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.     

美国黄松、班克松和油松的抗寒性比较

通过人工冰冻和电导率的测定,对黄土丘陵沟壑区引种载培的美国黄松、班克松和乡土树种油松的抗寒性进行了鉴定,并探讨其抗寒机理.结果表明,班克松的抗寒性比油松强,而美国黄松的抗寒性比油松稍弱.班克松的束缚水/自由水比值高达7.0,组织中ABA含量高达164.3 μg·g^-1 FW;但可溶性糖和K⁺含量较低,分别为12.0%和2 450 μg·g^-1 DW.油松则是可溶性糖、K⁺和ABA含量都较高,分别为18.68%、4 538 μg·g^-1 DW和95.8 μg·g^-1 FW;束缚水/自由水比值较低,为2.58.美国黄松的可溶性糖含量较高,18.05％;但束缚水/自由水比值、K⁺和ABA含量都较低,分别为2.18、2 275 μg·g^-1 DW和63.3 μg·g^-1 FW,可能是其抗寒性较弱的内在原因.班克松较低的叶绿素含量和较高的类胡萝卜素/叶绿素比值对其抗寒性也有贡献.说明3种树种虽然都是抗寒树种,但其内在机理仍有差异.     

Redox regulation of carbon storage and partitioning in response to light and sugars

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.     

Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.     

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     

荧光发光测定AGPase活力方法初步建立及应用

腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)活力传统上采用32P同位素标记反应底物来测定,但因测定条件的限制而极大地影响了其应用.该研究依据荧光素酶催化荧光素生成发光氧化荧光素的原理,在优化基本反应体系、确立反应体系ATP浓度和荧光强度线性关系等基础上,初步建立了以荧光发光反应测定AGPase活力的新方法,并运用新方法测定了含有不同glgC基因拷贝数菌株的AGPase活力.测定结果显示,不同菌株AGPase活力随glgC拷贝数不同存在显著差异,且其变化趋势与理论预期一致,即新方法可用于AGPase活力的体外测定,且具有更加安全、灵敏、简便和成本低的特点.     

Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves

Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.     

Induction of ApL3 expression by trehalose complements the starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the large subunit of ADP-glucose pyrophosphorylase

The disaccharide trehalose has strong effects on plant metabolism and development. In Arabidopsis seedlings, growth on trehalose-containing medium leads to an inhibition of root elongation, an accumulation of starch in the shoots, an increased activity of ADP-Glc pyrophosphorylase (AGPase), and an induction of the expression of the AGPase gene, ApL3 (A. Wingler, T. Fritzius, A. Wiemken, T. Boller, R.A. Aeschbacher [2000] Plant Physiol 124: 105-114). We used Arabidopsis mutants deficient in starch synthesis to examine whether the primary effect of trehalose was to affect carbohydrate allocation by the induction of AGPase in the photosynthetic tissue. In a mutant lacking the large AGPase subunit, ApL1, (adg2-1 mutant) growth on trehalose restored AGPase activity and led to a strong accumulation of starch in the shoots. In contrast, starch synthesis could not be induced in a mutant lacking the small AGPase subunit, ApS, (adg1-1 mutant) or in a mutant lacking plastidic phosphoglucomutase (pgm1-1 mutant). These results indicate that ApL3 can substitute for ApL1 in the AGPase complex. In addition, root elongation in the mutants, especially in the adg1-1 mutant, was partially resistant to trehalose, suggesting that the induction of ApL3 expression and the resulting accumulation of starch in the shoots were partially responsible for the effects of trehalose on the growth of wild-type plants.     

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.     

Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry

A search for naturally occurring aromatic cytokinins (ARCKs) in Arabidopsis thaliana plants and Populus x canadensis leaves led to the discovery of four new plant hormone substances: 6-(2-methoxybenzylamino)purine (ortho-methoxytopolin, MeoT), 6-(3-methoxybenzylamino)purine (meta-methoxytopolin, MemT) (Fig. 1) and their 9-beta-D-ribofuranosyl derivatives. These substances were identified by liquid chromatography electrospray ionization mass spectrometry [LC (+)ESI-MS] and capillary-liquid chromatography/frit-fast atom bombardment-mass spectrometry [CapLC/frit-FAB-MS] after pre-column derivatization. The chemical structures were subsequently confirmed by chemical synthesis. Because of lack of heavy labelled internal standards, the endogenous levels of methoxytopolins in A. thaliana plants, Populus x canadensis leaves and samples derived from cultures of Agrobacterium tumefaciens strain GV3101 were determined by enzyme-linked immunosorbent assay (ELISA) of HPLC-fractionated extracts. While the levels of MeoT, MemT and their ribosides in A. thaliana shoots and Populus x canadensis leaves were relatively low (approximately 0.25-10 pmol g-1 FW for MeoT and MemT, respectively), the A. tumefaciens strain produced up to 600 times more of the newly identified substances. Cytokinin activity of methoxytopolines was demonstrated in three bioassays testing their ability to stimulate tobacco callus growth, to delay chlorophyll degradation in excised wheat leaves, and to induce betacyanin synthesis in Amaranthus caudatus var. atropurpurea cotyledons. Notably, their anti-senescing activity in the wheat leaf assay exceeded that of BAP and Z by almost 200%. Methoxytopolins are proposed to be new members of the biologically active aromatic cytokinin family, which might have specific physiological functions.     

Modification of Sugar Composition in Strawberry Fruit by Antisense Suppression of an ADP-glucose Pyrophosphorylase

To modulate the soluble sugar content of strawberry fruits, we generated transgenic plants that incorporated an antisense cDNA of ADP-glucose pyrophosphorylase (AGPase) small subunit (FagpS) under the control of the strawberry fruit-dominant ascorbate peroxidase (APX) promoter (cv. Anther). Several independent transgenic lines were obtained and grown in the greenhouse for analysis of agronomic traits. Most transgenic fruit did not show significant differences in weight and hardness compared to control fruit. However, the starch content in fruit was decreased to 27–47% and the total soluble sugar content was increased to 16–37% in transgenic plants (analyzed by the HPLC of sugar composition at four different stages of fruit development). The sugar contents of fruits in transgenic lines were particularly higher than control fruits at the red stage. The results were consistent with northern analysis, which showed that the levels of AGPase mRNA drastically were reduced in the red stage of fruits in all the transgenic plants. In other tissues of transgenic plants, the FagpS mRNA expression level was similar to control plants. Our studies indicate that fruit-specific down-regulation of the AGPase gene might be an effective strategy for increasing sugar and decreasing starch content in strawberry.     

CO2 Assimilation and Partitioning of Carbon in Maize Plants Deprived of Orthophosphate

In order to study the effects of inorganic phosphate (P1) starvationon C₄plants, 3-week-old maize plants (Zea maysL cv. Brulouis)were grown in a growth chamber on a nutrient solution withoutP1 over 22 d During the first 2 weeks, Pi-starved plants grewas well as control plants The Pi concentration in the planttissue decreased rapidly with time, which suggests that normalbiomass production can be maintained at the expense of internalP1 In addition, photosynthetic CO2 assimilation measured 4-6h after dawn was not affected, but the concentration of glucose,sucrose, and starch in leaves was much higher than in the controls¹⁴CO₂ pulse-chase experiments earned out on the ninth day oftreatment showed that ¹⁴CO₂ assimilation was perturbed duringthis initial period, resulting in a larger flow of carbon toboth starch and sucrose At the beginning of the third week ofP1 starvation (15 d after treatment) ¹⁴C incorporation intosucrose stayed high relative to controls but this was not thecase for starch At the end of the third week of P1-deficiency,shoot growth was considerably reduced and fresh weight was onlyone-third of that of the control plants. The P1 concentrationof both the leaf and root tissues was less than 1.0 µmolg^–1 FW compared to 20-25µmol g¹ FW in the controls.Photosynthetic CO₂ assimilation was reduced and the leaf concentrationof sucrose and starch, which had begun to decrease after theend of the second week of P1 limitation, became lower than inthe controls. These results obtained on maize plants show thatphotosynthesis and carbon partitioning between sucrose and starchwere strongly affected by P1 deficiency, similar to C₃ species. Key words: CO₂ assimilation, corn, orthophosphate deficiency, starch, sucrose     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild-type and trehalose-6-phosphate synthase mutants of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Delta and tps2Delta (Tre6P phosphatase) strains. For the tps1Delta strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Delta strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Delta spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Delta strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein-protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity.     

Leaf Phosphate Status,Photosynthesis, and Carbon Partitioning in Sugar Beet (IV. Changes with Time Following Increased Supply of Phosphate to Low-Phosphate Plants)

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).     

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.     

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)     

ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It was recently shown that potato (Solanum tuberosum) tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular Cys bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farré, P. Geigenberger [2002] Plant Cell 14: 2191-2213). We show here that AGPase is also subject to posttranslational regulation in leaves of pea (Pisum sativum), potato, and Arabidopsis. Conversion is accompanied by an increase in activity, which involves changes in the kinetic properties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer when preilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves via the petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced in the starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPB monomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novel posttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels in the leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recycling of phosphate during photosynthetic carbon fixation and sucrose synthesis.