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.     

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.     

Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase

We have previously described the generation of transgenic potato ( Solanum tuberosum L. cv. Desiree) lines expressing the S. tuberosum plastidial phosphoglucomutase ( StpPGM) gene in the antisense orientation under the control of the 35S promoter and characterised heterotrophic metabolism in these lines [E. Tauberger et al. (2000) Plant J 23:43-53]. The aim of the current work was to examine the role of plastidial phosphoglucomutase (pPGM, EC 5.4.2.2) in photosynthetic carbon partitioning. Here we characterise the metabolism of leaves of the same lines and show that reducing the activity of this enzyme has profound effects on carbon partitioning, characterised by a strong (up to 50%) reduction in the rate of starch accumulation accompanied by a minor reduction in the rate of sucrose accumulation. Gas-exchange and (14)CO(2)-feeding experiments revealed that the transgenic lines exhibited a decreased rate of photosynthesis and a corresponding reduced assimilation of radiolabel into starch, even in lines exhibiting only a minor decrease in pPGM activity. In illuminated leaves, decreasing the amount of pPGM resulted in decreased amounts of triose-phosphates, hexose-phosphates and inorganic phosphate without changes in the level of 3-phosphoglycerate. Most importantly, the deduced ratio of phosphoesters to inorganic phosphate increased, indicating the likelihood that photosynthesis was phosphate-limited in these lines. Determination of a more complete metabolic profile of leaf material from these lines revealed a large number of changes in the levels of amino and organic acids, consistent with an inhibition of triose-phosphate export from the chloroplast, but little change in the energy status of the transformants. We discuss the implications of these changes with respect to both consequences of inhibiting starch synthesis and of inhibiting photosynthesis, and conclude that a high activity of pPGM is required both to prevent phosphate limitation of photosynthesis and for co-ordination of plastidially and cytosolically compartmented photosynthetic metabolism.     

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.     

Altered plastidic ATP/ADP-transporter activity influences potato ( Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch

The metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild-type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5–18.0% amylose, whereas starch from sense plants had levels of 22.7–27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.     

Alteration of photosynthate partitioning by high-level expression of phosphoglucomutase in tobacco chloroplasts

Plastidial phosphoglucomutase (PGM) plays an important role in starch synthesis and degradation. Nonetheless, the impact of enhanced plastidial PGM activity on metabolism in photosynthetic tissue is yet to be elucidated. In this study, we generated transplastomic tobacco plants overproducing Arabidopsis thaliana plastidial PGM (AtptPGM) in chloroplasts and analyzed the consequent metabolic and physiological parameters in the transplastomic plants. AtptPGM accumulated in the chloroplasts to up to 16% of total soluble protein in the leaves. PGM activity in leaves increased 100-fold relative to that of wild-type plants. The transplastomic plants were phenotypically indistinguishable in their growth rates, photosynthetic activities, and starch synthesis from wild-type plants, but hexose partitioning in the light period was dramatically different. Furthermore, alteration of extracellular invertase activity was observed in the lower leaves of the transplastomic plants. These observations suggest that high-level expression of plastidial PGM alters hexose partitioning in light periods via modification of extracellular invertase activity.     

A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.     

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.     

Expression of an <Emphasis Type="Italic">Escherichia coli</Emphasis> phosphoglucomutase in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting

The aim of this work was to evaluate the influence of elevating the cytosolic activity of phosphoglucomutase (PGM; EC 5.4.2.2) on photosynthesis, growth and heterotrophic metabolism. Here we describe the generation of novel transgenic plants expressing an Escherichia coli phosphoglucomutase (EcPGM) under the control of the 35S promoter. These lines were characterised by an accumulation of leaf sucrose, despite displaying no alterations in photosynthetic carbon partitioning, and a reduced tuber starch content. Determinations of the levels of a wide range of other metabolites revealed dramatic reductions in maltose and other sugars in leaves of the transformants, as well as a modification of the pattern of organic and amino acid content in tubers of these lines. Intriguingly, the transgenics also displayed a dramatically delayed rate of sprouting and significantly enhanced rate of respiration, however, it is important to note that the severity of these traits did not always correlate with the level of transgene expression. These results are discussed in the context of current understanding of the control of respiration and the breaking of tuber dormancy.     

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspectsof cell function, but their role in the regulation of biosyntheticprocesses is still unclear. It was previously reported thatdecreased expression of plastidial adenylate kinase, catalysingthe interconversion of ATP and AMP to ADP, leads to increasedadenylate pools and starch content in transgenic potato tubers.However, the underlying mechanisms were not elucidated. Here,it is shown that decreased expression of plastidial adenylatekinase in growing tubers leads to increased rates of respiratoryoxygen consumption and increased carbon fluxes into starch.Increased rates of starch synthesis were accompanied by post-translationalredox-activation of ADP-glucose pyrophosphorylase (AGPase),catalysing the key regulatory step of starch synthesis in theplastid, while there were no substantial changes in metabolicintermediates or sugar levels. A similar increase in post-translationalredox-activation of AGPase was found after supplying adenineto wild-type potato tuber discs to increase adenine nucleotidelevels. Results provide first evidence for a link between redox-activationof AGPase and adenine nucleotide levels in plants. Key words: Adenylate kinase, ADPglucose pyrophosphorylase, plastid, redox-regulation, potato, respiration, starch Received 18 September 2007; Revised 12 November 2007 Accepted 13 November 2007     

Plastidial localization of a potato 'Nudix' hydrolase of ADP-glucose linked to starch biosynthesis

Escherichia coli and potato (Solanum tuberosum) ADP-sugar pyrophosphatases (EcASPP and StASPP, respectively) are 'Nudix' hydrolases of the bacterial glycogen and starch precursor molecule, ADP-glucose (ADPG). We have previously shown that potato leaves expressing EcASPP either in the cytosol or in the chloroplast exhibited large reductions in the levels of starch, suggesting the occurrence of cytosolic and plastidial pools of ADPG linked to starch biosynthesis. In this work, we produced and characterized potato and Arabidopsis plants expressing EcASPP and StASPP fused with green fluorescent protein (GFP). Confocal fluorescence microscopy analyses of these plants confirmed that EcASPP-GFP has a cytosolic localization, whereas StASPP-GFP occurs in the plastid stroma. Both source leaves and potato tubers from EcASPP-GFP-expressing plants showed a large reduction of the levels of both ADPG and starch. In contrast, StASPP-GFP-expressing leaves and tubers exhibited reduced starch and normal ADPG contents when compared with control plants. With the exception of starch synthase in StASPP-GFP-expressing plants, no pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in EcASPP-GFP- and StASPP-GFP-expressing plants. The overall data (i) show that potato plants possess a plastidial ASPP that has access to ADPG linked to starch biosynthesis and (ii) are consistent with the occurrence of plastidic and cytosolic pools of ADPG linked to starch biosynthesis.     

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).     

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.     

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.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

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.     

The influence of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) on potato tuber metabolism

The aim of this work was to investigate the importance of cytosolic phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPC) in potato carbohydrate metabolism. For this purpose, the cytosolic isoform of phosphorylating GAPC was cloned and used for an antisense approach to generate transgenic potato plants that exhibited constitutively decreased GAPDH activity. Potato lines with decreased activities of phosphorylating GAPC exhibited no major changes in either whole-plant or tuber morphology. However, the levels of 3-phosphoglycerate were decreased in leaves of the transformants. A broad metabolic phenotyping of tubers from the transformants revealed an increase in sucrose and UDPglucose content, a decrease in the glycolytic intermediates 3-phosphoglycerate and phosphoenolpyruvate but little change in the levels of other metabolites. Moreover, the transformants displayed no differences in cold sweetening with respect to the wild type. Taken together these data suggest that phosphorylating GAPC plays only a minor role in the regulation of potato metabolism. The results presented here are discussed in relation to current models regarding primary metabolism in the potato tuber parenchyma.