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ADP-Glucose Pyrophosphorylase Is Activated byPosttranslational Redox-Modification in Response toLight and to Sugars in Leaves of Arabidopsis andOther Plant Species1[w]

Janneke H.M. Hendriks, Anna Kolbe, Yves Gibon, Mark Stitt, and Peter Geigenberger*

Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, 14476 Golm, Germany

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It wasrecently 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 heterotetramericholoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farre, P. Geigenberger [2002] Plant Cell14: 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 kineticproperties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidlyconverted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer whenpreilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves viathe petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced inthe starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPBmonomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novelposttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels inthe leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recyclingof phosphate during photosynthetic carbon fixation and sucrose synthesis.

During photosynthesis, triose-phosphates (triose-P)are exported to the cytosol where they are convertedto end products, including Suc. This releases inorganicorthophosphate (Pi), which is recycled to the chloro-plast in counterexchange with triose-P (Edwards andWalker, 1983). Some of the photosynthate is retainedin the chloroplast to synthesize starch. Leaf starchrepresents a transient store, which is remobilized dur-ing the night to support leaf metabolism, and contin-ued synthesis and export of Suc (Geiger and Servaites,1994). Its importance is demonstrated by the pheno-type of starch-deficient mutants, which grow poorlyor die in short-day conditions (Caspar et al., 1986;Schulze et al., 1991; Geiger et al., 1995; Sun et al., 2002).A consensus has developed that leaf starch synthesisis regulated by changes in the levels of phosphory-lated metabolites and Pi that are generated when therate of photosynthesis increases or when rising levelsof sugars lead to feedback regulation of Suc synthesis.

This paper presents evidence that light and sugarsalso regulate starch synthesis more directly via redox-dependent posttranslational activation of ADP-Glcpyrophosphorylase (AGPase).

AGPase catalyzes the first committed step in thepathway of starch synthesis (Preiss, 1988; Martin andSmith, 1995). The higher plant enzyme is a heterotet-ramer that contains two “regulatory” (AGPS, 51 kD)and two slightly smaller “catalytic” (AGPB, 50 kD;Morell et al., 1987; Okita et al., 1990) subunits. AG-Pase is exquisitely sensitive to allosteric regulation,with glycerate-3-phosphate (3PGA) acting as an acti-vator and Pi as an inhibitor (Sowokinos, 1981;Sowokinos and Preiss, 1982; Preiss, 1988). Studieswith isolated chloroplasts led to the concept thatstarch synthesis is stimulated when low Pi restrictscarbon export from the plastid (Heldt et al., 1977). Inthese conditions, ATP falls, leading to an inhibitionof 3PGA reduction. A rising 3PGA to Pi ratio pro-vides a sensitive signal that carbon fixation is exceed-ing the rate of export, and activates AGPase. Ananalogous situation arises in leaves when the rate ofend product synthesis falls below the rate of photo-synthesis. For example, feedback regulation of Sucsynthesis will lead to the accumulation of phosphor-ylated intermediates, depletion of Pi, and activationof AGPase by the rising 3PGA to Pi ratio, resulting ina compensatory stimulation of starch synthesis (seeHerold, 1980; Stitt et al., 1987).

1 This work was supported by the Deutsche Forschungsgemein-schaft (grant no. SFB 429 TP–B7 to A.K. and P.G.) and by theBundesministerium fur Bildung und Forschung (GABI; grant toY.G. and M.S.).

[w] The online version of this article contains Web-only data.* Corresponding author; e-mail geigenberger@mpimp-golm.

mpg.de; fax 49 –331–567– 8408.Article, publication date, and citation information can be found

at www.plantphysiol.org/cgi/doi/10.1104/pp.103.024513.

For more than a decade, this biochemical modelhas provided the framework to explain how the pho-tosynthate allocation between Suc and starch is reg-ulated. Support has been provided by biochemicalanalyses of changes of metabolites, enzyme activities,and fluxes during the diurnal cycle (Gerhardt et al.,1987; Stitt et al., 1988), by genetic evidence that AG-Pase colimits starch synthesis in leaves (Neuhaus etal., 1990), and by genetic evidence that reduced ex-pression of various enzymes in the pathway of Sucsynthesis (Neuhaus et al., 1989; Neuhaus and Stitt,1990; Zrenner et al., 1996; Geigenberger and Stitt,2000; Scott et al., 2000; Hausler et al., 2000; Draborg etal., 2001) or overexpression of Suc phosphate syn-thase (SPS; Baxter et al., 2001; Laporte et al., 2001)leads to the predicted stimulation and inhibition,respectively, of starch synthesis. Details of the bio-chemical mechanisms that contribute to the feedbackinhibition of Suc synthesis have been elucidated.In spinach (Spinacia oleracea), rising Suc leads toposttranslational inhibition of SPS (Stitt et al., 1988;Neuhaus and Stitt, 1990). In other species, Suc ishydrolyzed to reducing sugars, which are rephos-phorylated (Huber, 1989; Goldschmidt and Huber,1992). Rising cytosolic hexose phosphates (Gerhardtet al., 1987) lead via activation of Fru-6-phosphate2-kinase and inhibition of Fru-2,6-bisphosphatase(Stitt et al., 1987; Villadsen and Nielsen, 2001;Markham and Kruger, 2002) to an increase of Fru-2,6-bisphosphate, which inhibits cytosolic Fru-1,6-bisphosphatase (cFBPase; Stitt et al., 1987; Neuhauset al., 1989, 1990). The predicted decrease of ATP andincrease of 3PGA has been confirmed when Suc syn-thesis is inhibited by decreased expression of trans-porters and enzymes in the pathway (see above),agents that sequester phosphate (Stitt et al., 1987) andlow temperature (Stitt and Grosse, 1988).

Curiously, the evidence is less convincing for treat-ments that modify partitioning by altering sugar lev-els in the leaf. Starch synthesis was stimulated in theabsence of an increase of 3PGA when sugars weresupplied to detached spinach leaves (Krapp et al.,1991), when spinach leaves were cold-girdled to de-crease export (Krapp and Stitt, 1995), and whenphloem transport was inhibited by phloem-specificexpression of Escherichia coli pyrophosphatase in to-bacco (Nicotiana tabacum; Geigenberger et al., 1996).In at least some conditions, decreased expression ofSPS in Arabidopsis leads to decreased rather thanincreased starch synthesis (Strand et al., 2000). Fur-ther, transgenic potato (Solanum tuberosum) plantswith increased levels of 3PGA due to antisense inhi-bition of cytosolic phosphoglycerate mutase did notshow any increase of starch in their leaves (Westramet al., 2002). These results indicate that there are gapsin our understanding of the regulation of photosyn-thate partitioning.

When potato AGPB and AGPS are heterologouslyoverexpressed in E. coli, an intermolecular bridge

forms between the cys82 residues of the two AGPBsubunits. To obtain active enzyme, it was necessaryto incubate the complex with dithiothreitol (DTT) orthioredoxin to break this link (Fu et al., 1998; Bal-licora et al., 2000). It was recently shown that ananalogous process occurs in planta in potato tubers(Tiessen et al., 2002). In both cases, reduction of theintermolecular bridge leads to a dramatic increase ofactivity, due to a decrease of the Km(ATP) and in-creased sensitivity to activation by 3PGA. Activationof AGPase in planta correlated closely with the tuberSuc content across a range of physiological and ge-netic manipulations, indicating that redox modula-tion is part of a novel regulatory loop that channelsincoming Suc toward synthesis of storage starch(Tiessen et al., 2002). Crucially, it allows the rate ofstarch synthesis to be increased in response to exter-nal inputs and independently of any increase in thelevels of glycolytic intermediates. The following pa-per asks whether AGPase is regulated by an analo-gous mechanism in leaves and investigates its con-tribution to the regulation of photosyntheticmetabolism.

RESULTS

AGPB Expressed in Leaves Contains a ConservedN-Terminal Cys

Almost all dicotyl plant AGPB sequences contain aconserved SQTCLDPDAS motif at the N terminus,which includes the Cys shown by Fu et al. (1998) tobe involved in formation of the intermolecular Cysbridge in the potato enzyme (see Supplementary Ma-terial). The only exception is Sumatra orange (Citrusunshui). Monocots contain two types of AGPB tran-script: One encodes proteins that contain this motif,and the other encodes proteins that lack it. The lattermay represent cytosolic isoforms that occur in theendosperm of growing cereal seeds (Sikka et al.,2001; Burton et al., 2002).

The full genome sequence for Arabidopsis containsone reading frame (At5g48300) with a high homologyto AGPB in other higher plants. A second open read-ing frame, which is annotated as a putative AGPB(At1g05610), shows considerable deviations from allother AGPB sequences (see Supplementary Material).Diversification occurs throughout the sequence andincludes the loss of many highly conserved aminoacids. At1g05610 is the only gene that falls outside ofthe group for plant AGPB sequences when a tree iscalculated with ClustalX (Thompson et al., 1997),using only those stretches of the alignment selectedfor tree calculation by Gblocks 0.91b (Castresana,2000). It even does not group with plant AGPS andbacterial AGP sequences (Supplementary Material).We therefore suspect that this gene does not code fora functional AGP protein. Further, RNA primers de-signed to distinguish between transcripts for thesetwo genes detected At5g48300 but not At1g05610 in

Redox Regulation of Starch Synthesis

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Arabidopsis leaf extracts (data not shown). We con-clude that an AGPB protein that contains the con-served Cys is expressed in the leaves of most if not allplants.

AGPB Exists as a Dimer in the Dark and BecomesMonomerized after Illumination of Potato,Arabidopsis, and Pea (Pisum sativum) Leaves

Pea, potato, and Arabidopsis leaves were har-vested during the second half of the light period andtoward the end of the dark period to investigatewhether leaf AGPB undergoes reversible dimeriza-tion in vivo. Extracts were rapidly prepared in de-gassed SDS solutions and subjected to non-reducingSDS-PAGE, and AGPB protein was detected using arabbit-antibody raised against AGPB from potato(see Tiessen et al., 2002). When extracts from growingpotato tubers are analyzed in this way, they containa mixture of monomeric (molecular mass of approx-imately 50 kD) and dimeric (molecular mass of ap-proximately 100 kD) AGPB (Tiessen et al., 2002). Ininitial experiments with leaves, all of the AGPB pro-tein ran with an apparent molecular mass of about100 kD, irrespective of whether extracts were pre-pared from illuminated or darkened plants (data notshown). When extracts from leaves were mixed 1:1with extracts from growing potato tubers, the immu-nosignal was also obtained at only 100 kD (data notshown). These results indicated that leaf extracts con-tain unknown compounds that rapidly oxidize AGPBto a dimer. To prevent this, leaves were extracted ina trichloroacetic acid (TCA)-diethyl ether mixture torapidly denature AGPase and physically separateany AGPB subunits that were present as monomers.This new procedure revealed that pea leaf AGPB iscompletely dimerized in the dark, and partly con-verted to a monomer in the light (Fig. 1A). When theextracts were separated in a reducing gel (includingDTT), immunosignal was found only at 50 kD, show-ing that the intermolecular link involves a Cysbridge. Similar results were obtained for potato andArabidopsis (Fig. 1B) leaves. The proportion con-verted to a monomer was lower in Arabidopsis, pos-sibly reflecting the lower growth light intensity.

AGPB Dimerization Is Accompanied by a Decrease ofAGPase Activity

In potato tubers, dimerization increases theKm(ATP) and decreases sensitivity to activation by3PGA (Tiessen et al., 2002). This change is reversedby incubation with DTT. We investigated whetherdimerization of Arabidopsis leaf AGPase is also ac-companied by changes in AGPase activity. Appear-ance of the monomer was accompanied by an in-

crease in AGPase activity when assayed in absence of3PGA but not in the presence of saturating amountsof 3PGA (Fig. 2A). The ratio of activity in the twoconditions (�3PGA/�3PGA) changed from 0.15 inthe dark to 0.67 in the light. Illumination led to amarked increase of the affinity for ATP in the absenceof 3PGA, which could be overcome at high 3PGAconcentrations (Fig. 2, B–C). The sensitivity to activa-tion by 3PGA is also changed. Whereas AGPase fromilluminated leaves attained significant activities inthe absence of 3PGA and was stimulated 4- to 10-foldby 3PGA depending on the ATP level, activity ofAGPase from leaves at the end of the night was verylow in the absence of 3PGA and was stimulated10-fold by 3PGA in the presence of high ATP and upto 25-fold in the presence of low ATP (Fig. 2D). Thesmall increase of overall AGPase activity in the darkis frequently seen in Arabidopsis leaves and reflectschanges in the amount of AGPase protein (data notshown).

Figure 1. Dimerization of AGPB varies between the day and night inpotato, pea, and Arabidopsis leaves. A, Western of pea leaf (cvMarcia) tissue harvested during the second half of the day (day) andat the end of the night (night). Samples were prepared with TCA etherand run directly (non-reducing) or after adding 4 mM DTT to part ofthe sample (reducing). B, Non-reducing westerns of leaf material ofArabidopsis and potato harvested during the day and the night.

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Light-Dark Transitions Are Accompanied by RapidChanges in AGPB Dimerization

The appearance of AGPB monomer during the dayin leaves could be due to illumination or it could bean indirect effect due, for example, to leaves contain-

ing more sugars in the light. To investigate whetherthere are rapid light-dependent changes in AGPBmonomerization, we darkened pre-illuminatedplants. This treatment was chosen because it leads toan abrupt change in photosynthesis, whereas illumi-nation leads to only slow changes due to the need toinduce photosynthesis and increase stomatalconductance.

Arabidopsis plants were illuminated for 6.5 h, sam-ples were taken in the light, the remaining plantswere darkened, and samples taken 6, 15, and 60 minlater. In the light, a small proportion of AGPB waspresent as a monomer (Fig. 3A; see also Fig. 1B).After darkening, the monomer decreased within 6min and almost totally vanished within 15 min.Sugar levels were measured in the same leaf material(Fig. 3B). There were no significant changes of Suc,Glc, or Fru levels in the first 6 min and only smallchanges in the first 60 min after darkening. Similarresults were obtained for pea plants (data notshown).

Figure 2. Increased monomerization of AGPB in the light leads to achange in the kinetic properties of AGPase. A, AGPase activity inpresence of 0 or 1 mM 3PGA in presence of 1.5 mM ATP and 1.5 mM

G1P in Arabidopsis leaves harvested during the end of the night (f)or the second half of the day (�). B, ATP substrate saturation curvesof AGPase from Arabidopsis leaves harvested at the end of the day(ƒ, E) and end of the night (�, F) assayed in presence of no (E, F)or 3 mM 3PGA (ƒ, �) and 1.5 mM G1P. C, Lineweaver-Burk presen-tation of the data in B. D, The activation factor by 3 mM 3PGAcompared with 0 mM 3PGA for the day (�) and the night sample (f).The data points in presence of 3 mM 3PGA and 5 mM ATP are omittedfrom C and D, because they showed substrate inhibition. Leaveswere taken from 8-week-old plants.

Figure 3. Darkening rapidly reverses the light-dependent monomer-ization of AGPB. A, Non-reducing western blot of leaf samples of6.5-week-old Arabidopsis harvested 6.5 h into the day, and afterdarkening the plants for 6, 15, and 60 min. B, Sugar content in theseleaves (f, Glc; , Fru; and , Suc).

Light Leads to Monomerization of AGPB in IsolatedPea Chloroplasts

To provide independent evidence that light pro-motes monomerization of AGPB protein, we investi-gated the responses in isolated chloroplasts. Chloro-plasts do not contain or synthesize Suc or othersugars. These experiments were carried out withchloroplasts from young pea plants. Pea chloroplastshave the advantage that it is possible to manipulatethe adenylate content. Addition of inorganic pyro-phosphate (PPi) leads to the loss of adenylates fromthe chloroplast, which can be reversed by addingATP or ADP (Lunn and Douce, 1993).

AGPB occurred almost exclusively as a dimerwhen chloroplasts were incubated in the dark withPPi, ATP, and 3PGA (Fig. 4A). A large proportionwas converted to monomer after 6 min of illumina-tion. This paralleled the increase of plastid FBPaseactivity (Fig. 4B). Addition of 30 mm Suc to isolatedchloroplasts did not lead to monomerization ofAGPB in the dark over a 15-min period (data notshown).

In vitro experiments with heterologously ex-pressed potato tuber AGPase have shown that mo-nomerization can be mediated by thioredoxins (Bal-licora et al., 2000). In many cases, substrate levelsmodulate the activation of thioredoxin-regulated en-zymes (Scheibe, 1991: Stitt, 1996; Schurmann andJacquot, 2000). Activation of several Calvin cycle en-zymes is promoted by high substrate concentrations,and activation of NADP-malate dehydrogenase by ahigh NADPH to NADP ratio. We investigated theeffect of illuminating pea chloroplasts in full mediumand in the absence of 3PGA or ATP on AGPB mono-merization. Appearance of the monomer was sup-pressed when 3PGA was omitted and was stimulatedwhen ATP was omitted (Fig. 4C). In contrast, activa-tion of plastidic FBPase was high in the absence of3PGA but decreased when ATP was omitted (Fig.4D). Activation of NADP-malate dehydrogenase wasrelatively low in full medium, increased when 3PGAwas omitted, and rose further when ATP was omit-ted (Fig. 4E). These results indicate that light-dependent monomerization of AGPB does not re-quire high levels of ATP or a high NADPH to NADPratio, but is promoted when metabolites, in particular3PGA, are high.

Supplying Suc to Leaves in the Dark Leads toConversion of AGPB from a Dimer to a Monomer and toIncreased Rates of Starch Synthesis

A second set of experiments was carried out toinvestigate whether sugars promote AGPB mono-merization. Leaves were harvested from Arabidopsisplants at the end of the normal day and supplied viatheir petiole with zero, 50, 100, or 200 mm Suc for 13 hin the dark (Fig. 5). AGPB was present almost exclu-sively as a dimer in leaf material at the end of the

Figure 4. Changes in dimerization of AGPB also occur in isolatedpea chloroplasts. A and B, Non-reducing western-blot (A) and FBPaseactivity (B) of chloroplasts incubated in the dark, and 3 and 6 minafter turning the lamp on in presence of 0.67 mM NaPPi, 1 mM 3PGA,and 1 mM ATP. C through E, Non-reducing western-blot (C), FBPase(D), and NADP-malate dehydrogenase activity (E) in chloroplasts inthe dark (f) and after 6 min in the light (�) in the presence of 0.67mM PPi, 1 mM 3PGA, and 1 mM ATP (all), or when either 3PGA orATP was left out of the incubation medium.

Hendriks et al.

night and in leaves incubated in the dark withoutsugars. Suc led to the appearance of monomer. Theproportion converted by Suc in the dark was similarto that seen in the light under normal growth condi-tions (Fig. 5A).

Feeding sugars led to a progressive increase in thelevels of sugar (Fig. 5B), but 3PGA remained unal-tered (Fig. 5C). There was also an increase of starch(Fig. 5D). This might be due to a stimulation of starchsynthesis or to slower breakdown of starch duringthe 13-h dark treatment. To measure the rate of starchsynthesis, the unlabeled Suc was spiked with highspecific activity [14C]Glc. The rate of starch synthesiswas calculated by dividing the label incorporatedinto starch by the specific activity of the hexose phos-phate pool (for a detailed discussion of this approach,see Geigenberger et al., 1997). Suc feeding led to aconcentration-dependent stimulation of starch syn-thesis in the dark (Fig. 5E).

Time-of-Day Dependent Changes in AGPBDimerization in the Starch-DeficientPhosphoglucomutase Mutant (pgm) ShowThat Light and Leaf Sugar Levels Interact toRegulate AGPB Activation

To provide further evidence that sugars increasemonomerization of AGPB, we carried out a set ofexperiments comparing diurnal changes in wild-typeColumbia-0 (Col0) and the pgm mutant (Caspar et al.,1986). The pgm mutant is deficient in starch synthesis,due to a loss-of-function mutation in a unique geneencoding plastid phosphoglucomutase (Kofler et al.,2000). In leaves of wild-type Col0, sugars rise to aplateau soon after illumination (Fig. 6, A–B, Fru notshown). Starch accumulates in a linear mannerthrough the light period and is degraded during thenight (Fig. 6C). In the pgm mutant (see also Caspar etal., 1986), large amounts of sugars accumulate in theleaf during the light period. They are depleted dur-ing the night, falling to levels at the end of the nightthat are lower than in wild-type Col0 (Fig. 6, A–B).

In the same samples, the proportion of AGPBpresent as monomer was determined on westernblots and quantified after scanning the films (Fig.6D). Typical examples of immunoblots are shown inFigure 6, E through G. In wild-type Col0, AGPB ispresent almost exclusively as dimer at the end of thenight, did not show a marked shift after 15 minillumination, was gradually converted to a monomeras the day progressed, and rapidly reverted to dimerafter darkening (see also Fig. 3). The response wasmarkedly changed in pgm. AGPB became partly mo-nomerized within 15 min after illumination. Mono-merization increased further during the next 2 to 3 hand by the second part of the light period AGPB wasalmost totally converted to monomer. 3PGA levelswere comparable with those in Col0 (data notshown). After darkening, a substantial proportion of

Figure 5. AGPB is converted into the monomeric form by supplyingsugars to leaves in the dark. Leaves from 8-week-old Arabidopsisplants were fed via their petioles with buffer and varying concentra-tions of Suc in the dark during their natural night (0, 50, 100, and 200mM). For comparison, leaves harvested from intact plants at the start(end of day [ED]) and at the end of the experiment (end of night [EN])are also shown. A, Non-reducing western blot of AGPB; B, sugarcontent (f, Glc; , Fru; and , Suc); C, 3PGA content; and D, starchcontent of the leaves. E, In parallel incubations, high specific[U-14C]Glc was supplied together with the various concentrations ofunlabeled Suc, to investigate the rate of starch synthesis.

AGPB remained as monomer for the first 2 h of thenight.

The data in Figure 6, A, B, and D, are replotted inFigure 7 to show the relation between monomeriza-tion of AGPB and total sugars. When extracts fromdarkened leaves are compared, there is a correlationbetween leaf sugar levels and the appearance ofAGPB monomer. Illumination leads to increased mo-nomerization at a given sugar content in the dark.

AGPase activity was measured with limiting ATP(0.2 mm) in the presence and absence of 1 mm 3PGAin extracts from wild-type Col0 harvested at the endof the night and pgm harvested at midday. Theserepresent the most extreme changes obtained in ourexperiments. The shift from dimer to monomer wasaccompanied by a 7-fold stimulation of activity (from25 to 176 nmol min�1 g�1 fresh weight) in the absenceof 3PGA, whereas activity was not affected in thepresence of 3PGA (583 and 559 nmol min�1 g�1 freshweight, respectively).

DISCUSSION

A consensus has developed that starch synthesis isregulated in response to changes of metabolism inthe cytosol (see introduction). When the rate oftriose-P use for the synthesis of Suc and other endproducts is lower than the rate of photosynthesis,falling Pi is proposed to lead to a restriction of ATPsynthesis and 3PGA reduction. The resulting increaseof the 3PGA to Pi ratio activates AGPase, leading toan increased rate of starch synthesis and increasedrecycling of Pi within the chloroplast.

Tiessen et al. (2002) recently discovered thatAGPase is subject to posttranslational regulation in

Figure 6. Monomerization of AGPB is increased in the starch-deficient pgm mutant. A to C, Glc (A), Suc (B), and starch content (C)in Arabidopsis leaves of 6-week-old Col0 (F) and 11-week-old pgm(E) during a night/day cycle (indicated by black and white bar abovethe figures). D, The reduction state of the AGPase antigen in the samesamples. E and F, Non-reducing western of leaves of Col0 (E) andpgm (F) at the change of light. G, Non-reducing western of pgmsamples harvested at the indicated times after the start of theillumination.

Figure 7. Relation between AGPB dimerization and the leaf sugarcontent in the light and dark. The data from Figure 6 were replottedshowing the relation between the AGPB dimerization state and thetotal sugar content (sum of Fru, Glc, and Suc) for Col0 (F, E) and pgm(�, ƒ) during the night (F, �) and the day (E, ƒ).

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potato tubers. The mechanism involves formation ofan inter-molecular Cys bridge between the AGPBsubunits in the AGPase heteroteramer. Rising Sucleads to monomerization and activation of AGPase,channeling carbon toward starch and away from res-piration when the Suc supply increases. The experi-ments in the present paper show that AGPase is alsosubject to posttranslational redox regulation in leavesof Arabidopsis, potato, and pea. As in potato tubers,this involves reversible interconversion between aless active form in which AGPB is present as a dimerand an active form in which AGPB is present asmonomers. To routinely monitor the dimerizationstate, we used an extreme extraction method inwhich trichloroacetic acid is used to rapidly dissoci-ate the protein and physically separate AGPB mono-mers to prevent post extracto formation of a intermo-lecular Cys bridge. This was essential, because theAGPB monomer is rapidly converted to a dimer inleaf extracts. This lability probably explains why thisimportant posttranslational mechanism was over-looked in earlier studies.

The shift from a dimer to a monomer is accompa-nied by an increase in leaf AGPase activity. Preissand coworkers have shown for heterogeneouslyoverexpressed potato tuber AGPase that DTT or thi-oredoxin lead to monomerization of AGPB and aconcomitant increase in AGPase activity (Fu et al.,1998; Ballicora et al., 2000). DTT also led to mono-merization of AGPB and an increase of AGPasaeactivity in potato tuber extracts (Tiessen et al., 2002).We have not yet been able to provide direct evidencethat the shift from dimer to monomer causes thechange in leaf AGPase activity, because DTT seri-ously interferes with AGPase assay in leaf extracts(data not shown). However, it is a reasonable as-sumption that the changes in monomerization andAGPase activity are causally related.

The increase of AGPase activity involved a changein the kinetic properties, including an increased af-finity for ATP and altered sensitivity to regulation by3PGA. The increase in activity is less marked thanTiessen et al. (2002) reported for potato tuberAGPase. This may be due to technical difficulties inretaining AGPB in the in planta status in leaf extracts(see above). Alternatively, it may reflect a real differ-ence in sensitivity between AGPase in differentplants or organs due, for example, to association witha different AGPS isoform. It will be necessary to carryout detailed studies with purified AGPase to resolvethis point.

At least two inputs modulate the posttranslationalredox-activation of AGPase in leaves. The first inputis a light-dependent signal. This is analogous to theway that several Calvin cycle enzymes and otherproteins involved in photosynthesis are regulated(Scheibe, 1991; Schurmann and Jacquot, 2000). Evi-dence for a light-dependent input is provided by twoindependent observations: AGPB monomerization

decreases rapidly after darkening wild-type leaveseven though sugar levels do not change and in-creases rapidly after illumination of isolated chloro-plasts. The second input is a sugar-related signal,which is analogous to the situation in potato tubers.Evidence is provided by two independent observa-tions: AGPase monomerization is increased by sup-plying exogenous sugars to wild-type leaf material inthe dark and is also increased in starch-deficient pgmmutants in the light and at the start of the night whenthis mutant contains higher levels of sugars thanwild-type plants. Comparison of the relation betweenlight, internal sugar levels, and AGPase monomer-ization in wild-type Col0 and the starch-deficientpgm mutant indicates that light and sugars act in anadditive manner to increase AGPase activation.

The reductive activation of heterologously ex-pressed AGPase can be mediated in vitro by thiore-doxin (Fu et al., 1998; Ballicora et al., 2000). Arabi-dopsis contains a family of thioredoxins, of whichseveral are targeted to the plastid. Whereas Calvincycle enzymes are activated by thioredoxin-f, othertargets including NADP-malate dehydrogenase,cfATPase, and Rubisco activase are regulated prefer-entially by thioredoxin-m. Further studies are neededto identify which thioredoxin interacts with AGPB.The light-dependent redox activation of AGPase canbe envisaged to be a direct result of increased reduc-tion of thioredoxin. It is however not yet clear howincreased levels of Suc modify this process (for dis-cussion, see Tiessen et al., 2002). Intriguingly, anti-sense inhibition of a SNF1-homolog strongly attenu-ates the reductive activation of AGPase after addingSuc in potato tubers (Tiessen et al., 2003). This im-plies that the transduction pathway that regulates thereductive activation of AGPase in plastids and theregulatory network that controls the expression andphosphorylation of cytosolic enzymes have somecommon components.

The light-dependent activation of photosyntheticenzymes by thioredoxin is modulated by metabolites,which modify the mid-redox potential of the Cys inthe target protein (Scheibe, 1991; Schurmann andJacquot, 2000). This provides an elegant mechanismto fine-tune the activity of enzymes at different sitesaround the Calvin cycle and poise ATP and NADPHlevels production (Stitt, 1996). Monomerization ofAGPase in isolated chloroplasts is promoted by3PGA but not by ATP. This indicates that reductiveactivation of AGPase may be promoted by high3PGA in leaves. This could provide a mechanism toprevent depletion of phosphorylated intermediatesdue to excessive posttranslational activation ofAGPase. It should, however, be noted that an in-crease of 3PGA level is not involved in the posttrans-lational regulation of AGPase in response to sugarsin leaves (see Figs. 4 and 5; also comments in thepreceding paragraph) or potato tubers (Tiessen et al.,2002).

Posttranslational regulation of AGPase allowsstarch synthesis to be modulated in response to lightor the accumulation of sugars, without any require-ment for changes in the levels of phosphorylatedintermediates or Pi. Maintenance of an appropriatebalance between phosphorylated intermediates andPi is of crucial importance during photosynthesis.Triose-P are exported from the chloroplast and con-verted into Suc in the cytosol, and the Pi that isreleased is recycled to the chloroplast to supportfurther photosynthesis. Excessive triose-P export willinhibit photosynthesis because it depletes the levelsof Calvin cycle intermediates and inhibits regenera-tion of the CO2 acceptor ribulose-1,5-bisphosphate,and inadequate triose-P export will inhibit photosyn-thesis because Pi is sequestered in phosphorylatedintermediates leading to depletion of free Pi and aninhibition of ATP synthesis (Edwards and Walker,1983). Because the exchange of triose-P and Pi via thetriose phosphate:phosphate translocator (Hausler etal., 2000) is a passive process, the rate of triose-Pexport depends on the rate of consumption inthe cytosol (Stitt et al., 1987; Stitt, 1996). Sophisticatedmechanisms act on the cFBPase and SPS to coordi-nate the rate of Suc synthesis with the rate ofphotosynthesis (Stitt et al., 1987; Stitt, 1996). Risinglevels of triose-P and 3PGA and falling Pi inhibitFru-6-phosphate,2-kinase and stimulate Fru-2,6-bisphosphatase. The resulting decrease of Fru-2,6-bisphosphate (Stitt et al., 1987; Villadsen and Nielsen,2001; Markham and Kruger, 2002) in combinationwith rising Fru-1,6-bisphosphate, stimulates cFBPaseactivity (Stitt et al., 1987; Stitt, 1997), leading to in-creased synthesis of hexose phosphates. A rising Glc-6-phosphate to Pi ratio activates SPS allosterically(Stitt et al., 1987) and modulates protein kinase andphosphatase activities resulting in posttranslationalactivation of SPS (Toroser et al., 2000; Winter andHuber, 2000). Feedback regulation of Suc synthesis(see introduction) in effect reverses this chain ofevents. This carries a concomitant risk that it mayinhibit photosynthesis. Posttranslational regulationof AGPase by light and leaf sugar levels will stabilizethe regulation network, because it allows partition-ing between Suc and starch to be altered without thisnecessarily requiring changes in the levels of phos-phorylated intermediates and Pi. As pointed out inthe introduction, there are several reports in the lit-erature in which starch synthesis changed indepen-dently of overall AGPase activity and the levels ofphosphorylated intermediates. They all involved ma-nipulations that alter sugar levels in the leaf and,according to the results in the present paper, willtherefore probably have led to posttranslational re-dox regulation of AGPase.

In conclusion, three mechanisms interact to regu-late AGPase activity in leaves. (a) Allosteric regula-tion allows instantaneous changes of AGPase activitywhen the 3PGA to Pi ratio changes. Although it may

in some conditions be part of a regulatory sequencethat links sugar accumulation to an increase of starchsynthesis, its main significance is more likely to be torapidly increase the recycling of Pi in the stromawhen there is a transient imbalance between photo-synthesis and triose P export. (b) Posttranslationalredox regulation provides a mechanism that allowsdirect light activation of starch synthesis in leavesand also allows starch synthesis to be increased whensugars accumulate in the leaf. Crucially, this mecha-nism allows starch synthesis to be increased withoutan increase of the 3PGA to Pi ratio as a necessaryintervening step. This will increase the flexibility ofthe regulatory network, because it allows photosyn-thetic carbon allocation to be regulated indepen-dently of the poising of intermediary photosyntheticmetabolism. (c) Expression of AGPB and AGPS isincreased by sugars (Salanoubat and Belliard, 1989;Muller-Rober et al., 1990; Sokolov et al., 1998) anddecreased by nitrate (Scheible et al., 1997) and phos-phate (Nielsen et al., 1998). Transcriptional controlmay operate mainly to allow starch accumulation torespond to sustained changes in the carbon or nutri-ent status of the plant.

MATERIALS AND METHODS

All experiments were reproduced at least once with independent biolog-ical material. Data points are at least the average of duplicate measurementsof the same biological sample. When error bars are shown, they representthe sd of the average of the measurements on at least two biological samplesof the same experiment.

Plant Growth

Pea (Pisum sativum cv Marcia) was grown either in a greenhouse with a16-h day of 180 �E, 21°C/19°C (day/night), and 50% humidity or in ahigh-light phytotron with a 14-h day, 20°C/16°C, and 60%/75% humidity.The pea cv Kelvedon Wonder was grown in a short-day phytotron (8-h dayof 180 �E, 20°C/16°C, and 60%/75% humidity day/night). Arabidopsis varCol0, wild type, and a plastidic pgm (Caspar et al., 1986) were grown in thesame short-day phytotron. At least 2 weeks before their use, the plants weretransferred into a small growth cabinet with a 10-h day of 160 �E and 20°Cthroughout the day/night cycle. Potato (Solanum tuberosum cv Desiree)plants were grown in a greenhouse at 400 �E, 20°C/16°C day/night, and50% humidity throughout.

Harvesting Procedure, Sample Storage

Leaves were harvested while leaving the plants in place. Only sourceleaves that were not shaded by other leaves were selected. The leaves wereput directly into liquid nitrogen, and stored at �80°C until use.

Incubation of Leaves with Sugars in the Dark

At the end of the light period, plants were taken from the growth cabinet.Non-shaded source leaves were cut, and their petioles were recut underbuffer solution. The recut petioles were inserted into the feeding solution,containing 2 mm MES, pH 6.5, and varying concentrations of Suc. The leaveswere returned to the growth cabinet and incubated there during the night.At the end of the night, leaves were frozen immediately in liquid nitrogen,after excising that part of the petiole, which had been immersed in thefeeding solution.

Hendriks et al.

Chloroplast Preparation

Ten- to 16-d-old pea seedling were subjected to an extended night by 5 hto deplete the internal starch pools and subsequently were transferred tolight for about 30 min to induce photosynthesis. Chloroplasts were thenprepared essentially as described by Lunn et al. (1990), but using 10 mmMES, pH 6.5, as buffer in the blending medium. The chlorophyll content ofthe final preparation was determined in MeOH extracts (Porra et al., 1989).

Incubations of Chloroplasts, Measurement ofPhotosynthesis

Oxygen evolution was measured in an oxygen electrode at 25°C on achloroplasts suspension of 50 �g chlorophyll mL�1 in resuspension buffercontaining 4 mm HCO3 and additives as indicated in the figure legends. Thecuvette was darkened for 5 min before the dark sample was taken. Afterrestabilization of the evolution trace, the sample was illuminated using thebeam of a slide projector.

Extraction of AGPase for Blotting, Procedures for Gels

Frozen leaf material was homogenized using a liquid nitrogen cooledball-mill, and 50 mg of leaf material was extracted in cold 16% (w/v) TCAin diethyl ether, mixed, and stored at �20°C for at least 2 h. The pellet wascollected by centrifugation at 13,000 rpm for 5 min at 4°C. The pellet waswashed three times with ice-cold acetone, dried briefly under vacuum, andresuspended in 1� Laemmli sample buffer containing no reductant (Lae-mmli, 1970). After heating the sample for 3 min at 95°C, the insolublematerial was settled by a 1-min spin, and the supernatant was used for gelelectrophoresis on 10% (w/v) acrylamide gels in presence of SDS. Proteinscoming from 0.5 or 1 mg of fresh weight were loaded per small or broadlane, respectively. The gels were blotted onto polyvinylidene difluorideaccording to standard procedures. AGPase antigen was detected using aprimary rabbit antibody raised against the His-tagged AGPB of potato(Tiessen et al., 2002) and a peroxidase-conjugated secondary goat anti-rabbitantibody (Bio-Rad Laboratories, Hercules, CA). The peroxidase was de-tected on film using the ECL kit of Amersham Biosciences (Uppsala). Toquantify the amount of AGPase present as monomer, the films were scannedwith standardized settings, saved as tif files, and analyzed with Tina 2.10isoftware (Raytest Isotopenmessgerate GmbH, Straubenhardt, Germany).Gel samples from chloroplasts were prepared by mixing 1 volume of chlo-roplasts, taken directly from the oxygen electrode, with 1 volume of 2�Laemmli sample buffer without reductant. The samples were heated at 95°Cfor 3 min and stored at room temperature until use. Proteins coming from6.25 �g of chlorophyll were loaded per lane. Electrophoresis, blotting, andimmunolabeling procedures were as described above. For detection of theperoxidase, the ECL or ECL advance kit was used (Amersham Biosciences).

Extraction and Assay of AGPase

Activity measurements were performed essentially as described (Tiessenet al., 2002): Fifty milligrams of material was extracted with 0.5 mL ofextraction buffer (50 mm K-HEPES, pH 7.5, 5 mm MgCl2, 1 mm EGTA, 1 mmEDTA, 1 mm benzamidine, and 1 mm �-aminocaproic acid). The sample wascentrifuged for 30 s at 4°C. The supernatant was used directly at 1/10 or1/20 of the volume in the activity assay containing 50 mm K-HEPES, pH 7.5,5 mm MgCl2, 1.5 mm G1P, and varying amounts of ATP and 3PGA. After 10min at 30°C, the reaction was stopped by boiling for 5 min. After a 5-mincentrifugation, the supernatant was stored at 4°C or �80°C until the ADP-Glc content was determined by HPLC as described (Tiessen et al., 2002).

Extraction and Assay of FBPase and NADPMalate Dehydrogenase

FBPase and NADP-malate dehydrogenase activities in chloroplasts weremeasured by mixing 20 �L of the chloroplasts solution from the oxygenelectrode with 180 �L of reaction mixture containing 50 mm K-Tricine, pH8.0, 5 mm MgCl2, and 0.1% (v/v) Triton X-100. For FBPase, the mixtureadditionally contained 0.1 mm NADP�, 40 �m Fru-1,6-bisphosphate, and1.75 mm EDTA. The reaction was stopped by the addition of 20 �L of 1 m

NaOH either directly or after a 3- or 10-min incubation at room temperature.The reaction mix for NADP-malate dehydrogenase assays instead contained0.1 mm NADPH, and 0 or 2 mm oxaloacetate additionally. It was stoppedafter 10 min by addition of 20 �L 1 m HCl/0.1 m Tricine, pH 9. In both cases,the difference in NADP(H) content of the two samples was taken as ameasure for enzyme activity. The samples were stored at 4°C until furtherprocessing. Heating of the samples for 5 min at 95°C ensured the completedisrupture of all unused nucleotide-adenine substrate (NADP or NADPH).Five or 10 �L of the reaction was brought to pH 9 by the addition of 25 mmHCl/50 mm Tricine, pH 9, for FBPase or 0.1 m NaOH for MDH. TheNADP(H) content was determined directly after the pH adjustment by anenzymatic cycling assay (Gibon et al., 2002). Both assays were shown to belinear with time for over 10 min.

Extraction and Assay of Suc, Reducing Sugars, Starch,Hexose Phosphates, and 3PGA

Suc, Glc, Fru, and starch were determined in ethanol extracts as describedby Geigenberger et al. (1996), and hexose phosphates as by Gibon et al.(2002). For starch determination, the pellets of the ethanol extraction weresolubilized by heating them to 95°C in 0.1 m NaOH for 30 min. Afteracidification to pH 4.9 with an HCl/sodium-acetate, pH 4.9, mixture, part ofthe suspension was digested overnight with amyloglucosidase and�-amylase. The Glc content of the supernatant was then used to assess thestarch content of the sample. 3PGA was determined in perchloric acidextracts using an enzymatic cycling assay (Gibon et al., 2002).

Labeling Experiments and Label Separation

Labeling experiments were carried out with whole Arabidopsis leavescut directly from the plant, with ends of petioles re-cut under water. Leaveswere incubated in the dark for 13 h at 20°C (humidity of 60%) in mediumcontaining 2 mm MES-KOH (pH 6.5) and 0.66 mm or 0.33 mm [U-14C]Glc(specific activity, 111 mBq mm�1; Amersham-Buchler, Braunschweig, Ger-many) together with various concentrations of Suc (see legends to figuresfor details). Incubations were done in petri dishes (5-mL volume). Wet endsof petioles of incubated leaves were cut and discarded, and leaves werefrozen immediately in liquid nitrogen. After ethanol extraction, the solublefraction was further separated into neutral, anionic, and cationic compo-nents by ion-exchange chromatography as by Geigenberger et al. (1997), andthe insoluble material left after ethanol extraction was resuspended anddigested overnight as described above and counted for starch. Label in thehexose phosphate pool was analyzed as by Geigenberger et al. (1997), andtotal carbon in the hexose phosphate pool was determined in ethanolextracts as described above using non-radioactive replicates incubated inparallel.

ACKNOWLEDGMENTS

We are grateful to Axel Tiessen for doing preliminary experiments, forvaluable discussions, and for preparation of the His-tagged AGPB proteinused for the rabbit immunization; to John Lunn for his advice concerningthe chloroplast work; to Christian Scherling for help with sample analysis;and to Sam Zeeman (Bern, Switzerland) for providing the pgm mutant.

Received April 1, 2003; returned for revision May 9, 2003; accepted July 10,2003.

LITERATURE CITED

Ballicora MA, Frueauf JB, Fu Y, Schurmann P, Preiss J (2000) Activation ofthe potato tuber ADP glucose pyrophosphorylase by thioredoxin. J BiolChem 275: 1315–1320

Baxter CJ, Foyer CH, Rolfe SA, Quick WP (2001) A comparison of thecarbohydrate composition and kinetic properties of sucrose phosphatesynthase (SPS) in transgenic tobacco (Nicotiana tabacum) leaves express-ing maize SPS protein with untransformed controls. Ann Appl Biol 138:47–55

Burton RA, Johnson PE, Beckles DM, Fincher GB, Jenner HL, Naldrett MJ,Denyer K (2002)Characterisation of the genes encoding the cytosolic and

plastidial forms of ADP-glucose pyrophosphorylase in wheat en-dosperm. Plant Physiol 130: 1464–1475

Caspar T, Huber SC, Somerville C (1986) Alterations in growth, photosyn-thesis and respiration in a starch deficient mutant of Arabidopsis thaliana(L.) Heynh deficient in chloroplast phosphoglucomutase activity. PlantPhysiol 79: 1–7

Castresana J (2000) Selection of conserved blocks from multiple alignmentsfor their use in phylogenetic analysis. Mol Biol Evol 17: 540–552

Draborg H, Villadsen D, Nielsen TH (2001) Transgenic Arabidopsis plantswith decreased activity of fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning. Plant Physiol 126:750–758

Edwards G, Walker DA (1983) C3, C4, Mechanisms, and Cellular andEnvironmental Regulation of Photosynthesis. Blackwell Scientific,Oxford

Fu Y, Ballicora MA, Leykam JF, Preiss J (1998) Mechanism of reductiveactivation of potato tuber ADP-glucose pyrophosphorylase. J Biol Chem273: 25045–25052

Geigenberger P, Lerchl J, Stitt M, Sonnewald U (1996) Phloem-specificexpression of pyrophosphatase inhibits long distance transport of carbo-hydrates and amino acids in tobacco plants. Plant Cell Environ 19: 43–55

Geigenberger P, Reimholz R, Geiger M, Merlo L, Canale V, Stitt M (1997)Regulation of sucrose and starch metabolism in potato tubers in responseto short-term water deficit. Planta 201: 502–518

Geigenberger P, Stitt M (2000) Diurnal changes in sucrose, nucleotides,starch synthesis and AGPS transcript in growing potato tubers that aresuppressed by decreased expression of sucrose phosphate synthase. PlantJ 23: 795–806

Geiger DR, Servaites JC (1994) Diurnal regulation of photosynthetic carbonmetabolism in C3 plants. Annu Rev Plant Phys Plant Mol Biol 45: 235–256

Geiger DR, Shieh WJ, Yu X-M (1995) Photosynthetic carbon metabolismand translocation in wild-type and starch-deficient mutant Nicotianasylvestris L. Plant Physiol 107: 507–514

Gerhardt R, Stitt M, Heldt HW (1987) Subcellular metabolite levels inspinach leaves: regulation of sucrose synthesis during diurnal alterationsin photosynthesis. Plant Physiol 83: 399–407

Gibon Y, Vigeolas H, Tiessen A, Geigenberger P, Stitt M (2002) Sensitiveand high throughput metabolic assays fro inorganic pyrophosphate,ADPglc, nucleotide phosphates, and glycolytic intermediates based on anovel enzymic cycling system. Plant J 30: 221–235

Goldschmidt EE, Huber SC (1992) Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, andhexose sugars. Plant Physiol 99: 1443–1448

Hausler RE, Schlieben NH, Flugge UI (2000) Control of carbon partitioningand photosynthesis by the triose phosphate/phosphate translocator intransgenic tobacco plants (Nicotiana tabacum): II. Assessment of controlcoefficients of the triose phosphate/phosphate translocator. Planta 210:383–390

Heldt HW, Chon CJ, Maronde D, Herold A, Stankovic ZS, Walker DA,Kraminer A, Kirk MR, Heber U (1977) Role of orthophosphate and otherfactors in the regulation of starch formation in leaves and isolated chlo-roplast. Plant Physiol 59: 1146–1155

Herold A (1980) Regulation of photosynthesis by sink activity: the missinglink. New Phytol 86: 131–144

Huber SC (1989) Biochemical mechanism for regulation of sucrose accumu-lation in leaves during photosynthesis. Plant Physiol 91: 656–662

Kofler H, Hausler RE, Schulz B, Groner F, Flugge UI, Weber A (2000)Molecular characterisation of a new mutant allele of the plastid phos-phoglucomutase in Arabidopsis, and complementation of the mutantwith the wild-type cDNA. Mol Gen Genet 263: 978–986

Krapp A, Quick WP, Stitt M (1991) Rubisco, other Calvin cycle enzymesand chlorophyll decrease when glucose is supplied to mature spinachleaves via the transpiration stream. Planta 186: 58–69

Krapp A, Stitt M (1995) An evaluation of direct and indirect mechanisms forthe “sink regulation” of photosynthesis of spinach: changes in gas ex-change, carbohydrates, metabolites, enzyme activities and steady-statetranscript levels after cold girdling of source leaves. Planta 195: 313–323

Laemmli UK (1970) Cleavage of structural proteins during assembly of thehead of bacteriophage T4. Nature 227: 680–685

Laporte MM, Galagan JA, Prasch AL, Vanderveer PJ, Hanson DT, Shew-maker CK, Sharkey TD (2001) Promoter strength and tissue specificityeffects on growth of tomato plants transformed with maize sucrose-phosphate synthase. Planta 212: 817–822

Lunn JE, Douce R (1993) Transport of inorganic pyrophosphate across thespinach chloroplast envelope. Biochem J 290: 375–379

Lunn JE, Droux M, Martin J, Douce R (1990) Localisation of ATP sulfury-lase and O-acetylserine(thiol)lyase in spinach leaves. Plant Physiol 94:1345–1352

Markham JE, Kruger NJ (2002) Kinetic properties of bifunctional6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase from spinachleaves. Eur J Biochem 269: 1267–1277

Martin C, Smith AM (1995) Starch biosynthesis. Plant Cell 7: 971–985Morell MK, Bloom M, Knowles V, Preiss J (1987) Subunit structure of

spinach leaf ADPglucose pyrophosphorylase. Plant Physiol 85: 182–187Muller-Rober BT, Kossmann J, Hannah LC, Willmitzer L, Sonnewald U

(1990) One of two different ADP-glucose pyrophosphorylase genes frompotato responds strongly to elevated levels of sucrose. Mol Gen Genet224: 136–146

Neuhaus H, Stitt M (1990) Control analysis of photosynthate partitioning:impact of reduced activity of ADP-glucose pyrophosphorylase or plastidphosphoglucomutase on the fluxes to starch and sucrose in Arabidopsisthaliana L. Heynh. Planta 182: 445–454

Neuhaus HE, Kruckeberg AL, Feil R, Gottlieb L, Stitt M (1989) Dosagemutants of phosphoglucose isomerase in the cytosol and chloroplasts ofClarkia xantiana: II. Study of the mechanisms which regulate photosyn-thate partitioning. Planta 178: 110–122

Neuhaus HE, Quick WP, Siegl G, Stitt M (1990) Control of photosynthatepartitioning in spinach leaves: analysis of the interaction between feed-forward and feedback regulation of sucrose synthesis. Planta 181:583–592

Nielsen TH, Krapp A, Roper-Schwarz U, Stitt M (1998) The sugar-mediated regulation of genes encoding the small subunit of Rubisco andthe regulatory subunit of ADP glucose pyrophosphorylase is modified bynitrogen and phosphate. Plant Cell Environ 21: 443–455

Okita TW, Nakata PA, Anderson JM, Sowokinos J, Morell M, Preiss J(1990) The subunit structure of potato tuber ADPglucose pyrophospho-rylase. Plant Physiol 93: 785–790

Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurateextinction coefficients and simultaneous equations for assaying chloro-phylls a and b extracted with four different solvents: verification of theconcentration of chlorophyll standards by atomic absorption spectros-copy. Biochim Biophys Acta 975: 384–394

Preiss J (1988) Biosynthesis of starch and its regulation. In J Preiss, ed, TheBiochemistry of Plants, Vol 14. Academic Press, San Diego, pp 181–254

Salanoubat M, Belliard G (1989) The steady-state level of potato sucrosesynthase mRNA is dependent on wounding, anaerobiosis and sucrose.Gene 84: 181–185

Scheibe R (1991) Redox-modulation of chloroplast enzymes: a commonprinciple for individual control. Plant Physiol 96: 1–3

Scheible WR, Gonzalez-Fontes A, Lauerer M, Muller-Rober B, CabocheM, Stitt M (1997) Nitrate acts as a signal to induce organic acid metab-olism and repress starch metabolism in tobacco. Plant Cell 9: 783–798

Schulze W, Stitt M, Schulze ED, Neuhaus HE, Fichtner K (1991) A quan-tification of the significance of assimilatory starch for growth of Arabi-dopsis thaliana L. Heynh. Plant Physiol 95: 890–895

Schurmann P, Jacquot J-P (2000) Plant thioredoxin systems revisited. AnnuRev Plant Physiol Plant Mol Biol 51: 371–400

Scott P, Lange AJ, Kruger NJ (2000) Photosynthetic carbon metabolism inleaves of transgenic tobacco (Nicotiana tabacum L.) containing decreasedamounts of fructose 2,6-bisphosphate. Planta 211: 864–873

Sikka VK, Choi SB, Kavakli IH, Sakulsingharoj C, Gupta S, Hioyuki I,Okita TW (2001) Subcellular compartmentation and allosteric regulationof the rice endosperm ADPglucose pyrophosphorylase. Plant Sci 161:461–468

Sokolov LN, Dejardin A, Kleczkowski LA (1998) Sugars and light/darkexposure trigger differential regulation of ADP-glucose pyrophosphory-lase genes in Arabidopsis thaliana (thale cress). Biochem J 336: 681–687

Sowokinos JR (1981) Pyrophosphorylases in Solanum tuberosum: II. Cata-lytic properties and regulation of ADP-glucose and UDP-glucose pyro-phosphorylase activities in potatoes. Plant Physiol 68: 924–929

Sowokinos JR, Preiss J (1982) Pyrophosphorylases in Solanum tuberosum III:purification, physical, and catalytic properties of ADPglucose pyrophos-phoryase in potatoes. Plant Physiol 69: 1459–1466

Stitt M (1996) Metabolic regulation of photosynthesis. In N Baker, ed,Advances in Photosynthesis, Vol 3: Environmental Stress and Photosyn-thesis. Academic Press, New York, pp 151–190

Hendriks et al.

Stitt M, Grosse H (1988) Interactions between sucrose synthesis and CO2

fixation 4: temperature-dependent adjustment of the relation betweensucrose synthesis and CO2 fixation. J Plant Physiol 133: 392–400

Stitt M, Huber S, Kerr P (1987) Control of photosynthetic sucrose synthesis.In MD Hatch, NK Boardman, eds, The Biochemistry of Plants, Vol 10.Academic Press, New York, pp 327–409

Stitt M, Wilke I, Feil R, Heldt HW (1988) Coarse control of sucrosephosphate synthase in leaves: alterations in the kinetic properties inresponse to the rate of photosynthesis and the accumulation of sucrose.Planta 174: 217–230

Strand A, Zrenner R, Stitt M, Gardestrom P (2000) Antisense inhibition oftwo key enzymes in the sucrose biosynthesis pathway, cytosolic fruc-tosebisphosphatase and sucrose phosphate synthase, has different con-sequences for photosynthetic carbon metabolism in transgenic Arabidop-sis thaliana. Plant J 23: 751–770

Sun JD, Gibson KM, Kiirats O, Okita TW, Edwards GE (2002) Interactionsof nitrate and CO2 enrichment on growth, carbohydrates, and Rubisco inArabidopsis starch mutants. Plant Physiol 130: 1573–1583

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997)The CLUSTAL_X windows interface: flexible strategies for multiple se-quence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM,Geigenberger P (2002) Starch synthesis in potato tubers is regulated bypost-translational redox-modification of ADP-glucose pyrophosphory-

lase: a novel regulatory mechanism linking starch synthesis to the sucrosesupply. Plant Cell 14: 2191–2213

Tiessen A, Prescha K, Branscheid A, Palacios N, McKibbin R, HalfordNG, Geigenberger P (2003) Evidence that SNF1-related kinase and hex-okinase are involved in separate sugar-signalling pathways modulatingpost-translational redox activation of ADP-glucose pyrophosphorylase inpotato tubers. Plant J 35: 490–500

Toroser D, Plaut Z, Huber SC (2000) Regulation of a plant SNF1-relatedprotein kinase by glucose-6-phosphate. Plant Physiol 123: 403–411

Villadsen D, Nielsen TH (2001) N-terminal truncation affects the kineticsand structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana. Biochem J 359: 591–597

Westram A, Lloyd JR, Roessner U, Riesmeier JW, Kossmann J (2002)Increases of 3-phosphoglyceric acid in potato plants through antisensereduction of cytoplasmic phosphoglycerate mutase impairs photosynthe-sis and growth, but does not increase starch contents. Plant Cell Environ25: 1133–1143

Winter H, Huber SC (2000) Regulation of sucrose metabolism in higherplants: localization and regulation of activity of key enzymes. Crit RevBiochem Mol Biol 35: 253–289

Zrenner R, Krause KP, Apel P, Sonnewald U (1996) Reduction of thecytosolic fructose-1,6-bisphosphatase in transgenic potato plants limitsphotosynthetic sucrose biosynthesis with no impact on plant growth andtuber yield. Plant J 9: 671–687

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