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.     

Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis

The original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose. We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase-expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild-type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two- to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three- to fivefold increases in carbon dioxide production. These changes occur in the lines expressing invertase, and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.     

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 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     

Overriding the co-limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.     

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.     

The development of a high-yield recombinant protein bioreactor through RNAi induced knockdown of ATP/ADP transporter in Solanum tuberosum

There is an increased need for high-yield protein production platforms to meet growing demand. Tuber-based production in Solanum tuberosum offers several advantages, including high biomass yield, although protein concentration is typically low. In this work, we investigated the question whether minor interruption of starch biosynthesis can have a positive effect on tuber protein content and/or tuber biomass, as previous work suggested that partial obstruction of starch synthesis had variable effects on tuber yield. To this end, we used a RNAi approach to knock down ATP/ADP transporter and obtained a large number of transgenic lines for screening of lines with improved tuber protein content and/or tuber biomass. The initial screening was based on tuber biomass because of its relative simplicity. We identified a line, riAATP1-10, with minor (less than 15%) reduction in starch, that had a nearly 30% increase in biomass compared to wild-type, producing both more and larger tubers with altered morphological features compared to wild-type. riAATP1-10 tubers have a higher concentration of soluble protein compared to wild-type tubers, with nearly 50% more soluble protein. We assessed the suitability of this line as a new bioreactor by expressing a human scFv, reaching over 0.5% of total soluble protein, a 2-fold increase over the highest accumulating line in a wild-type background. Together with increased biomass and increased levels in total protein content, foreign protein expression in riAATP1-10 line would translate into a nearly 4-fold increase in recombinant protein yield per plant. Our results indicate that riAATP1-10 line provides an improved expression system for production of foreign proteins.     

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.     

The effect of exogenous sugars on the control of flux by adenosine 5′-diphosphoglucose pyrophosphorylase in potato tuber discs

The aim of this work was to investigate the effect of exogenous sugars on the extent to which starch synthesis in potato ( Solanum tuberosum L.) is controlled by adenosine 5'-diphosphoglucose pyrophosphorylase (EC 2.7.7.27; AGPase). Tuber discs were incubated in the presence of a range of concentrations of glucose and sucrose, and metabolic fluxes measured following the supply of [U-14C]glucose and measurement of the specific radioactivity of the hexose phosphate pool. In the presence of glucose there was a marked increase in the flux through glucose-phosphorylating hexokinase, and at high concentrations of external glucose this led to a stimulation of the rate of starch and sucrose synthesis relative to those measured in the presence of sucrose. In the presence of glucose the ratio of the rate of starch synthesis to the rate of glycolysis was higher than in the presence of sucrose. Similar effects of glucose were observed at two stages of tuber development. We conclude that the presence of glucose perturbs the carbohydrate metabolism of tuber discs so that starch synthesis is favoured. In order to determine the extent to which AGPase controls flux, we measured fluxes in wild-type plants and transgenic plants with reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. In the presence of sucrose a reduction in AGPase activity had a greater impact on the rate of starch synthesis than in the presence of glucose. The flux control coefficient of AGPase over starch synthesis was higher in the presence of sucrose (0.7-0.9) than in the presence of glucose (0.4-0.6). Conversely, the impact of reduced AGPase activity on the rate of sucrose synthesis was lower in the presence of sucrose than glucose. In the presence of 200 mM sucrose the flux control coefficient of AGPase over the rate of sucrose synthesis was not significantly different from zero. This demonstrates that the nature of the sugar supplied to potato tuber discs can have a major influence on the distribution of control within metabolism. These data were also used to investigate the relationship between demand for ATP and the rate of hexose phosphate entry into glycolysis. A very strong correlation between ATP demand and glycolytic flux was demonstrated.     

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1

Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.     

Expression of a bacterial sucrose phosphorylase in potato tubers results in a glucose-independent induction of glycolysis

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.     

Effect of gibberellic acid on growth and carbohydrate metabolism of developing tubers of potato (Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Ostara) were grown in aerated water culture in a controlled environment. When the tubers had reached a diameter of 1–3 cm. ¹⁴C-labelled or unlabelled gibberellic acid (GA₃) was applied to the surface of the stolons at points approximately 1 crn from the developing tubers, and treatment continued for 10 days. - Significant quantities of GA₃ moved into tuber tissue within 2–4 days of hormone application. This influx of GA₃ was accompanied by a marked reduction in both the activity of ADPG-pyrophospharylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase and an increase in the activity of UDPG-pyrophosphorylase. Starch phosphorylase activity initially increased slightly but then fell, whereas the activity of starch synthase remained constant throughout the experiment. The soluble sugar composition of the tubers changed qualitatively towards a pattern characteristic of growing stolon tips prior to tuber initiation, but there was no clear evidence of net starch degradation. Changes in the activities of the enzymes were observed prior to noticeable effects of the hormone on tuber growth rate or the development of new stolons at the tuber eyes. - GA₃- treated tubers imported more ¹⁴C from labelled photosynthate than expected on the basis of growth rate. However, the capacity to convert solub#e-¹⁴C to ethaTiol-insoluble-¹⁴C (predominantly starch) was reduced in comparison with non-treated tubers. - The observed changes in carbohydrate composition and enzyme activities indicate that GA₃ induces a drastic change in potato tuber metabolism towards a pattern characteristic for the termination of the storage process.     

The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers

In the present paper we investigated the effect of the sucrose (Suc) analog palatinose on potato (Solanum tuberosum) tuber metabolism. In freshly cut discs of growing potato tubers, addition of 5 mM palatinose altered the metabolism of exogenously supplied [U-14C]Suc. There was slight inhibition of the rate of 14C-Suc uptake, a 1.5-fold increase in the rate at which 14C-Suc was subsequently metabolized, and a shift in the allocation of the metabolized label in favor of starch synthesis. The sum result of these changes was a 2-fold increase in the absolute rate of starch synthesis. The increased rate of starch synthesis was accompanied by a 3-fold increase in inorganic pyrophosphate, a 2-fold increase in UDP, decreased UTP/UDP, ATP/ADP, and ATP/AMP ratios, and decreased adenylate energy charge, whereas glycolytic and Krebs cycle intermediates were unchanged. In addition, feeding palatinose to potato discs also stimulated the metabolism of exogenous 14C-glucose in favor of starch synthesis. In vitro studies revealed that palatinose is not metabolized by Suc synthases or invertases within potato tuber extracts. Enzyme kinetics revealed different effects of palatinose on Suc synthase and invertase activities, implicating palatinose as an allosteric effector leading to an inhibition of Suc synthase and (surprisingly) to an activation of invertase in vitro. However, measurement of tissue palatinose levels revealed that these were too low to have significant effects on Suc degrading activities in vivo. These results suggest that supplying palatinose to potato tubers represents a novel way to increase starch synthesis.     

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.     

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.     

Identification of the major starch synthase in the soluble fraction of potato tubers.

The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. Immunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for approximately 80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.     

Tuber-specific cytosolic expression of a bacterial phosphoglucomutase in potato (Solanum tuberosum L.) dramatically alters carbon partitioning

Constitutive antisense inhibition of the cytosolic isoform of phosphoglucomutase in the potato (Solanum tuberosum L.) results in restriction of photosynthesis, growth inhibition and modified tuber morphology, and a severe restriction of tuber starch synthesis. Here we describe the consequences of the tuber-specific expression of an Escherichia coli phosphoglucomutase in the cytosol. Analysis of [14C]glucose metabolism by tuber discs isolated from wild type and transformants revealed that the rates of sucrose and starch synthesis were unaltered but that the rate of glycolysis was depressed in the transgenics. The transformant tubers also contained dramatically reduced amino acid content and significantly higher levels of ADP, but were characterized by elevated levels of Krebs cycle intermediates and an unaltered rate of respiration. In addition to these metabolic consequences of the overexpression of the E. coli enzyme, we observed morphological changes in tubers, with the transformants having a smaller number of larger tubers which exhibited delayed rates of sprouting with respect to the wild type. These results are discussed with respect to current models of the regulation of central plant metabolism and tuber dormancy.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.