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     

Cell-type specific,coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12–15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mRNA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.Abbreviations AGPase ADP-glucose pyrophosphorylase - DAF days after fertilization - Glc1P glucose-1-phosphate - 3-PGA 3-phosphoglycerate - VfAGPC AGPase subunit of Vicia faba mainly expressed in cotyledons - VfAGPP AGPase subunit of Vicia faba mainly expressed in leaves and cotyledons - pVfAGPC, pVfAGPP plasmids containing VfAGPC and VfAGPP, respectively This work was supported by the Bundesministerium für Forschung und Technologie BCT 0389, Molekular- und Zellbiologie von höheren Pflanzen und Pilzen. U.W acknowledges additional support by the Fonds der chemischen Industrie. We thank Elsa Fessel for excellent technical assistance.     

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.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Evidence that SNF1-related kinase and hexokinase are involved in separate sugar-signalling pathways modulating post-translational redox activation of ADP-glucose pyrophosphorylase in potato tubers

We recently discovered that post-translational redox modulation of ADP-glucose pyrophosphorylase (AGPase) is a powerful new mechanism to adjust the rate of starch synthesis to the availability of sucrose in growing potato tubers. A strong correlation was observed between the endogenous levels of sucrose and the redox-activation state of AGPase. To identify candidate components linking AGPase redox modulation to sugar supply, we used potato tuber discs as a model system. When the discs were cut from growing wild-type potato tubers and incubated for 2 h in the absence of sugars, redox activation of AGPase decreased because of a decrease in internal sugar levels. The decrease in AGPase redox activation could be prevented when glucose or sucrose was supplied to the discs. Both sucrose uptake and redox activation of AGPase were increased when EDTA was used to prepare the tuber discs. However, EDTA treatment of discs had no effect on glucose uptake. Feeding of different glucose analogues revealed that the phosphorylation of hexoses by hexokinase is an essential component in the glucose-dependent redox activation of AGPase. In contrast to this, feeding of the non-metabolisable sucrose analogue, palatinose, leads to a similar activation as with sucrose, indicating that metabolism of sucrose is not necessary in the sucrose-dependent AGPase activation. The influence of sucrose and glucose on redox activation of AGPase was also investigated in discs cut from tubers of antisense plants with reduced SNF1-related protein kinase activity (SnRK1). Feeding of sucrose to tuber discs prevented AGPase redox inactivation in the wild type but not in SnRK1 antisense lines. However, feeding of glucose leads to a similar activation of AGPase in the wild type and in SnRK1 transformants. AGPase redox activation was also increased in transgenic tubers with ectopic overexpression of invertase, containing high levels of glucose and low sucrose levels. Expression of a bacterial glucokinase in the invertase-expressing background led to a decrease in AGPase activation state and tuber starch content. These results show that both sucrose and glucose lead to post-translational redox activation of AGPase, and that they do this by two different pathways involving SnRK1 and an endogenous hexokinase, respectively.     

Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518) proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [¹⁴C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress. Received: 24 February 1999 / Accepted: 28 May 1999     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Leaf-Specific Antisense Inhibition of Starch Biosynthesis in Transgenic Potato Plants Leads to an Increase in Photoassimilate Export from Source Leaves during the Light Period

In an attempt to study the importance of starch synthesis inleaves with respect to sink-source interactions, we investigateddaily turnover of carbohydrates in leaves of transgenic potatoplants inhibited for ADP-glucose pyrophosphorylase (AGPase).Down-regulation of AGPase has been performed using two differentpromoters: the near-constitutive CaMV 35S promoter, and theSTLSI promoter which is active in photosynthetic cells only.Residual AGPase activity in leaves was between 6 and 30% inindividual transformants as compared to wild-type potato plants.We found that: (i) photosynthesis is not significantly alteredrelative to wild-type plants; (ii) levels of starch are markedlyreduced in leaves of transgenic plants; (iii) levels of solublesugars and malate are largely unaffected by the inhibition ofAGPase; (iv) the reduction of starch synthesis leads to a higherportion of assimilated carbon being transported from leavesto sink tissues during the light period; (v) altered leaf exportcharacteristics do not change tuber yield under greenhouse conditions.Collectively, these data demonstrate a striking flexibilityof the potato plant with respect to day/night rhythms of carbonexport from leaves and utilization by the major storage sinks,i.e. developing tubers. (Received November 1, 1994; Accepted March 2, 1995)     

Nucleotide sequence and regulated expression of a wound-inducible potato gene (wun1)

Summary Mechanical wounding of potato leaves, stems, roots and tubers leads to a rapid increase of wun1 mRNA. In potato leaves, the wound-induced accumulation of wun1 mRNA is inhibited by the addition of sucrose or other osmotically active agents. This inhibition is organ specific since sucrose does not prevent wun1 mRNA accumulation in wounded tubers. In contrast, expression of patatin was shown to be repressed in tubers by wounding and this repression was reversed by increasing osmotic pressure. Sequence data obtained from the analysis of a wun1 cDNA and a wun1 genomic clone show no homology to any gene known so far. Histochemical data demonstrate a striking analogy in cell specific expression of chimeric genes expressed under the control of the wun1 promoter and the cell specific production of callose in wounded tobacco leaves.     

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)⁶⁰, and Asn⁹⁰, but the third glycosylation site (Asn²⁰²) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn asparagine - ConA concanavalin A - EndoH endoglycosidase H - Fuc fucose - GlcNAc N-acetylglucosamine - HPLC high-performance liquid chromatography - Man mannose - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulfate - Ser serine - TFMS trifluoromethanesulfonic acid - Thr threonine - Xyl xylose     

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.     

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.     

Increased fatty acid production in potato by engineering of acetyl-CoA carboxylase

In contrast to oil seeds, potato (Solanum tuberosum L.) is characterized by a high amount of starch stored in the tubers. To assess the capacity for oil synthesis in potato tubers, the changes in lipid content and flux into lipid synthesis were explored in transgenic potatoes altered in carbohydrate or lipid metabolism. A strong decrease in the amount of starch observed in antisense lines for ADP-glucose pyrophosphorylase or plastidic phosphoglucomutase had no effect on storage-lipid content. Similarly, potato lines over-expressing the Arabidopsis thaliana (L.) Heynh. plastidic ATP/ADP transporter that contained an increased amount of starch were not altered in oil content, indicating that the plastidic ATP level is not limiting fatty acid synthesis in potato tubers. However, over-expression of the acetyl-CoA carboxylase from Arabidopsis in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of triacylglycerol. Taken together, these data demonstrate that potato tubers have the capacity for storage-lipid synthesis and that malonyl-CoA, the substrate for elongation during fatty acid synthesis, represents one of the limiting factors for oil accumulation.Abbreviations AATP Plastidic ADP/ATP transporter - ACCase Acetyl-CoA:carboxylase - DGAT Acyl-CoA:diacylglycerol acyltransferase - FW Fresh weight - TLC Thin-layer chromatography - WT Wild typeSource for transgenic plant material. Upon request, transgenic potato lines altered in ACCase activity can be obtained from Peter Dörmann. For potato lines with alterations in AATP transporter activity, please refer to H. Ekkehard Neuhaus. Transgenic AGP and PGM lines are available from A. Fernie (Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).     

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K _m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed. Received: 2 February 1999 / Accepted: 25 March 1999