Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis (original) (raw)
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Plant, Cell and Environment, 1999
The aim of this work was to define the metabolic factors which regulate the respiratory pathways in trangenic potato tubers. We previously found that respiration is enhanced in transgenic tubers which express a yeast invertase and a glucokinase from Zymomonas mobilis. In this study we investigated glycolysis in three further transgenic potato lines with profound changes in the mobilization of sucrose. We studied antisense ADPglucose pyrophosphorylase lines which are characterized by a reduction in starch accumulation and a significant build up of sucrose and related metabolic intermediates. We also report the generation of two novel double transgenic lines where the yeast invertase is expressed specifically in tubers of the ADPglucose pyrophosphorylase antisense line, targeted to either the cytosol or apopolast. We evaluated whether the localization of sucrose cleavage had an impact on the glycolytic induction, and assessed if invertase expression in the high-sucrose background had any further effects on glycolysis. We found that induction of the glycolytic enzymes only occurs when the invertase is targeted to the cytosol, and that the extent of this induction was comparable in the wild type and antisenseADPglucose pyrophosphorylase backgrounds. We conclude that the signal regulating glycolysis is directly linked to cytosolic sucrose hydrolysis.
Planta, 1999
Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-speci®c patatin promoter either alone (EC 3.2.1.26; or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [ 14 C]sucrose, [ 14 C]glucose or [ 14 C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [ 14 C]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. Labelling kinetics did not reveal whether this was due to changes in the¯uxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a signi®cant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enhanced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineer-ing without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation.
Plant Molecular Biology, 2004
The constitutive cytosolic expression of a yeast (Saccharomyces cerevisiae) invertase within potato (Solanum tuberosum) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis. These transgenic lines were further characterised by a massive cycle of sucrose degradation and resynthesis via sucrose-phosphate synthase. We have recently developed a B33 patatin driven alc gene construct allowing tight chemical control of gene expression following supply of acetaldehyde with minimal pleiotropic effects of the inducing agent on metabolism. This construct was used for chemical induction of the yeast invertase gene after 10-weeks growth to dissect the complex metabolic phenotype obtained after constitute expression. Inducible expression led to increased invertase activity within 24 h in well-defined areas within growing tubers. Although the sucrose levels were reduced, there was no effect on the levels of starch whilst levels of many amino acids decreased. Labelling experiments revealed that these lines exhibited increased rates of sucrose cycling, whereas rates of glycolysis and of starch synthesis were not substantially changed. From these results we conclude that sucrose cycling is stimulated in response to a short-term increase in the rate of sucrose mobilisation, providing evidence for a role of sucrose cycling as a buffering capacity that regulates the net rate of sucrose usage. In contrast, the dramatic increase in hexosephosphate levels and the switch from starch synthesis to respiration seen on the constitutive expression of the invertase was not observed in the inducible lines, suggesting that this is the result of cumulative pleiotropic effects that occurred when the transgene was expressed throughout development.
Plant, Cell and Environment, 2004
Starch is of great importance both as a carbon storage reserve in plants and as a biotechnologically important product. The potato tuber is an attractive model system for the study of starch metabolism, because it is a relatively homogenous tissue in which conversion of sucrose to starch represents the dominant metabolic flux. All the major genes of the potato tuber sucrose to starch pathway have been cloned in recent years, allowing the generation of a suite of antisense transgenic lines to be produced in which the activity of each individual enzyme in the pathway is progressively decreased. Investigations of these plants have provided a complete picture of the distribution of control in this important pathway. Sucrose synthase, UGPase, hexokinase, cytosolic phosphoglucomutase, plastidial phosphoglucomutase, the amyloplastidial adenylate translocator, AGPase, starch synthase and starch branching enzyme have flux control coefficients (FCCs) of 0.10, approximating 0.00, approximating 0.00, 0.15, 0.23, 0.98, 0.35, 0.12 and approximating 0.00 for starch accumulation. These results show that the majority of the control on starch accumulation in potato tubers resides in the transfer of adenylate between the cytosol and the amyloplast, with a minor contribution being made by the first two steps of the plastidial starch synthesis pathway (the reactions catalysed by plastidial phosphoglucomutase and AGPase). This contrasts with leaves, in which the majority of the control has been found to reside in the reactions catalysed by plastidial phosphoglucomutase and AGPase. In leaves, ATP for starch synthesis is generated within the plastid via photophosphorylation. Several studies have attempted to increase the rate of starch synthesis by overexpressing pathway enzymes in tubers. The results of these studies and the role of other ATP producers in the starch synthetic process are reviewed. In the same time period methods of nonaqueous fractionation have been adapted to potato tuber tissue in order to ascertain subcellular metabolite levels. Results obtained from these studies allow the calculation of mass action ratios of the constitutive enzymes of the sucrose to starch transition. When taken together with the known regulatory properties of these enzymes the combination of broad control analysis studies and assessment of the mass action ratios of the respective enzymes allows a comprehensive description of this important metabolic network. Some illustrative examples of how this network responds to environmental change are presented. Finally implications of this whole pathway evaluation for more general studies of plant metabolic pathways and networks are discussed.
PLANT PHYSIOLOGY, 2003
Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1--glucuronidase reporter gene construct was more strongly induced in the invertaseoverexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of 14 C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible. .
Plant, Cell and Environment, 2001
Sugars are not only metabolic substrates: they also act as signals that regulate the metabolism of plants. Previously, we found that glycolysis is induced in transgenic tubers expressing a yeast invertase in the cytosol but not in those expressing invertase in the apoplast. This suggests that either the low level of sucrose, the increased formation of cytosolic glucose or the increased levels of metabolites downstream of the sucrose cleavage is responsible for the induction of glycolysis in storage organs. In order to discriminate between these possibilities, we cloned and expressed a bacterial sucrose phosphorylase gene from Pseudomonas saccharophila in potato tubers. Due to the phosphorolytic cleavage of sucrose, formation of glucose was circumvented, thus allowing assessment of the importance of cytosolic glucose -and, by implication, flux through hexokinase -in glycolytic induction. Expression of sucrose phosphorylase led to: (i) a decrease in sucrose content, but no decrease in glucose or fructose; (ii) a decrease in both starch accumulation and tuber yield; (iii) increased levels of glycolytic metabolites; (iv) an induction of the activities of key enzymes of glycolysis; and (v) increased respiratory activity. We conclude that the induction of glycolysis in heterotrophic tissues such as potato tubers occurs via a glucose-independent mechanism.
Journal of Experimental Botany, 2003
To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desire  e) plants under control of the rolC promoter. Vascular tissue speci®c expression of the transgene was veri®ed 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 invertaseexpressing 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-speci®c 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.
Czech Journal of Genetics and Plant Breeding
Cold-stored potato tubers gradually accumulate reducing sugars. A proposed reason is a cold-induced blocking of glycolysis. The introduction and expression of the bacterial gene Lbpfk coding for cold-tolerant phosphofructokinase might counteract this effect. We have recently introduced this gene into several Czech potato cultivars. The obtained transgenic lines were then tested for three years in field trials. In 17 transgenic lines derived from two of the cultivars we have investigated the accumulation of reducing sugars during two and four months of cold storage. Although in all transgenic lines the sugar content still increased between the 2 nd and the 4 th month of cold-storage, the level of reducing sugars was in all transgenic lines after both two and four months of cold storage considerably lower than in the original cultivars. The extent of sugar accumulation was also influenced by the parental genotype. No significant differences in sugar accumulation were observed between the transgenic lines from the same parent.