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American Society of Plant Biologists Starch Synthesis in Potato Tubers Is Regulated by Post-Translational Redox Modification of ADP-Glucose PyrophosphorylaseA Novel Regulatory Mechanism Linking Starch Synthesis to the Sucrose SupplyMax Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany 1 To whom correspondence should be addressed. E-mail geigenberger{at}mpimp-golm.mpg.de; fax 49-331-5678408
Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.
ADP-Glc pyrophosphorylase (AGPase) catalyzes the conversion of Glc-1-P and ATP to ADP-Glc and inorganic pyrophosphate (PPi), which is the first committed step in the pathway of starch synthesis (Figure 1) (Preiss, 1988  ; Martin and Smith, 1995; Smith et al., 1997). The higher plant enzyme is a heterotetramer, consisting of two "regulatory" subunits (AGPS; 51 kD) and two slightly smaller "catalytic" subunits (AGPB; 50 kD) (Okita et al., 1990). AGPase plays a major role in the regulation of starch synthesis. Studies with an Arabidopsis AGPS mutant have demonstrated that AGPase is a key site for the control of starch synthesis in leaves (Neuhaus and Stitt, 1990). In potato tubers expressing an antisense AGPB construct, starch synthesis is decreased when activity is decreased to <50% of the wild-type level (Müller-Röber et al., 1992; Geigenberger et al., 1999a).
Two mechanisms are known to regulate AGPase activity. First, AGPase is subject to transcriptional regulation, with expression being increased by sugars (Salanoubat and Belliard, 1989 ; Müller-Röber et al., 1990; Sokolov et al., 1998) and decreased by nitrate (Scheible et al., 1997) and phosphate (Nielsen et al., 1998). This may allow starch accumulation to respond to changes in the carbon and nutritional status (Scheible et al., 1997; Stitt and Krapp, 1999). Second, AGPase is exquisitely sensitive to allosteric regulation, being activated by glycerate-3-phosphate (3PGA) and inhibited by Pi (Sowokinos, 1981; Sowokinos and Preiss, 1982; Preiss, 1988).
Increasing levels of phosphorylated intermediates typically lead to a marked increase of the 3PGA/Pi ratio. Therefore, activation of AGPase by an increasing 3PGA/Pi ratio allows the rate of starch synthesis to be adjusted in response to changes in the balance between photosynthesis and Suc synthesis in leaves (Heldt et al., 1977
Several situations have been reported in which starch synthesis changes independently of overall AGPase activity and reciprocally to the levels of phosphorylated intermediates (Geigenberger et al., 1994
Because only the overall metabolite levels were measured, there are various explanations for these observations. One is that AGPase is being inhibited by a novel mechanism. Another is that the substrate supply for AGPase may be changing as a result of regulation of the envelope hexose phosphate:phosphate transporter (Kammerer et al., 1998 Here, we show that the inhibition of starch synthesis after tuber detachment occurs via a mechanism that depends on the properties of the plant AGPase; additionally, we used nonaqueous fractionation to investigate the response of cytosolic and plastidic metabolite levels and define a unique crossover point at AGPase. We also demonstrate that detachment does not lead to a decrease of the plastid 3PGA/Pi ratio and has no effect on AGPB protein or overall AGPase activity; instead, it leads to post-translational inactivation of AGPase via a reversible mechanism that involves redox-dependent dimerization of the ABPB subunits. Finally, we present evidence that this novel mechanism contributes to the regulation of starch synthesis in response to a range of treatments that modify the Suc level in tubers.
Inhibition of Starch Synthesis in Response to Tuber Detachment Is Abolished in Transgenic Tubers That Express a Heterologous AGPase We used two independent approaches to determine the step or steps in the pathway of starch synthesis that are the targets for the novel mechanism that inhibits starch synthesis when tubers are detached from the mother plant. The first approach asked whether specific regulatory features of potato tuber AGPase are essential for the inhibition of starch synthesis. To answer this question, the response was compared in wild-type tubers and in transformants in which endogenous AGPase was replaced largely by a nonplant AGPase.
The introduced construct encodes a form of the monomeric Escherichia coli enzyme (glgC16) that is different in its kinetic properties from the plant enzyme (Stark et al., 1992
To measure the rate of starch synthesis, low concentrations of 14C-Glc were injected into a fine borehole in growing tubers that were attached to the mother plant or 1 and 3 days after detaching them from the mother plant by severing the stolon (for details, see Geigenberger et al., 1994 ). After 1 h, the area around the injected label was removed and analyzed to determine how much of the injected label had been converted to starch (Figure 2B). Metabolite levels were measured in the same material (Figures 2C and 2D). Similar results were obtained in an independent experiment in which 14C-Glc was supplied for 30 min to discs cut from attached or detached tubers (data not shown).
In wild-type tubers, 34% of the label was incorporated into starch in attached tubers, decreasing to 21 and 15% in tubers that had been detached for 1 and 3 days, respectively (Figure 2B). The inhibition of starch synthesis was accompanied by an increase of hexose phosphates (Figure 2C) and a slight increase of 3PGA (data not shown). Suc decreased gradually, declining by 24 and 70% after 1 and 3 days, respectively (Figure 2D). This finding confirms the results described previously (Geigenberger et al., 1994 In the antisense line AGP93, 19% of the injected label was incorporated into starch in attached tubers, decreasing to 8% in detached tubers (Figure 2B). Hexose phosphates were high in attached tubers and remained high after detachment (Figure 2C), and Suc decreased even more slowly than in wild-type tubers (Figure 2D). In the double transformants AF1-20 and AF1-28, 24 to 26% of the label was incorporated into starch in attached tubers (Figure 2B). Detachment did not lead to a rapid inhibition of starch synthesis in these lines (Figure 2B) but instead led to a marked decline of hexose phosphates (Figure 2C) and a rapid 70 to 80% decrease of Suc during the first day after detachment (Figure 2D). There were no substantial changes in the specific activities of the internal hexose phosphate pools in the various genotypes after detachment (data not shown), demonstrating that the different response in the double transformants is not attributable to isotopic dilution of the incoming label by internal pools. These results provide strong genetic evidence that starch synthesis is inhibited after detachment by a regulatory mechanism that requires the presence of native potato tuber AGPase.
Separation of Tuber Material into Subcellular Compartments
This was performed by nonaqueous fractionation, a technique developed to analyze the subcellular compartmentation of metabolites in leaves (Gerhardt and Heldt, 1984
Marker enzyme activities are measured to reveal how materials from different cellular compartments distribute across the gradient (Figure 3A). The vacuolar marker mannosidase (Boller and Kende, 1979
Metabolites are measured in each fraction, and their subcellular distribution are estimated by more-dimensional linear regression. The estimated distribution can be compared with the overall content to estimate the metabolite content in each compartment (per gram of total dry weight) and with empirically determined values for the volume of each compartment (Farré et al., 2001 ) to estimate the subcellular concentrations. This analysis yields estimates for the vacuole, plastids, and cytosol. The estimated values for the cytosol include the mitochondrial metabolites because the cytosol and mitochondria are not separated (see above).
Changes in Subcellular Levels of Its Substrates Reveal That AGPase Is Involved in the Inhibition of Starch Synthesis
ATP is imported from the cytosol via the envelope adenylate exchanger (Figure 1). A substantial proportion of the adenine nucleotides are located in the plastid (Figures 3E and 3F), whereas uridine and guanidine nucleotides are located mainly in the cytosol of tubers (data not shown) (Farré et al., 2001 ), as is found for leaves (Dancer et al., 1990; Riens et al., 1991). Detachment led to a 50% increase of ATP in the plastid and a 50% decrease of ATP in the cytosol (Figure 3E). ADP levels were not affected substantially after detachment (Figure 3F).
The ATP/ADP ratio was lower in the plastid than in the cytosol in attached tubers (1.2 compared with 3.3; data calculated from Figures 3E and 3F), again resembling the ratio in leaves (Stitt et al., 1982
The estimated plastidic concentrations of Glc-1-P and ATP were in the range of the values determined in vitro for the substrate affinity S0.5 Glc-1-P (40 to 140 µM) and S0.5 ATP (120 to 190 µM) of AGPase (Sowokinos and Preiss, 1982
Estimation of Mass-Action Ratios for Each Enzyme-Catalyzed Reaction and Transport Step between Suc and Starch Identifies AGPase as the Unique Site Involved in the Inhibition of Suc-to-Starch Interconversion after Tuber Detachment
These results (Table 1) were used to calculate the ratio between the in vivo concentrations of the products and the substrates (termed the mass-action ratio) for every step between Suc and ADP-Glc (Table 2). The theoretical equilibrium constant (Keq; the ratio of product and substrate concentrations at which the reaction is at its thermodynamic equilibrium and net flux is zero) is listed for comparison. Keq for the transport steps is set at unity. In attached tubers, the mass-action ratios of the reactions catalyzed by SuSy, UGPase, and cytosolic PGM, the transport exchanges catalyzed by the Glc-6-P/Pi and ATP/ADP transporters, and the reaction catalyzed by plastidic PGM are close to their Keq. The mass-action ratios of fructokinase, AGPase, and inorganic pyrophosphatase are displaced from their Keq (Table 2). These results resemble those reported previously for cytosol and plastids in leaves (Stitt et al., 1982
The inhibition of starch synthesis after detachment was accompanied by a 40-fold decrease of the mass-action ratio for AGPase (Table 2). This was the result of an increase in the concentration of the substrates Glc-1-P and ATP and a decrease in the concentration of the products ADP-Glc and PPi in the plastid (Table 1) (Geigenberger et al., 1994 ). The mass-action ratios for all of the other steps were unaltered or even increased slightly after tuber detachment (Table 2). This finding provides strong biochemical evidence that AGPase is the only site at which significant regulation occurs after detachment.
Inhibition of Starch Synthesis in Response to Tuber Detachment Does Not Involve Changes in the Plastidic Concentrations of 3PGA and Pi
To explain why there is a 30 to 50% inhibition of starch synthesis after tuber detachment (Figure 2B) (Geigenberger et al., 1994
Changes in AGPase Expression Are Not Reflected Rapidly at the Protein Level and Do Not Explain the Inhibition of Starch Synthesis in Wild-Type Tubers after 1 Day of Detachment
AGPase activity was measured using a standard assay in which DTT was included in the extraction and assay buffer and activity was determined in the presence of saturating substrate levels. AGPase activity was unaltered at 1 day after detachment and started to decrease at 3 days after detachment (Figure 4C and data not shown) (Geigenberger et al., 1994 ). These results show that the inhibition of starch synthesis during the first day after tuber detachment is not the result of changes in AGPase expression.
Tuber Detachment Leads to Reversible Oxidation and Dimerization of AGPase To determine whether analogous changes occur in planta, growing potato tubers were extracted in degassed SDS extraction buffer, separated immediately by nonreducing SDS-PAGE to maintain the in vivo redox status of the enzyme, and immunoblotted with antibodies raised against maize or potato AGPB. To ensure the specificity of the antibodies, His-tagged potato AGPB and AGPS were overexpressed in E. coli and purified; their identities were confirmed by matrix-assisted laser-desorption ionization time-of-flight analysis. Then, they were applied in a concentration series, separated by SDS-PAGE, and immunoblotted. The antibody raised against maize brittle-2 was highly specific for the potato AGPB subunit (Figure 5A).
Extracts from tubers that were attached to the plant gave immunopositive signals with the AGPB-specific maize antibody at 50 and 100 kD (Figure 5B, lane 1). When 1-day-detached tubers were analyzed (Figure 5B, lane 2), the 50-kD band disappeared almost completely and the 100-kD band became more intense. Incubation of the extracts in vitro with DTT before SDS-PAGE completely converted the 100-kD band to the 50-kD band (Figure 5B, cf. lanes 3 and 4 with lanes 1 and 2). Similar results were obtained in repeated experiments over a period of months with separate batches of tubers. These results reveal that AGPB is present as a mixture of dimer and monomer in growing tubers and becomes completely dimerized when the tubers are detached and Suc import from the mother plant is interrupted.
Establishment of a New Protocol to Extract and Measure AGPase Activity Reveals That the Changes in Redox State Are Accompanied by Changes in Activity and Kinetic Properties
To detect the large changes of activity of redox-modulated Calvin cycle enzymes that occur when chloroplasts or leaves are illuminated or darkened, it was necessary to develop very fast assay and extraction procedures (Laing et al., 1981 When an extract from growing tubers was prepared in this way and subjected to nonreducing SDS-PAGE, a mixture of bands at 50 and 100 kD (Figure 5C, lane 2) was found that resembled a parallel sample that was extracted in SDS (Figure 5C, lane 3). Large differences in AGPase activity were found between extracts from attached tubers and tubers that had been detached for 1 day (Figure 6). The differences were stable for at least 10 min in the assay conditions (data not shown) but were lost rapidly if extract was incubated in the absence of substrate (data not shown).
For routine determination of AGPase activity, the assay was performed in the reverse direction (Figures 6A to 6C) using PGM and Glc-6-P dehydrogenase to couple Glc-1-P formation in real time to NADP+ reduction. Detachment led to a decrease of AGPase activity and the affinity for ADP-Glc when activity was assayed in the absence of allosteric effectors (Figure 6A). This inhibition was reversed completely when 5 mM DTT was included during extraction and assay (Figure 6A). Detachment also led to a decrease of activity and the affinity for ADP-Glc when activity was assayed in the presence of 300 µM 3PGA and 1200 µM Pi (Figure 6B), which lie in the range of estimated concentrations in amyloplasts (Table 1). When assayed with limiting substrate concentrations, AGPase from detached tubers was strongly dependent on the presence of 3PGA and was not activated until high concentrations of 3PGA were present (Figure 6C). It also was more sensitive to inhibition by Pi than AGPase extracted from attached tubers (Figure 6C). Maximum activity in the presence of high 3PGA and zero Pi was similar for attached and detached tubers. We also investigated whether AGPase activity was modified when it was assayed in the ADP-Glc–producing direction. A stopped assay was used, in which each individual sample was analyzed by HPLC to determine ADP-Glc (Figures 6D to 6F). Control experiments showed that ADP-Glc was not degraded during the incubation. Detachment led to a very marked inhibition of activity. The affinity for ATP was decreased when assayed in the presence of 200 µM 3PGA (Figure 3D) or 300 µM 3PGA plus 1200 µM Pi (Figure 6E). AGPase from detached tubers showed a strongly reduced sensitivity to 3PGA activation at physiological concentrations (Figure 6F). To provide a routine microplate-compatible test for changes in redox regulation in subsequent experiments, AGPase activity was measured in the ADP-Glc–cleaving direction in the presence of 600 µM ADP-Glc in the absence (Vsel) or presence of 5 mM DTT (Vred). Similar results were obtained at a range of ADP-Glc concentrations between 200 and 1000 µM (Figure 6A and data not shown). The ratio between the activities in these two assays (Vsel/Vred) is termed "activation."
Incubation of Tuber Discs with DTT to Redox Activate AGPase Leads to a Stimulation of Starch Synthesis and a Decrease of Phosphorylated Metabolites Increasing concentrations of DTT led to a progressive increase of AGPase activation (Figure 7A) and the proportion of AGPB that was present as a monomer (data not shown). There was a parallel increase of label incorporation into starch (Figure 7B). AGPase activation (Figure 7A) and starch synthesis (Figure 7B) were lower in discs from detached tubers than in discs from attached tubers in the absence of DTT, and this difference was reversed partly by DTT. When the results for all of the treatments were combined, a strong correlation was found between AGPase activation and label incorporation into starch (Figure 7C).
The levels of hexose phosphates and label in the phosphorylated intermediates were measured and used to calculate the specific activity of the hexose phosphate pool and the absolute rate of starch synthesis. This calculation confirmed that there is a strong correlation between AGPase activation and the rate of starch synthesis (data not shown). The stimulation of starch synthesis was accompanied by a marked decrease of the levels of hexose phosphates (Figure 7D) and 3PGA (data not shown). This finding shows that the stimulation of starch synthesis by DTT is not caused by an additional action besides redox activation of AGPase, such as a stimulation of Suc breakdown or a restriction of respiration.
Incubation of Tuber Discs Leads to a Decrease in AGPase Activation State, Which Is Prevented by Suc
Geiger et al. (1998)
Inhibition of Starch Synthesis in Transgenic Tubers Overexpressing Suc Phosphorylase Involves the Inactivation of AGPase In the second approach, heterologous expression of bacterial Suc phosphorylase in the cytosol was used as a tool to decrease Suc levels in planta (Trethewey et al., 2001 ). Suc phosphorylase converts Suc and Pi to Fru and Glc-1-P. This resembles the endogenous pathway for Suc breakdown via SuSy and UGPase in not producing Glc but differs because it does not require PPi or produce UDP-Glc as an intermediate. Critically, Suc phosphorylase has a far lower Km(Suc) ( 1 mM; Silverstein et al., 1967) than SuSy (40 to 200 mM; Avigad, 1982).
Introduction of Suc phosphorylase led to a threefold to fourfold decrease of Suc (Figure 9A). Glc did not change (data not shown) (Trethewey et al., 2001
Redox Inactivation of AGPase Correlates with the Decrease in Suc and the Inhibition of Starch Accumulation as Potato Plants Age The experiments shown in Figures 2 to 9 used tubers from 8-week-old plants, which contain high levels of Suc. A third approach investigated changes in wild-type tubers as the mother plant ages. Tuber starch levels increased on a fresh weight basis between 6 and 10 weeks but did not increase further between 10 and 12 weeks (Figure 10A). Suc levels decreased slightly between 6 and 8 weeks and decreased markedly at 10 and 12 weeks (Figure 10B) (Mares and Marschner, 1980 ; Merlo et al., 1993). Hexose phosphate levels increased between 10 and 12 weeks, revealing that starch synthesis is inhibited more strongly than Suc breakdown (Figure 10C). As shown previously (Mares and Marschner, 1980; Merlo et al., 1993), SuSy activity decreased sharply (Figure 10D) and overall AGPase activity decreased markedly (assayed with DTT; Figure 10E) as the plant aged. There also was a marked decrease of AGPase activation (Figure 10F).
A Novel Mechanism Involving Reductive Post-Translational Regulation of AGPase Inhibits Starch Synthesis after Detachment of Tubers from the Mother Plant The starting point for the experiments presented here was the observation (Geigenberger et al., 1994 ) that removal of growing potato tubers from the mother plant leads within 1 day to an inhibition of starch synthesis that cannot be explained easily by allosteric regulation or by changes in the expression of AGPase. This led us to suspect that there might be a major gap in our understanding of the regulation of starch synthesis. The first step in identifying the missing regulatory mechanism was to determine the step(s) at which flux was being inhibited. Substitution of higher plant AGPase with a heterologous bacterial AGPase provided genetic evidence that the inhibitory mechanism required the presence of native potato AGPase (Figure 2). To provide biochemical evidence that AGPase is the unique site at which flux is regulated, we performed a systematic investigation of the subcellular levels of every metabolite in the pathway between Suc and starch. This allowed the mass-action ratio (the product/substrate ratio) to be estimated for every enzyme and transport step between Suc in the cytosol and starch synthesis in the plastid. AGPase was the only step at which this ratio decreased (by 2 orders of magnitude) after detachment of the tubers (Table 2). These genetic and biochemical experiments demonstrate (1) that the regulatory mechanism acts at AGPase and (2) that AGPase is the sole step involved in the inhibition of starch synthesis after tuber detachment.
The second step was to exclude the possibility that known mechanisms are responsible for the inhibition of AGPase activity. Measurements of 3PGA and Pi levels in the plastid showed that the 3PGA/Pi ratio increased after detachment of tubers. Although there was a marked decrease in the transcript levels for AGPB and AGPS1, there were no changes of AGPB protein or AGPase activity. AGPase activity also changes much more slowly than transcript levels during the photoperiod in Arabidopsis leaves (Sokolov et al., 1998
The last step was to identify the missing mechanism. Since Sowokinos (1981) We found that AGPB exists as a mixture of monomers and dimers in growing tubers and is converted almost completely to dimers in detached tubers. Furthermore, dimerization led to inactivation, as a result of a strong decrease of the substrate affinity, an increased requirement for and decreased sensitivity to activation by 3PGA, and an increased sensitivity to inhibition by Pi. The change in the kinetic properties was especially dramatic when AGPase activity was assayed in the forward reaction. Dimerization and inactivation both were reversed by incubating extracts with DTT. We interpret these results as evidence that the redox changes identified by Preiss and co-workers in heterologously overexpressed AGPase operate as a regulatory mechanism in planta. However, biochemical analyses of the position of the Cys bridges in AGPase holoenzyme isolated from plants is needed to confirm this and to exclude the possibility that other intermolecular or intramolecular Cys bridges also form in planta. Final genetic proof that this mechanism is responsible for the inhibition of starch synthesis after detachment of tubers would require the production of transformants in which the native AGPB is replaced by a modified form of AGPB in which Cys-82 is modified to prevent the formation of the intermolecular bridge. This experiment would require a mutant line in which transcription of the native AGPB is blocked to allow effective overexpression without cosuppression of the modified construct. At present, this is not possible in potato.
Redox Modulation of AGPase Allows Starch Synthesis to Be Increased, Whereas the Levels of Phosphorylated Metabolites Decrease
Post-Translational Redox Modulation of AGPase Also Makes an Important Contribution to the Regulation of Starch Synthesis in Tubers in Other Conditions These results can be explained by post-translational redox modulation of AGPase. Feeding Suc to discs led to increased activation of AGPase and stimulated the rate of starch synthesis (Figure 8, Table 3), the heterologous expression of Suc phosphorylase led to redox inactivation of AGPase and a lower tuber starch content (Figure 9), and the gradual changes in the sink-source balance as the mother plant aged led to a decrease of AGPase activation. In the latter two cases, there also was a 30 to 40% decrease of overall AGPase activity, but this alone (see above) is not large enough to explain the increase in hexose phosphates and the decrease in the rate of starch synthesis.
Post-Translational Redox Modulation of AGPase Represents a Component in a Novel Regulatory Pathway That May Link the Rate of Starch Synthesis to Suc Supply
When tuber discs were incubated in the absence of Suc, the internal Suc level of the discs decreased and there was a decrease of AGPase activation that could be reversed by the addition of Suc (Figure 9, Table 3). Overexpression of Suc phosphorylase to alter the Suc level and the precise way in which Suc was metabolized in the tuber also led to redox inactivation of AGPase (Trethewey et al., 2001 Figure 11 summarizes the data on tuber Suc content and AGPase activation from these different experiments, which were performed over a period of 2 years with many separate batches of plants grown under different conditions. By comparing the two parameters, a correlation coefficient of 0.85 was found. These results provide very strong correlative evidence that post-translational redox modulation of AGPase responds to changes in Suc availability, although it is not possible at this time to determine whether a specific influx, a particular Suc pool, or changes in related metabolites provide the immediate stimulus for the transduction pathway.
Candidate Components of Signal Transduction That Lead to the Redox Modulation of AGPase Have Yet to Be Identified and Confirmed The light-dependent redox modulation of Calvin cycle enzymes and enzymes involved in ATP synthesis and NADPH export in chloroplasts is mediated by thioredoxin f and thioredoxin m, respectively (Schümann and Jacquot, 2000 ). Photosynthetic electron transport leads to a reduction of ferredoxin, and reducing groups then are transferred by ferredoxin:thioredoxin reductase to the thioredoxins, which react with their targets. Oxidation is thought to occur primarily by a reversal of this process. The reaction between the thioredoxins and their individual targets is modulated by changes in pH, Mg2+, and the levels of their substrates or products, which alter the mid-redox potential of the Cys groups on enzymes (Scheibe, 1991; Schümann and Jacquot, 2000).
Ballicora et al. (2000)
Finally, our results raise the question of whether the post-translational regulation of AGPase contributes to the regulation of starch synthesis in other organs. Two lines of evidence indicate that this may be the case. First, anomalies similar to those that prompted our investigations on tubers have been reported for other tissues. Starch synthesis changed independently of overall AGPase activity and the levels of phosphorylated intermediates after phloem transport was inhibited by detaching cotyledons of germinating Ricinus seedlings (Geigenberger and Stitt, 1991
Second, the QTCL motif is conserved in the N-terminal region of almost all sequenced AGPB genes of dicots and in some of the sequences available for monocots. Interestingly, the QTCL motif is absent in the major AGPB transcript in cereal endosperm (Thorbjornsen et al., 1996
The QTCL motif is absent in E. coli AGPase (glgC) and in cyanobacterial AGPase and also is absent in the small subunit of the heterotetrameric Chlamydomonas reinhardtii AGPase (STA6) (Zabawinski et al., 2001
This comparison also implies that it may be necessary to reevaluate the interpretation of experiments in which the overexpression of glgC16 in higher plants was shown to lead to a strong stimulation of starch synthesis (Stark et al., 1992
Regulation of AGPase at the Level of Expression, Post-Translational Regulation, and Allosteric Regulation
Allosteric control by 3PGA and Pi operates in a time frame of seconds to stimulate starch synthesis when phosphorylated intermediates increase and to inhibit it when they decrease. Existing physiological data indicate that allosteric control does not play a major role in coordinating the rate of starch synthesis with the Suc supply. Instead, it adjusts the rate of starch synthesis to the balance between Suc breakdown and carbon use. Conditions in which changes in the 3PGA/Pi ratio correlate with starch synthesis include high temperature, during which an increasing rate of respiration leads to a depletion of phosphorylated intermediates and an inhibition of starch synthesis (Geigenberger et al., 1998a ), and mild water stress, during which post-transcriptional activation of Suc phosphate synthase stimulates the resynthesis of Suc and leads to a decrease of phosphorylated intermediates and an inhibition of starch synthesis (Geigenberger et al., 1997, 1999b).
Post-translational redox modulation leads to changes of activity in a time frame of
First, lower levels of phosphorylated intermediates will favor Suc breakdown via the reversible reactions catalyzed by SuSy and UGPase (Geigenberger and Stitt, 1993
Transcriptional regulation in response to changes in Suc allows more gradual changes in AGPase activity that may require days to develop. For example, during diurnal rhythms (Geigenberger and Stitt, 2000 |
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Abstract
INTRODUCTION
