalents g FW−1 25min−1) Genotype/ HP speciﬁc activity treatment (dpm nmol−1) Starch synthesis Glycolytic Sucrose synthesis Total

Wild type 25 mM Man 151 ± 14 286 ± 54 150 ± 26 396 ± 48 832 25 mM Suc 133 ± 9 388 ± 54 212 ± 22 444 ± 54 1044 100 mM Suc 110 ± 11 698 ± 140b 240 ± 48 532 ± 56 1470 SP-11 25 mM Man 39 ± 2a 408 ± 100 388 ± 74a 2578 ± 170a 3374 25 mM Suc 33 ± 1a 436 ± 48 480 ± 98a 3076 ± 174a 3992 100 mM Suc 37 ± 2a 440 ± 42 400 ± 44a 2996 ± 152a 3836 SP-2 25 mM Man 36 ± 2a 642 ± 94a 502 ± 84a 3130 ± 284a 4274 25 mM Suc 44 ± 4a 536 ± 126 474 ± 96a 2714 ± 336a 3724 100 mM Suc 33 ± 2a 848 ± 150 534 ± 112a 3874 ± 384a 5254 SP-29 25 mM Man 30 ± 1a 724 ± 190 412 ± 42a 4014 ± 204a 5150 25 mM Suc 28 ± 2a 504 ± 130 432 ± 68a 4290 ± 344a 5226 100 mM Suc 29 ± 1a 744 ± 174 496 ± 70a 5622 ± 408ab 6862

ester fraction by about 30% in discs from wild-type tubers, altered in SP-2 and SP-29, and decreased in SP-11. The compared with discs incubated with 25 mM sucrose or man- unidirectional rate of sucrose breakdown was calculated as nitol, resulting in a decrease of the specific activity of the the difference between the net rate of sucrose degradation hexose phosphate pool (Table 2). This presumably reflects and the rate of unidirectional sucrose synthesis. Expression an increased rate of sucrose mobilization when sucrose is of sucrose phosphorylase led to 1·6- to 2·3-fold increase in entering the discs from the medium. The specific activity of the hexose phosphate pool was much (and significantly) lower in the transformants than in wild-type tuber discs, probably reflecting an increased rate of carbohydrate mobi- Table 4. Direct measurement of the net rate of sucrose degradation and estimation of unidirectional rates of sucrose synthesis and lization in these lines (Table 3). degradation within tuber discs excised from wild-type and sucrose The specific activities were then used to estimate abso- phosphorylase-expressing lines following incubation in 25 mM lute fluxes within the tuber discs (Table 3). When discs incu- mannitol as in Table 2. Net sucrose degradation was measured as bated in 25 mM mannitol were compared, the expression of the difference of the sucrose content of the discs before incubation sucrose phosphorylase led to a 2·6- to 3·3-fold significant (see Table 1) minus the sucrose content of the discs at the end of increase in glycolytic flux, a 1·4- to 2·5-fold increase in the the incubation (see Fig. 2). Unidirectional (UD) sucrose degrada- absolute rate of starch synthesis (only significant for line tion was estimated as the sum of net sucrose degradation plus UD sucrose synthesis (see Table 3) SP-2), and also a 6·5- to 10-fold significant increase in the unidirectional rate of sucrose re-synthesis. Thus, the trans- Fluxes (nmol hexoses g FW−1 25 min−1) formants partitioned less label from 14C glucose to starch, but the calculated absolute carbon flux towards starch was, Net sucrose UD sucrose UD sucrose surprisingly, higher than in the wild type. Genotype degradation synthesis degradation The net rate of sucrose degradation was measured Wild type 2186 ± 199 396 ± 48 2582 ± 548 directly by subtracting the sucrose levels in the discs before SP-11 1303 ± 133a 2578 ± 170a 3881 ± 652 and at the end of the incubation (Table 4). This approach SP-2 2264 ± 418 3130 ± 284a 5394 ± 996a is only valid in the case in which no external sucrose is SP-29 1902 ± 171 4014 ± 204a 5916 ± 832a supplied, and we therefore only present data for discs incubated in 25 mM mannitol. Unexpectedly, the expression of Data presented are the mean ± SE, n = 4. aValues that were sucrose phosphorylase did not increase the net rate of determined by the t-test to be significantly different from wild type sucrose degradation, with the rate not being significantly (P < 0·05).

Table 5. Estimation of the ATP demand of sucrose cycling. The wastage of ATP during sucrose cycling was calculated for wild type and transformants by using the metabolic flux data of Table 3. In the case of the wild type we assumed that the cycle involves operation of sucrose synthase and SPS and that one molecule of ATP is required per molecule of sucrose which is degraded and re-synthesized (to phosphorylate the fructose formed by sucrose synthase). In the case of the transformants expressing sucrose phosphorylase we assumed that two molecules of ATP are required per molecule of sucrose during the cycle (as no UTP is synthesized when sucrose is converted to Glc-1-P by sucrose phosphorylase). The rate of sucrose degradation was estimated as the sum of the rates of starch synthesis, glycolysis and sucrose re-synthesis. The total gain of ATP was estimated from the glycolytic flux assuming that (i) flux into glycolysis equals respiration, that (ii) each glucose-equivalent entering glycolysis is fully oxidized, and that (iii) the total yield of a fully oxidized glucose is 36 ATP (for the latter see Stryer 1988)

Wild type SP-11 SP-2 SP-29

Estimated rates of ATP production and consumption [µmol (g FW)−1 (25min)−1] Total ATP produced 5·40–8·64 13·9–17·3 17·0–19·2 14·84–17·86 ATP required for sucrose cycling 0·20–0·26 2·98–3·54 3·22–4·57 4·58–6·26 Sucrose degradation 0 1·69–2·00 1·86–2·63 2·58–3·44 Sucrose re-synthesis 0·20–0·26 1·29–1·54 1·36–1·94 2·00–2·82 ATP required for sucrose cycling (in % of total produced) 3–4 20–21 19–24 31–35

the unidirectional rate of sucrose breakdown (significant is consumed in this cycle, because not every glucose mole- for SP-2 and SP-29). cule that enters glycolysis will be respired, and it is unlikely that respiratory electron transport and ATP production are perfectly coupled. These calculations demonstrate that Estimation of ATP consumption during sucrose expression of sucrose phosphorylase leads to a substantial cycling and determination of adenylate levels loss of cellular energy in potato tubers. This is consistent Although a cycle of sucrose synthesis and degradation with the significant decrease of the ATP/ADP ratio and the allows sensitive regulation of sucrose breakdown, it also adenylate energy charge in intact tubers following sucrose represents a ‘futile cycle’ (Dancer, Hatzfeld & Stitt 1990; phosphorylase expression (Fig. 1) and furthermore also Geigenberger & Stitt 1991) leading to consumption of ATP. provides an explanation for the elevated rate of respiration We used the data in Table 3 to calculate the wastage of ATP in these tubers. during sucrose cycling. In the case of wild-type tubers we assumed that the cycle involves operation of sucrose syn- Influence of high external sucrose on the thase and SPS and that one molecule of ATP is required metabolism of labelled glucose per molecule of sucrose, which is degraded and re-synthesized (to phosphorylate the fructose formed by sucrose syn- In agreement with the results of previous studies (Geiger thase). In the case of the transformants expressing sucrose et al. 1998; Loef, Stitt & Geigenberger 2001) there was a phosphorylase we assumed that two molecules of ATP are 2·5-fold significant increase in the rate of starch synthesis required per molecule of sucrose during the cycle (as no and a smaller increase in the rate of sucrose synthesis and UTP is generated when sucrose is converted to Glc-1-P by glycolysis when external sucrose was supplied to wild-type sucrose phosphorylase). Table 5 summarizes the loss of discs. In contrast, starch synthesis was not significantly ATP during the cycle as well as the maximum rate of ATP changed in response to high external sucrose in discs from production, assuming that (1) flux into glycolysis equals transgenic tubers (Table 3). This might be because the respiration, that (2) each glucose-equivalent entering glyc- transformants have lost the capacity to respond to sucrose, olysis is fully oxidized, and that (3) the total yield of a fully or because the introduced sucrose cleaving activity mobi- oxidized glucose is 36 ATP (for the latter see Stryer 1988). lizes sucrose so efficiently that it does not accumulate in the In wild-type tubers, ATP production was estimated to discs, despite being supplied at high concentrations in the be 5·4–8·7 µmol ATP (g FW)−1 (25 min)−1, whereas 0·20– medium. To distinguish between these possibilities we 0·26 µmol ATP (g FW)−1 (25 min)−1 were consumed during determined the internal sucrose level in the discs at the end sucrose cycling (Table 5). Therefore only 3–4% of the total of the incubation (Fig. 2). The discs were washed three ATP produced was wasted in the cycle, which is similar to times to remove sucrose from the apoplast. A separate the proportion estimated in germinating Ricinus seedlings experiment showed that 94% of the labelled sucrose was (Geigenberger & Stitt 1991). A different picture emerged removed on the first wash, another 5% on the second and in the transgenic lines in which ATP production and ATP up to 1% by the third, and that the fourth to tenth washes wastage during cycling was estimated to be 14–19 and 3– only yielded trace levels of label. The sucrose content in − − 6·3 µmol (g FW) 1 (25 min) 1, respectively, revealing that wild-type discs doubled following incubation in 100 mM 20–35% of the total ATP produced in respiration is being sucrose. The level of sucrose within the transgenic lines was wasted during operation of this cycle. This represents a very low, and although it doubled when 100 mM sucrose minimum estimate of the proportion of the total ATP that was included in the medium it remained far below wild-

15 ) 1 –

10 mol gFW m

5 Sucrose content (

0 0 255075100

External sucrose (mM)

Figure 2. Internal sucrose concentrations of wild-type and transgenic tuber discs following incubation in varying concentrations of sucrose. Samples in which the sucrose levels were determined are the same as those used for the analysis of the redistribution of radiolabel presented in Table 2. Sucrose levels in the transgenic lines were determined to be signiﬁcantly different from wild type (t-test, P < 0·05). , Wild type (WT); , SP-11; , SP-2; , SP-29.

type levels. These results show that supplying exogenous sucrose does not lead to a substantial enough increase of the internal sucrose pool in the transgenic discs to allow complementation of the metabolic phenotype (see below for further experiments).

External amino acid supply to tuber discs leads to feed-back inhibition of glycolysis, without influencing starch synthesis Expression of sucrose phosphorylase led to a significant and up to two-fold increase in the level of asparagine, which is the major amino acid in potato tubers (Table 6). There were also significant changes in the levels of minor amino acids, including a significant increase in alanine, tyrosine, arginine and serine, whereas GABA, valine and isoleucine decreased. The total level of amino acids increased, but this was only significant in line SP-2 (Table 6). This accumulation of amino acids is consistent with the increased rate of

Figure 1. Nucleotide levels and adenylate energy status in tubers expressing a bacterial sucrose phosphorylase. Tuber slices from developing tubers were frozen immediately in liquid nitrogen and then extracted in trichloroacetic acid to analyse nucleotide levels. (a) ATP; (b) ADP; (c), AMP; (d) ATP/ADP ratio; (e) adenylate 1 × energy charge, calculated as (ATP + /2ADP) (ATP + ADP + AMP)−1; (f) sum of adenylates; and (g) sum of uridinylates. Values presented are the mean ± SE for determinations on ﬁve individual plants per line. *Values that were determined by the t-test to be signiﬁcantly different from wild type (P < 0·05).

Table 6. Amino acid contents of transgenic tubers. Amino acid contents were determined in samples from developing tubers

Wild type SP-11 SP-2 SP-29 SP-12

Alanine 0·8 ± 0·1 1·7 ± 0·2a 2·5 ± 0·1a 3·4 ± 0·5a 1·7 ± 0·3a Arginine 2·4 ± 0·1 5·5 ± 0·4a 6·6 ± 0·1a 3·9 ± 0·1a 6·0 ± 1·0a Asparagine 12·8 ± 1·0 20·8 ± 2·3a 22·9 ± 2·8a 15·1 ± 2·2 25·0 ± 3·9a Aspartate 1·4 ± 0·1 1·8 ± 0·2 1·5 ± 0·2 1·7 ± 0·1a 1·5 ± 0·1 Glutamate 2·9 ± 0·3 2·9 ± 0·2 2·4 ± 0·2 2·5 ± 0·1 2·7 ± 0·4 Glutamine 9·4 ± 0·3 8·5 ± 0·9 7·9 ± 1·1 6·6 ± 0·7a 8·0 ± 1·5 Glycine 0·3 ± 0·0 0·3 ± 0·0 0·4 ± 0·1 0·3 ± 0·1 0·2 ± 0·0 Histidine 0·5 ± 0·1 0·6 ± 0·1 0·7 ± 0·1 1·5 ± 0·1a 0·5 ± 0·1 Isoleucine 1·0 ± 0·1 0·6 ± 0·1a 0·8 ± 0·1 0·6 ± 0·1a 0·6 ± 0·1a Leucine 0·1 ± 0·0 0·1 ± 0·0 0·1 ± 0·0 0·1 ± 0·0 0·1 ± 0·0 Lysine 0·7 ± 0·1 0·9 ± 0·2 0·8 ± 0·1 0·6 ± 0·1 0·5 ± 0·1 Methionine 1·1 ± 0·0 1·2 ± 0·1 1·2 ± 0·1 1·0 ± 0·1 0·8 ± 0·0a Phenylalanine 1·0 ± 0·1 1·2 ± 0·2 1·4 ± 0·1 1·4 ± 0·1 1·0 ± 0·1 Proline 0·5 ± 0·0 0·5 ± 0·0 0·5 ± 0·0 0·5 ± 0·0 0·5 ± 0·0 Serine 1·0 ± 0·1 1·7 ± 0·2a 1·9 ± 0·2a 2·3 ± 0·2a 1·7 ± 0·1a Threonine 0·9 ± 0·0 0·7 ± 0·1 0·7 ± 0·1 0·0 ± 0·1 0·6 ± 0·1 Tyrptophan 0·2 ± 0·0 0·3 ± 0·1 0·4 ± 0·0 0·3 ± 0·1 0·2 ± 0·0 Tyrosine 0·9 ± 0·1 1·9 ± 0·2a 2·2 ± 0·3a 1·5 ± 0·2a 1·8 ± 0·2a Valine 2·6 ± 0·2 1·3 ± 0·2a 1·4 ± 0·2a 1·2 ± 0·1a 1·4 ± 0·2a Gaba 3·9 ± 0·2 2·3 ± 0·2a 2·8 ± 0·1a 3·3 ± 0·3 2·3 ± 0·2a Total amino acids 44·8 ± 2·6 54·9 ± 5·3 59·3 ± 7·5a 47·8 ± 3·7 57·2 ± 9·2

− The data presented are the mean ± SE for determinations on ﬁve individual plants per line, and are given as µmol g FW 1. Gaba = γ-amino- butyric acid. aValues that were determined by the t-test to be signiﬁcantly different from wild type (P < 0·05).

glycolysis in the transgenic tuber discs. Similar results were large pool within the tuber (see Table 6) and is mainly obtained in invertase and invertase/glucokinase overex- located in the cytosol (A. Tiessen and P. Geigenberger, pressing lines (Trethewey et al. 1998). The higher amino unpubl. results). Proline was chosen as it often increases acid content in tubers expressing sucrose phosphorylase or inside cells in response to osmotic stress (Roosens et al. invertase and containing decreased levels of sucrose is con- 1999). The uptake rates of sucrose, proline and asparagine sistent with the proposal that sucrose plays a role in the were equivalent in all cases. Fractionation revealed that the maintenance of the cellular osmotic status (Winter, Robin- amino acids were relatively inert, with 15–20% of the label son & Heldt 1993; Hare et al. 1998; Geigenberger et al. being recovered in protein after 2 h of incubation, and the 1997, 1999b). Interestingly, transgenic lines with decreased remainder staying in the amino acid pool (data not shown). expression of AGPase or plastidial PGM that contain Tuber discs were incubated in tracer levels of labelled increased levels of sucrose are characterized by a reduction glucose with and without supplementary 50 mM proline or in amino acid levels (Trethewey et al. 1999a; Fernie et al. 100 mM asparagine for a 2 h period (Fig. 3). When discs 2001b). incubated without external amino acids were compared, One possible explanation for the lower starch content in expression of sucrose phosphorylase led to an increase in the sucrose phosphorylase-expressing lines could be that the proportion of label incorporation into sucrose (Fig. 3c) there is increased allocation of carbon towards glycolysis whereas the proportion of label incorporated into starch and amino acid biosynthesis, to compensate for the low (Fig. 3b), phosphate-ester (Fig. 3d), organic acid (Fig. 3e) sucrose and meet the requirement for cellular osmotica. To and especially that in amino acids (Fig. 3f) decreased. Fur- investigate this possible interplay between amino acid and thermore, the specific activities of the phospho-ester pool starch synthesis, we investigated whether externally sup- of the transgenic lines decreased sharply with respect to the plied amino acids influence metabolic fluxes in tuber discs. wild type (Fig. 3g). These results resemble those already In this experiment (Fig. 3) and also in the following exper- presented above. In discs from wild-type tubers external iment of Fig. 4 we used only two transgenic lines SP-11 and amino acids led to decreased labelling of organic acids and SP-29. These lines have been shown to be representative amino acids from 14C-glucose (Fig. 3e & f, changes were not lines in the present study (see above) and during extensive significant at the 5% level, but a consistent trend is investigations in a previous study (Trethewey et al. 2001). observed), whereas labelling of starch remained unchanged In an initial experiment the relative uptake rates of pro- (Fig. 3b). The same trend was observed after feeding proline, asparagine and sucrose were compared by incubating line or asparagine to discs of tubers expressing sucrose wild-type tuber discs in 50 mM of [U-14C] labelled substrate phosphorylase (significant in three cases with respect to the (100 mM in the case of asparagine). Asparagine was chosen decrease in organic acids, and in one case with respect to because it is the major amino acid representing a fairly amino acids). The sum of label in organic acids and amino

acids provides an estimate of the glycolytic flux. In wild- type and transformant discs, label entering glycolysis decreased after feeding asparagine or proline. Label in glycolytic products decreased to 64 ± 8*, 86 ± 11 and 69 ± 9%* of control levels after feeding proline and 79 ± 9, 60 ± 8* and 47 ± 4%* of control levels after feeding asparagine to wild type, SP-11 and SP-29, respectively (* denotes values that are significantly different from the control). The results indicate that amino acid biosynthesis and glycolysis are feed-back inhibited when amino acids are supplied. Cru- cially, feed-back inhibition of glycolysis did not lead to a corresponding increase in the flux to starch. Further, similar changes were seen for discs from wild-type tubers and transformant lines. It is therefore unlikely that the decreased partitioning to starch in response to sucrose phosphorylase expression is a direct consequence of changes in glycolytic flux.

The partitioning of carbon toward starch in the transgenic lines can be considerably increased on incubation with palatinose In a second approach to investigate whether the low sucrose levels per se restrict starch synthesis in the sucrose phosphorylase-expressing tubers, we supplied the sucrose analogue palatinose. This approach was taken because addition of external sucrose did not lead to an increase of the internal sucrose content of discs from the transformants (see above), and sucrose feeding would anyway not distinguish between possible effects due to signalling, sucrose supply and osmotic status. Palatinose cannot be cleaved by SuSy, invertase or any other sucrose-degrading activity present in wild-type tubers (Fernie et al. 2001a), however, there are no reports in the literature of whether palatinose inﬂuences the activity of sucrose phosphorylase. For this reason we assayed sucrose phosphorylase activity in a desalted enzyme extract from a sucrose phosphorylase- expressing tuber in the presence of various palatinose and sucrose concentrations. Palatinose could not be metabolized by sucrose phosphorylase but it was a weak competi- tive inhibitor of the enzyme: sucrose phosphorylase

exhibited a Ki of 37 mM (data not shown). Since the esti-

Figure 3. Effect of amino acids on the metabolism of 14C-glucose by potato tuber slices. Freshly cut slices of growing potato tubers of wild type and transformants were incubated for 2 h in the presence of 10 mM Mes-KOH (pH 6·5) and 2 mM [U-14C]glucose (specific activity 1·4 kBq µmol−1) with and without the addition of 50 mM proline or 100 mM asparagine before they were washed and extracted to determine label distribution. (a) [14C]glucose absorbed by the tissue. Incorporation of 14C into (b) starch; (c) sucrose; (d) phosphate esters; (e) organic acids; and (f) amino acids is expressed as a percentage of the label metabolized. The specific activity of the hexose phosphate pool (g) was estimated by dividing the label retained in the phosphate ester pool by the summed carbon of the hexose phosphates. The results are means ± SE (n = 3). *Values that were determined by the t-test to be significantly different from buffer-only control (P < 0·05).

Figure 4. Addition of palatinose affects the metabolism of 14C-glucose by potato tuber slices. Freshly cut slices of growing potato tubers were incubated for 2 h in the presence of 2 mM [U-14C]glucose (specific activity 1·4 kBq µmol−1) supplemented with 100 mM mannitol or 100 mM palatinose, before they were washed and extracted to determine label distribution. (a) [14C]glucose absorbed by the tissue (b) absorbed label that is metabolized to other compounds. Incorporation of 14C into (c) starch; (d) sucrose; (e) phosphate ester; (f) organic acids; (g) amino acids; (h) maltose; and (i) soluble glucans is expressed as a percentage of the label metabolized. The specific activity of the hexose phosphate pool (j) was estimated by dividing the label retained in the phosphate ester pool by the summed carbon of the hexose phosphates, and was used to calculate absolute fluxes to starch (k); sucrose (l); and glycolysis (m). (n) The ratio between starch and glycolyis. The results are means ± SE (n = 3). Fluxes are given as nmol gFW−1 2 h−1. *Values that were determined by the t-test to be significantly different in response to sugar supplied (P < 0·05).

© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1219–1232 Metabolic fluxes in sucrose phosphorylase-expressing tubers 1229 mated cellular concentration of palatinose following a 2 h composition occur even though the unidirectional rate of incubation with 5–100 mM of the analogue is 0·1–1 mM (see sucrose breakdown is only increased about two-fold, and Fernie et al. 2001a) it is approximately three orders of mag- this increase is largely offset by a large stimulation of nitude lower than estimated concentrations of sucrose and sucrose re-synthesis, with the result that the net rate of is therefore far too low to inhibit sucrose phosphorylase in sucrose mobilization is not markedly stimulated (Table 4). vivo. This relatively small stimulation of sucrose breakdown is In both wild-type and transgenic discs, the rates of 14C- consistent with the relatively low activity of sucrose phos- glucose uptake and metabolism were slightly increased by phorylase in the transformants, which was 10-fold lower including 100 mM palatinose in the medium (Fig. 4a & b). than the activity of sucrose synthase (Table 1). Palatinose led to a change in the metabolism of labelled This comparison indicates that the changes we observe glucose in both wild type and transformants. In both cases, in tuber growth and metabolism may be due to specific there was a consistent trend of palatinose to increased par- features of sucrose phosphorylase, or the reaction which it titioning of radiolabel into starch (Fig. 4c). When absolute catalyses, rather than a major change in the overall rate of fluxes are calculated it becomes apparent that palatinose sucrose breakdown per se. One difference between sucrose stimulates the absolute rate of starch synthesis in transgenic phosphorylase and sucrose synthase is that the differences as well as wild-type discs (Fig. 4k, significant at the 5% level in their kinetic properties mean that sucrose phosphorylase only for the wild type, in SP-29 significant at the 10% level). will allow it to drive sucrose concentrations much lower The relative stimulation of starch synthesis was similar in than sucrose synthase. Sucrose phosphorylase has a much wild-type and transgenic lines. The ratio between starch higher affinity for sucrose (Km(sucrose) approximates 1 mM, synthesis and glycolysis increased significantly from 3·2 to see Silverstein et al. 1967) than sucrose synthase (Km(sucrose) 4·8 when palatinose was supplied to wild-type discs, from 40–200 mM, see Avigad 1982), which is also subject to inhi- 2·8 to 3·7 when palatinose was supplied to SP-11, and sig- bition by fructose and glucose (Doehlert 1987; Dancer et nificantly from 3·8 to 5·9 when palatinose was supplied to al. 1990). Thermodynamic factors will also contribute to the SP-29 (Fig. 4n). These results show that the capacity for the depletion of sucrose, as the equilibrium constant of the transgenics to respond to the sucrose analogue palatinose reaction is further displaced towards sucrose degradation is not impaired. As in previous experiments, the absolute for sucrose phosphorylase than sucrose synthase, and the rates of metabolic fluxes are higher in the transformants Pi concentration in the cytosol of tubers is about 100-fold than in wild-type discs. higher than the UDP concentration (Loef, Stitt & Geigen- Labelling of starch degradation products was also analy- berger 1999; and data not shown). Another major differ- sed in the experiment of Fig. 4. In discs incubated with 14C- ence is that sucrose phosphorylase leads directly to glucose and mannitol, label in maltose (Fig. 4h) was not formation of Glc-1-P, and does not require the reaction detectable in the wild type but represented 5 and 10% of catalysed by UGPase, which will remove the requirement the total 14C absorbed in SP-11 and SP-29, respectively. for PPi and uridine nucleotides and increase the energy Label in soluble glucans (Fig. 4i) was not detectable in wild requirement for sucrose mobilization. Expression of type and SP-11, but represented approximately 2% of the sucrose phosphorylase indeed resulted in a dramatic total label in SP-29. Similar results were obtained when decrease of the steady-state sucrose content (Table 1), and palatinose was included in the incubation medium, except in increased ATP consumption (Table 5) and a decreased in the case of soluble glucans where palatinose feeding led ATP/ADP ratio and adenylate energy charge (Fig. 1). to a decrease in the labelling of soluble glucans in SP-29. These results imply that the metabolite levels maintained The results indicate that expression of sucrose phosphory- in growing tubers as a consequence of sucrose degradation lase leads to a stimulation of starch degradation. via sucrose synthase are important for normal metabolic function. These include the maintenance of a relatively large sucrose pool which has functions in signalling and/or DISCUSSION in cellular osmotic regulation, and the minimization of ATP Introduction of sucrose phosphorylase leads to consumption. depletion of sucrose and increased energy consumption by only a small increase of sucrose breakdown Introduction of sucrose phosphorylase had a complex effect on starch metabolism, leading Introduction of sucrose phosphorylase has widespread con- to contradicting changes in intact tubers and sequences for many aspects of tuber metabolism, including tuber discs depletion of sucrose by only a moderate increase in the rate of sucrose breakdown, higher rates of glycolysis and respi- Sucrose phosphorylase had a complex effect on starch syn- ration and an activation of SPS and consequent increase in thesis. On the one hand, the higher rates of sucrose break- the rate of sucrose re-synthesis. These changes are coupled down and elevated levels of glycolytic intermediates should to reduction in the starch content and the activities of starch allow allosteric activation of AGPase and stimulate starch synthesizing enzymes (Table 1) and larger pools of amino synthesis. This was indeed observed in short-term labelling acids (Table 6). These marked changes in flux and tuber experiments with tuber discs. Nevertheless, allocation to

© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1219–1232 1230 A. R. Fernie et al. starch was decreased relative to other major fluxes (Table 3), the specific activities of the hexose phosphate pool that the and the starch content of intact tubers was reduced in com- transgenics have increased rates of isotopic dilution. It is parison with wild type (Table 1). Clearly, the introduction not immediately clear why starch degradation should of sucrose phosphorylase leads to a series of changes in be increased, although an attractive explanation would be metabolism that interact to counteract the effects of high that it is connected with the low level of sucrose in the levels of hexose-phosphates and 3PGA, resulting in reduced transformants. rather than increased levels of starch in intact tubers. The results of the present paper therefore indicate that First, consumption of ATP in the sucrose substrate cycle a combination of factors decrease allocation to starch syn- leads to a decrease of the cellular energy status (Fig. 1), thesis in intact tubers that overexpress sucrose phosphory- which may restrict starch synthesis. There is mounting evi- lase. These include a shift in the relative activities of enzymes dence that the rate of starch synthesis is restricted by the in the starch synthesis pathway and glycolysis, a greatly ATP supply, even in wild-type tubers. Studies with trans- accelerated sucrose cycling leading to increased consump- genic potato tubers with decreased and increased expres- tion of ATP, and depletion of sucrose to low levels which sion of the plastidic adenylate transporter indicate that may lead to an inhibition of starch synthesis by a novel starch synthesis is limited by the availability of ATP in the mechanism that has not yet been characterized. However, plastid (Tjaden et al. 1998; Geigenberger et al. 2001), and the absolute flux to starch in short-term feeding experiments incubation of wild-type tuber discs with adenine to increase with tuber discs was nevertheless still higher in the trans- ATP levels led to a stimulation of starch synthesis from formants than in wild-type tissue, indicating that further sucrose (Loef et al. 2001). factors will also be required to explain the reduced starch Second, sucrose phosphorylase leads to a dramatic content of the transgenic tubers. One possible explanation decrease of the cellular sucrose concentration. It has been for this could be that growing potato tubers are essentially shown elsewhere that sucrose stimulates starch synthesis hypoxic (Geigenberger et al. 2000), whereas oxygen supply via a mechanism that operates independently of changes in is not limiting in discs. 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