Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.     

Zebra chip disease incidence on potato is influenced by timing of potato psyllid infestation, but not by the host plants on which they were reared

Abstract  The Zebra chip (ZC) syndrome is an emerging disease of potato and a major threat to the potato industry. The potato psyllid, Bactericerca cockerelli (Sulc) is believed to be a vector of the ZC pathogen, which is now thought to be Candidatus Liberibacter, a bacterium. To further understand the relationship between potato psyllid infestation and ZC disease expression, healthy potato plants at different growth stages (4, 6 and 10 weeks after germination) were exposed separately to potato psyllids that were separately reared on four solanaceous hosts plants (potato, tomato, eggplant or bell pepper) for more than 1 year. ZC symptoms, leaf rates and total nonstructural carbohydrate accumulation in leaves and tubers of healthy and psyllid-infested plants were monitored and recorded. Typical ZC symptoms were observed in leaves and tubers of all plants exposed to potato psyllids regardless of the host plant on which they were reared. This was also accompanied by significant reductions in net photosynthetic rate. Caged potato plants without exposure to potato psyllids (uninfested controls) did not show any ZC symptom in both foliage and in harvested tubers. Foliage damage and ZC expression were most severe in the potato plants that were exposed to potato psyllids 4 weeks after germination compared to plants infested at later growth stages. Tubers from potato psyllid-infested plants had significantly higher levels of reducing sugars (glucose) and lower levels of starch than those in healthy plants, indicating that potato psyllid infestation interfered with carbohydrate metabolism in either leaves or tubers, resulting in ZC expression.     

Manipulation of sink-source relations in transgenic plants

Since 1980, the use of transgenic plants in modern plant science has become a powerful tool to study whole plant physiology. In this review, we try to summarize the data obtained in the field of photoassimilate partitioning. Attempts to study sink-source interactions concern factors which might limit sink strength and source capacity. Transgenic plants have been used to manipulate the sucrose to starch ratio in order to produce plants with higher sucrose levels in their source leaves. Alterations in partitioning were achieved by manipulating Calvin cycle enzymes, transport proteins and sucrose biosynthetic enzymes. The ability of sink tissues to attract photoassimilates has been altered by either increasing or decreasing sucrose hydrolytic activities. The increase of sucrose hydrolysis was achieved by creating transgenic potato plants with tuber specific yeast-derived invertase. Decreased sucrose utilization was achieved by antisense inhibition of sucrose synthase in potato tubers.     

Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development

Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield.     

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum)

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.     

Overcompensating plants: their expression of resistance traits and effects on herbivore preference and performance

Overcompensation is a plant tolerance response in which plants have higher fitness after herbivory than without damage. Although it has been demonstrated that plants are able to simultaneously express resistance and tolerance traits, it remains unclear whether overcompensating plants are also inducing resistance‐mediating secondary metabolite production and how herbivores perform on plants that overcompensate. Our previous work has shown that a potato variety [Solanum tuberosum L. cv. Pastusa Suprema (Solanaceae)] from Colombia can express overcompensatory responses to damage by larvae of the Guatemalan potato moth, Tecia solanivora Povolny (Lepidoptera: Gelechiidae). Here we investigated (1) whether potatoes that express overcompensatory responses also induce resistance traits and (2) how the previous damage affects Guatemalan potato moth preference and performance. Our results show that larval feeding not only systemically induces higher tuber biomass but also an increased production of resistance‐related compounds, such as phenolics and proteinase inhibitors. Pupal mass increased with increasing tuber size, whereas changes in tuber secondary metabolism did not correlate with any metric of larval performance. Oviposition preference did not change between induced and undamaged plants. Our data show that potato plants expressing overcompensatory responses also induce secondary compounds known to increase resistance against herbivores. However, the induced response was relatively small, reducing the opportunities for a negative effect on the herbivore. Hypotheses for why larvae perform better in larger tubers and are not affected by the secondary metabolism are discussed. From an ecological and agricultural point of view, our results suggest that the expression of overcompensatory traits could have positive effects on herbivore performance.     

Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

Decreased expression of sucrose phosphate synthase strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.     

The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber

The aim of this work was to evaluate the extent to which plastidial phosphoglucomutase (PGM) activity controls starch synthesis within potato (Solanum tuberosum L. cv. Desirée) tubers. The reduction in the activity of plastidial PGM led to both a correlative reduction in starch accumulation and an increased sucrose accumulation. The control coefficient of plastidial PGM on the accumulation of starch was estimated to approximate 0.24. The fluxes of carbohydrate metabolism were measured by investigating the metabolism of [U-14C]glucose in tuber discs from wild-type and transgenic plants. In tuber discs the control coefficient of plastidial PGM over starch synthesis was estimated as 0.36, indicating that this enzyme exerts considerable control over starch synthesis within the potato tuber.     

Stable isotope distribution in the major metabolites of source and sink organs of Solanum tuberosum L.: a powerful tool in the study of metabolic partitioning in intact plants

A method was developed for the purification of main intermediates and storage products of leaves and tubers of potato for analysis of their ¹³C content. The method was tested for recovery of metabolites and carbon isotope discrimination during the purification process. Leaf metabolite δ¹³C values showed an enrichment of starch relative to sucrose and citrate. This result is in agreement with previous findings in other higher plants and indicates the existence of isotope discrimination steps during transport and metabolism of triose-phosphates in potato leaf mesophyll cells. Active anaplerotic replenishment of the tricarboxylic acid cycle in the leaves of the plants investigated was also deduced from the significant ¹³C enrichment of malate relative to citrate and asparagine/aspartate relative to glutamine/glutamate. Analysis of tuber metabolite δ¹³C values showed no difference between starch and sucrose. However, tuber sucrose appeared significantly enriched compared with leaf sucrose and also relative to tuber citrate and malate. This finding suggests the existence of sites of isotopic discrimination during sucrose processing in developing tubers. It also confirms that metabolic cycles of sucrose synthesis and breakdown and of hexose-phosphate/triose-phosphate interconversion, which have been described in excised tuber tissue, also occur in intact organs. The δ¹³C values were also used to estimate the metabolic rate of carbon oxidation in developing tubers on the assumption that pyruvate dehydrogenase is the main site of isotopic discrimination in the tuber cells. The result obtained was in agreement with the available literature, suggesting that analyses of natural isotopic distribution in plant products may be a useful tool for the study of metabolic processes and sink-source relationships in intact plants. Received: 21 May 1998 / Accepted: 10 July 1998     

Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants

An important goal in biotechnological research is to improve the yield of crop plants. Here, we genetically modified simultaneously source and sink capacities in potato (Solanum tuberosum cv. Desirée) plants to improve starch yield. Source capacity was increased by mesophyll‐specific overexpression of a pyrophosphatase or, alternatively, by antisense expression of the ADP‐glucose pyrophosphorylase in leaves. Both approaches make use of re‐routing photoassimilates to sink organs at the expense of leaf starch accumulation. Simultaneous increase in sink capacity was accomplished by overexpression of two plastidic metabolite translocators, that is, a glucose 6‐phosphate/phosphate translocator and an adenylate translocator in tubers. Employing such a ‘pull’ approach, we have previously shown that potato starch content and yield can be increased when sink strength is elevated. In the current biotechnological approach, we successfully enhanced source and sink capacities by a combination of ‘pull’ and ‘push’ approaches using two different attempts. A doubling in tuber starch yield was achieved. This successful approach might be transferable to other crop plants in the future.     

The contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-¹⁴C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato. Received: 24 February 1999 / Accepted: 8 April 1999     

Effects of Russian wheat aphid infestation on barley plant response to drought stress

The influence of Russian wheat aphid ( Diuraphis noxia Mordvilko) infestation on the response of barley ( Hordeum vulgare L. ev Hazen) plants to drought stress was investigated. Fourteen-day-old plants were infested with eight apterous adult aphids, which were removed 7 days later with systemic insecticide. Leaves previously infested with aphids had lower relative water content, reduced stomatal conductance, more negative water potential, lower levels of chlorophyll and higher levels of amino-N, proline and glycinebetaine than corresponding leaves from uninfested plants. When water was withheld for a period of 7 days after aphids were removed, the relative water content of previously infested plants dropped steadily from 0.89 to 0.60, while the relative water content of uninfested plants remained at about 0.94 for the first 4 days of the drought stress period followed by a steady drop to about 0.77 by the end of the drought stress period. Leaf water potentials dropped steadily during the drought stress period in both previously infested (-1.14 to -1.91 MPa) and unin-fested (-0.54 to -1.52 MPa) plants. Analysis of glycinebetaine and proline levels at the end of the drought stress period indicated that leaves of previously infested plants accumulated lower levels of these solutes than leaves from uninfested plants. Upon alleviation of drought stress, plants previously infested with aphids showed little increase in dry weight while younger leaves and tillers from uninfested plants showed large increases. It is concluded that Russian wheat aphids cause drought-stress symptoms in leaves of infested plants even in the presence of ample root moisture. The observations of low levels of glycinebetaine and proline present in leaves after water was withheld from roots and lack of leaf growth upon alleviation of drought stress in previously-infested plants, suggest that aphid infestation limits the capacity of barley plants to adjust successfully to drought stress.     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.     

Symplastic connection is required for bud outgrowth following dormancy in potato (Solanum tuberosum L.) tubers

To gain greater insight into the mechanism of dormancy release in the potato tuber, an investigation into physiological and biochemical changes in tuber and bud tissues during the transition from bud dormancy (immediately after harvest) to active bud growth was undertaken. Within the tuber, a rapid shift from storage metabolism (starch synthesis) to reserve mobilization within days of detachment from the mother plant suggested transition from sink to source. Over the same period, a shift in the pattern of [U-(14)C]sucrose uptake by tuber discs from diffuse to punctate accumulation was consistent with a transition from phloem unloading to phloem loading within the tuber parenchyma. There were no gross differences in metabolic capacity between resting and actively growing tuber buds as determined by [U-(14)C]glucose labelling. However, marked differences in metabolite pools were observed with large increases in starch and sucrose, and the accumulation of several organic acids in growing buds. Carboxyfluorescein labelling of tubers clearly demonstrated strong symplastic connection in actively growing buds and symplastic isolation in resting buds. It is proposed that potato tubers rapidly undergo metabolic transitions consistent with bud outgrowth; however, growth is initially prevented by substrate limitation mediated via symplastic isolation.     

Effect of sink isolation on sugar uptake and starch synthesis by potato-tuber storage parenchyma

Import into potato (Solarium tuberosum L. cv. Record) tubers was terminated by removing the sink at its connection with the stolon. The ability of discs of storage tissue from the excised tubers to take up exogenous sugars and convert them to starch was compared with that of discs from untreated tubers from the same plant population. In rapidly-growing control tubers, glucose and fructose were taken up to a greater extent than sucrose, 77% of the glucose being converted to starch within 3 h (compared with 64% and 27% for fructose and sucrose, respectively). These values fell as the tubers aged but the ranking (glucose > fructose > sucrose) was maintained, emphasising a severe rate-limiting step following the import of sucrose into the growing tuber. Sink isolation had little effect on the ability of the storage cells to take up exogenous sucrose across the plasmalemma for up to 7 d after sink isolation. However, the ability of the same cells to convert the sucrose to starch was severely inhibited within 24 h, as was the sensitivity of starch synthesis to turgor. In the case of glucose, sink isolation inhibited both the uptake and the conversion to starch, the latter being inhibited to a greater degree. A detailed metabolic study of tubers 7 d after excision showed that, with sucrose as substrate, 94% of the radioactivity in the soluble sugar pool was recovered in sucrose following sink isolation (92% in control tubers). However, with glucose as substrate, 80% of the radioactivity was recovered as sucrose following tuber excision (28% in control tubers), providing evidence that sucrose synthesis acts as a major alternative carbon sink when starch synthesis is inhibited. In the same tubers, sucrose-synthase activity decreased by 70% following sink isolation, compared with a 45% reduction in ADP-glucose pyrophosphorylase. Activities of UDP-glucose pyrophosphorylase, starch phosphorylase, starch synthase nd both PPi- and ATP-dependent phosphofructokinases remained unchanged. Acid-invertase activity increased fivefold.