John E Lunn
email: lunn@mpimp-golm.mpg.de

Regina Feil

Janneke Hm Hendriks

Yves Gibon

Rosa Morcuende

Daniel Osuna

Wolf-Rüdiger Scheible

Petronia Carillo

Mohammad-Reza Hajirezaei

Mark Stitt

Janneke H M Hendriks

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Keywords: liquid chromatography-mass spectrometry, redox regulation, starch, sucrose, trehalose 6-phosphate ADPGlc, ADPglucose, AGPase, ADPglucose pyrophosphorylase, LC, liquid chromatography, MS-Q3, triple quadrupole mass spectrometry, 3PGA, 3-phosphoglycerate, Pi, inorganic orthophosphate, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SPP, sucrose-phosphatase, SPS, sucrose-phosphate synthase, Suc6P, sucrose-6 Fphosphate, TPP, trehalose-phosphatase, TPS, trehalose-phosphate synthase, Tre6P, trehalose 6-phosphate, UDPGlc, UDPglucose

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Trehalose 6-phosphate (Tre6P) is the intermediate in the classical pathway of trehalose biosynthesis, via trehalose-phosphate synthase (TPS) and trehalose-phosphatase (TPP), in bacteria, fungi, invertebrates and plants [START_REF] Cabib | The biosynthesis of trehalose phosphate[END_REF]. In yeast (Saccharomyces cerevisiae), Tre6P is also an essential signalling molecule, involved in regulating the entry of glucose and fructose into glycolysis via inhibition of hexokinase [START_REF] Blázquez | Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases[END_REF]. With the exception of a few "resurrection" plants that use trehalose to help them survive extreme desiccation [START_REF] Iturriaga | New desiccation-tolerant plants, including a grass, in the central highlands of Mexico, accumulate trehalose[END_REF], most Angiosperms, including Arabidopsis thaliana, contain only vanishingly small amounts of trehalose. However, evidence is accumulating that Tre6P has an essential signalling function in plants. Disruption of the TPS1 gene in A. thaliana was found to be embryo lethal, with growth being arrested at the torpedo stage of embryo development [START_REF] Eastmond | Trehalose-6phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation[END_REF]. Although the tps1 mutant can be rescued through embryogenesis by inducible expression of TPS, the resulting plants show retarded vegetative growth and are unable to flower when TPS expression is no longer induced [START_REF] Van Dijken | Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering[END_REF]. Plants expressing TPS or TPP genes from yeast or bacteria also show complex morphological and biochemical phenotypes [START_REF] Romero | Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance[END_REF][START_REF] Goddijn | Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants[END_REF][8][START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF][START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF]. The contrasting phenotypic effects of TPS and TPP overexpression in A. thaliana [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF] indicated that changes in the level of Tre6P, rather than trehalose, were responsible for the altered phenotypes of the plants. This was corroborated by the phenotypic similarity of plants expressing TPP or phosphotrehalase, an enzyme that cleaves Tre6P to Glc6P and glucose without producing trehalose [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF].

It is clear that Tre6P plays a vital role in the life of plants, but the exact nature of this role is unknown, as the complex pleiotropic phenotypes of plants with altered TPS or TPP activity make it difficult to distinguish between primary and secondary responses.

Nevertheless, several lines of evidence point to a link between Tre6P and signalling of nutrient status, in particular sugars, in plants. For example, in A. thaliana, the transcript abundances of several TPS and TPP genes respond strongly to changes in sugar levels [START_REF] Scheible | Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen[END_REF][START_REF] Thimm | MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes[END_REF][START_REF] Price | Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis[END_REF][14]. Expression of some of these genes is also affected by nitrate starvation and resupply [15]. Growth of tps1 embryos in culture can be partially rescued by reducing the sucrose concentration in the medium [START_REF] Eastmond | Trehalose-6phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation[END_REF], while constitutive overexpression of heterologous TPS [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF] or AtTPS1 [START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF] overcomes inhibition of germination by high glucose or ABA, and promotes growth on high sucrose [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF]14]. In contrast, transformants with constitutive overexpression of TPP show reduced growth on high sugar [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF]14]. Furthermore, overexpression of AtTPS1 [START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF] or addition of trehalose [14] affects the expression of genes that respond to other sugars. The A. thaliana genome encodes 11 TPS proteins. Several of these are targets for phosphorylation by SNF1-related or calcium-dependent protein kinases and binding of 14-3-3 proteins, and these processes are influenced by sugar status [17,[START_REF] Glinski | Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana -a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis[END_REF]. Taken together, these observations indicate that the level of Tre6P in plants is likely to be influenced by sugar-dependent transcriptional and post-translational regulation of TPS and TPP.

Little is known about the downstream targets of Tre6P signalling in plants. Unlike some hexokinases from yeast and other fungi, HXK1 and HXK2 from A. thaliana were found to be insensitive to Tre6P [START_REF] Gonzali | Arabidopsis (HXK1 and HXK2) and yeast (HXK2) hexokinases overexpressed in transgeic lines are characterized by different catalytic properties[END_REF]. However, Tre6P has recently been implicated in the redox regulation of ADPglucose pyrophosphorylase (AGPase), which plays a major role in control of flux through the pathway of starch synthesis [START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF]. Redox regulation of AGPase involves reversible formation of an intermolecular disulphide bridge between the two small subunits of the heterotetrameric holoenzyme [START_REF] Fu | Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase[END_REF]. The reduced, monomeric form has higher affinities for its substrates, and increased sensitivity to allosteric activation by 3PGA, than the oxidized, dimeric form [21]. Monomerisation is triggered by sugars in potato tubers [21,[START_REF] Tiessen | 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[END_REF], and by light or sugars in potato, pea and A. thaliana leaves [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF].

Several lines of evidence suggest a role for Tre6P in the signaling pathway that mediates the sucrose-dependent redox activation of AGPase [START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF]. First, feeding of 100 mM trehalose, which inhibits TPP, to potato tuber discs leads to an SNRK1-dependent activation of AGPase, similar to that seen with sucrose. Secondly, AGPase activation is increased in A. thaliana plants with constitutive overexpression of the Escherichia coli TPS, but decreased in TPP-overexpressors. Finally, addition of 100 µM Tre6P to intact pea leaf chloroplasts promotes activation of AGPase. It was proposed that rising sucrose in the cytosol leads to increased synthesis of Tre6P, which either enters the plastid or binds to some factor in the chloroplast envelope to trigger redox activation of AGPase [START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF]. Although such a signalling pathway would provide a simple explanation for sucrose-induced activation of AGPase, as yet we have no direct evidence that changes in sugar levels lead to changes in Tre6P, or that these are accompanied by redox modulation of AGPase.

One of the greatest obstacles to discovering the exact roles of Tre6P in plants is the lack of a reliable Tre6P assay that is sensitive and specific enough to measure this metabolite in plant tissues. None of the currently available methods for assaying Tre6P is really sensitive enough to reliably measure the small amounts of Tre6P in plant cells, which appear to be several orders of magnitude lower than in yeast cells [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF]14,[START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF][START_REF] Van Vaeck | Analysis and modification of trehalose 6-phosphate levels in the yeast Saccharomyces cerevisiae with the use of Bacillus subtilis phosphotrehalase[END_REF][START_REF] Blázquez | Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate[END_REF].

The available methods are also prone to interference by other metabolites in plant extracts, and need tedious sample preparation, limiting the number of samples that can be analyzed. In the present paper we describe a simple extraction procedure for Tre6P, and the development and validation of a highly specific assay for this metabolite using liquid chromatography (LC) coupled to triple quadrupole mass spectrometry (MS-Q3). The new assay is up to 100-fold more sensitive than previously used methods, and allowed us to investigate how the level of Tre6P in A. thaliana responds to alterations in the sugar status of the plants, and to directly compare the levels of Tre6P with changes in the posttranslational redox activation of AGPase. (from E. coli), in 25 mM Hepes-Na + , 5 mM MgCl 2 , 0.5 mM EDTA, pH 7.2, in a final volume of 100 µl. After incubation at 30°C for 3 h, the reaction was stopped by heating at 99°C for 2 min, and precipitated proteins were removed by centrifugation at 18,000 g for the manufacturer's instructions. The enzyme was further purified by size exclusion chromatography on a HiLoad 16/60 Superdex 200 gel filtration column (Amersham Biosciences Europe GmbH, Freiburg, Germany), equilibrated with buffer B (50 mM Hepes-Na + , 150 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA, pH 7.5). The flow rate was 2 ml min -1 , and 2-ml fractions were collected. TPS peak fractions were pooled, concentrated to a volume of approx. 100 µl using a Centricon Plus-20 (30,000 cutoff) centrifugal concentrator (Millipore, Billerica, MA, USA) and stored at -80°C. TPS activity was assayed as described in [29] for sucrose-phosphate synthase, except Fru6P was replaced by 10 mM Glc6P. One unit of activity represents 1 µmol UDP produced per minute.

Experimental

TPP was extracted and purified as described above for TPS. Superdex 200fractions containing TPP were pooled, diluted five-fold with buffer C (50 mM Hepes-Na + , 5 mM MgCl 2 , 0.5 mM EDTA, pH 7.5) and applied to an HR 5/5 MonoQ® column (Amersham Biosciences), equilibrated with buffer D, at a flow rate of 1 ml min -1 . TPP was eluted with a linear gradient of 0-0.5 M NaCl (30 ml) in buffer C and 0.5-ml fractions were collected. Samples (1 µl) were analysed by SDS-PAGE on 12 % (w/v) polyacrylamide gels, and fractions containing a single Coomassie-staining protein band (32 kDa) were pooled, concentrated to a volume of approx. 100 µl using a Centricon Plus-20 (10,000 cutoff) centrifugal concentrator, and stored at -80°C. TPP activity was assayed colorimetrically by measuring Pi production as described in [30]. One unit of activity represents 1 µmol Pi produced per min.

Phosphotrehalase was over-expressed in E. coli strain DH5 and purified as described in [28]. The -glucosidase activity of the enzyme was assayed with pnitrophenyl--D-glucopyranoside as substrate [28] during the purification. The purified enzyme was stored as a suspension in 80% saturated (NH 4 ) 2 SO 4 at 4°C. The Synechocystis sp. PCC 6803 SPP was expressed in E. coli and purified as described in [31].

Assay of Tre6P standards

Stock solutions of Tre6P were prepared in ultra-pure water (Elga Labwater, Celle, Germany) from unopened bottles of the dipotassium salt of Tre6P (approx. 95% pure, containing 0.2% methanol). The nominal concentration was calculated from the amount of Tre6P weighed, without correction for the indicated purity or solvent content. The actual concentration of Tre6P was determined spectrophotometrically using phosphotrehalase. The reaction contained: 100 mM Tricine-Na + , pH 8.0, 5 mM MgCl 2 , 0.25 mM NADP + , 0.2 U glucose-6-phosphate dehydrogenase and 0-10 nmol Tre6P, in a final volume of 1 ml, and was started by addition of 2.5 mU of phosphotrehalase (dissolved in 100 mM Tricine-Na + , 5 mM MgCl 2 , 1 mM DTT, pH 8.0). Glc6P production was calculated from the increase in absorbance at 334 nm ( = 6180 M -1 cm -1 ), measured using a ZFP 22 dual wavelength photometer (Sigma Instruments GmbH, Berlin, Germany).

The concentration of Tre6P in standard solutions was also assayed by measuring the release of Pi after complete hydrolysis by TPP. The reaction contained: 25 mM Hepes-Na + , pH 7.2, 5 mM MgCl 2 , 0.5 mM EDTA, 0-10 nmol Tre6P and 0.15 U TPP in a final volume of 100 µl. After incubation at 30°C for 10 min the amount of Pi released was assayed as described in [30].

Extraction of Tre6P

Tre6P was extracted from plant tissues by a modification of the method in [32]. Frozen plant material was ground to a fine powder at liquid nitrogen temperature using a ball mill (Retsch Technology, Haan, Germany). Aliquots (12-13 mg FW) of the frozen powder were transferred to pre-cooled, 2-ml Safe-Lock microcentrifuge tubes (Eppendorf Vertrieb Deutschland GmbH, Wesseling-Berzdorf, Germany) and quenched by adding 250 µl of ice-cold CHCl 3 :CH 3 OH (3:7 v/v). The frozen mixture was warmed to -20°C with vigorous shaking, and incubated at -20°C for 2 h with occasional mixing. Watersoluble components, including Tre6P, were extracted from the CHCl 3 phase by adding 200 µl of water and warming to 4°C with repeated shaking. After centrifugation at 420 g for 4 min, the upper, aqueous-CH 3 OH phase was transferred to a new tube, and kept at 4°C. The lower, CHCl 3 phase was re-extracted with 200 µl of cold water, centrifuged as described above, and the second aqueous-CH 3 OH extract was added to the first. The combined aqueous-CH 3 OH extract was evaporated to dryness using a centrifugal vacuum dryer at 20°C and redissolved in 250 µl of water. Viscous, high molecular weight components were removed from the samples by applying individual extracts to separate wells on a Multiscreen Ultracel-10 (Millipore) filter, and centrifugation at 2300 g for 2-3 h, 20°C. If this step was omitted, recoveries of Tre6P were decreased by about 30%.

Recovery experiments were carried out by addition of known amounts of authenticated Tre6P standards to the frozen tissue slurry immediately after addition of the cold CHCl 3 -CH 3 OH.

Assay of Tre6P and Suc6P by LC/MS-Q3

Tre6P and Suc6P were assayed in plant extracts by liquid chromatography (LC) using a Dionex HPLC system (Sunnyvale, CA, U.S.A.), coupled to a Finnigan TSQ Quantum triple quadrupole mass spectrometer (MS-Q3) (ThermoFinnigan, Waltham, MA, U.S.A.).

Aliquots (100 µl) of the extract were passed through a 2×50 mm AG11-HC pre-column (Dionex), before separation of anionic compounds on a 2×250 mm IonPac AS11-HC column (Dionex) at 25°C. The column was equilibrated with a mixture of 95% solution A (5% (v/v) methanol) and 5% solution B (100 mM NaOH in 5% (v/v) methanol) for 15 minutes before each sample run. Solutions were sparged with helium. Anionic compounds were eluted with a multi-step gradient as follows: 0-5 min, 5% B; 5-25 min, 5-29% B; 25-26 min, 29-100% B; 26-31 min 100% B; 31-32 min, 5% B, at a flow rate of 0.2 ml min -1 . Peak detection in the eluate from the Dionex system was done with suppressed conductivity (ASRS-Ultra, 2 mm, 50 mA, external water mode), after which the eluate entered directly into the MS-Q3, which was operated in a multiple reaction monitoring mode, with an ESI source in negative ionization mode and centroid data acquisition [33]. Nitrogen was used as a sheath and auxiliary gas, and the Q2 collision gas (argon) was set to 1.1 mTorr. The capillary voltage ranged from -1 to -56 V, the capillary temperature was 320 °C and the scan time was 0.

Assay of other metabolites

Glucose, fructose, sucrose, starch, UDPGlc, hexose phosphates, 3PGA and ATP were extracted and measured as in [35]. ADPGlc was assayed by LC/MS-Q3 in the same CHCl 3 /CH 3 OH extracts as Tre6P and Suc6P. For ADPGlc measurements the parent and product ions selected were 588.5 m/z and 346.1 m/z, respectively, and the collision energy was 28 eV.

Quantification of ADPglucose pyrophosphrylase redox status

The redox status of AGPase was assessed by monitoring the shift in electrophoretic mobility of the small subunit protein under non-reducing conditions, as described in [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF].

The protein was visualised by immunoblotting and chemiluminescent detection with CDP-Star® reagent (New England Biolabs, Ipswich, MA) and BioMax XAR film (Eastman Kodak Company, Rochester, NY, U.S.A.). Exposed films were digitally scanned, and signal intensity was quantified using Tina 2.10i software (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). (Yarrowia lipolytica) hexokinase inhibition assay [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF], HPLC [START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF], and enzymatic dephosphorylation and hydrolysis of Tre6P followed by determination of glucose [14].

All three methods involve lengthy sample preparation and suffer from other drawbacks.

The first method requires chemical destruction of other metabolites in the plant extracts, and treatment of reference samples with alkaline phosphatase, prior to the inhibition assay, and is susceptible to over-estimation of Tre6P if the plant extracts contain other phosphatase-sensitive inhibitors of the yeast hexokinase. The HPLC method also requires sample pretreatment with alkali, and is susceptible to over-estimation of Tre6P if other compounds in the extract co-elute with the Tre6P. For the enzymatic assay, a preliminary ion-exchange chromatography step is needed to remove sugars, followed by successive incubations with alkaline phosphatase, glucose oxidase and trehalase before the final measurement of glucose. This approach is highly dependent on the purity and specificity of the enzymes, and susceptible to over-estimation of Tre6P if any of the preceding treatments to remove glucose and trehalose is not 100% effective.

In preliminary experiments to test the hexokinase inhibition and enzymatic assays, as well as an HPLC approach linked to detection by single quadrupole mass spectrometry, we were able to detect Tre6P standards at comparable sensitivities to those reported previously for these methods. However, when we applied these methods to extracts from A. thaliana, the Tre6P signals were barely distinguishable from background, especially for darkened or carbon-starved material (data not shown). We therefore decided to develop a more sensitive assay with stringent specificity for Tre6P.

The approach we took was to couple liquid chromatography (LC) to detection with a triple quadrupole mass spectrometer (MS-Q3), providing three sequential filters to separate, identify and quantify different compounds. The anion-exchange LC step removes all neutral and basic components in the extract, and separates the acidic components (phosphorylated intermediates, nucleotides, organic acids) according to their charge and molecular weight. The eluate from the LC is fed on-line to the triple quadrupole mass spectrometer, which provides exquisite specificity and allows detection of metabolites that are present even in trace amounts. Fig. 1 shows the raw data from a typical measurement of an extract from A. thaliana seedlings grown in sterile liquid culture. To detect Tre6P, parent ions with a mass/charge ratio corresponding to a disaccharide-monophosphate (421.1 m/z) were selected in the first quadrupole, fragmented in the second quadrupole, and the product ion (phosphate, 79 m/z) was detected in the third quadrupole. These values were optimized using an authentic Tre6P standard, which eluted from the LC with a retention time of about 21 min (Fig. 1A). A distinct signal is obtained at this position when extract from 4.5 mg of plant material was injected into the LC (Fig. 1B). The area of this peak doubled when extract from 9 mg plant material was injected (data not shown). The signal increased in a linear fashion upon addition of authentic Tre6P to plant extracts in the range of 0 to 40 pmol Tre6P (data not shown). The only other disaccharidemonophosphate known to be widely present in plants is sucrose 6 F -phosphate (Suc6P), the intermediate of sucrose biosynthesis. Co-injection of authentic Suc6P showed that this metabolite is clearly resolved from Tre6P during the LC step, with a retention time of about 23 min, although it shares the same parent ion and fragment ion masses as Tre6P (Fig. 1A). Peaks corresponding to Suc6P were clearly detectable in plant extracts. Their identity was confirmed by pre-incubation of the extract with excess SPP, which led to specific loss of the Suc6P signal but did not affect the Tre6P signal (Fig. 1C). The identity of the Tre6P peak was confirmed by similar pre-incubation of plant extracts with a highly purified preparation of TPP, which led to specific loss of the signal attributed to Tre6P (Fig. 1D). The TPP preparation was shown to be highly specific for Tre6P, having little or no ( 1%) hydrolytic activity with Suc6P, Glc6P, Glc1P or Fru6P as substrates. Recovery experiments were carried out to check that Tre6P was quantitatively extracted from the plant tissue, and to test for matrix effects that might interfere with detection. Separate aliquots of the same plant material were extracted in parallel, with or without addition of known amounts of Tre6P to the frozen plant material prior to extraction. As will be described in more detail below, we found endogenous levels of Tre6P in A. thaliana tissues ranging from 20 to 500 pmol g -1 FW. We took samples from the low end of this range (approx. 106 pmol g -1 FW, equivalent to 1.1 pmol per 100 µl of extract) and added 1. Copyright 2006 Biochemical Society resupply. Seedlings were grown in axenic liquid culture with 15 mM sucrose under continuous low light [START_REF] Scheible | Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen[END_REF]27], transferred to medium without sugar for 2 days, and then re-supplied with 15 mM sucrose. Sugars and starch decreased 5 to 8-fold in starved seedlings, compared to control seedlings maintained with full nutrition throughout (Fig. 2). Sucrose re-addition to the medium led to a rapid 3-fold increase in the sucrose content of the seedlings within 12 min, and a 6-fold increase within 30 min (Fig. 2). Sucrose peaked at 3 h, and subsequently declined (Fig. 2). Hexose sugars (glucose and fructose) showed a marked rise from 75 min onwards (Fig. 2). Starch also accumulated from 75 min after sucrose re-addition, and accounted for over 60% of the carbohydrate present in the seedlings by 8 h (Fig. 2).

Tre6P was 6-fold lower in carbon-starved seedlings (18 pmol g -1 FW) than in control seedlings with full nutrition (121 pmol g -1 FW) (Fig. 3). Re-addition of sucrose led to a dramatic increase in Tre6P within 30 min, peaking at 3 h (482 pmol g -1 FW), when the level was over 26-fold higher than before sucrose re-addition, and substantially higher than in full nutrition. Controls that received 15 mM mannitol instead of sucrose contained similar levels of Tre6P to carbon-starved seedlings (data not shown).

Glc6P, Glc1P, Fru6P, UDPGlc and ATP were 2-to 3-fold lower in carbon-starved seedlings than controls, whereas 3PGA was slightly higher (Fig. 2). After sucrose readdition, the hexose-phosphates showed a marked increase, and UDPGlc and ATP a small increase, in the first 12-30 min. There was a further and marked increase in all these metabolites between 75 min and 3 h, reaching levels close to those found in control seedlings with full nutrition (Fig. 2). 3PGA showed a slight decrease between 75 min and 3 h, but then increased between 3h and 8 h (Fig. 2).

The redox activation state of AGPase (Fig. 4) was monitored in the same samples, using non-reducing SDS-PAGE and immunoblotting to measure the amount of AGPase small subunit protein that was in the reduced, monomeric (50 kDa) form [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF]. This approach is preferred over activity measurements to assess the activation state of AGPase, because there are compounds in plant extracts that rapidly oxidise and inactivate the enzyme [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF]. Representative immunoblots are shown in Fig. 4A, along with values for the proportion (Fig. 4B) and absolute amount (Fig. 4C) of the enzyme in the AGPase in the monomeric form (Fig. 4B) showed a similar time course to the changes in Tre6P (Fig. 3), rising steadily during the first 3 h after sucrose re-addition, although it remained fairly constant thereafter, rather than declining slightly as observed for Tre6P.

By 75 min there was substantial activation of AGPase. This matched the onset of marked increases in the level of ADPGlc and the rate of starch accumulation (Fig. 2). The absolute amount of AGPase in the monomeric form also rose during the first 3 h after sucrose readdition, but showed a further increase between 3h and 8h after sucrose was resupplied (Fig. 4C). It should be noted that this measure of AGPase activation is likely to be less accurate than measuring the proportion of enzyme in the monomeric form, because it is more sensitive to technical variation in signal strength between different blots. The apparent differences between the proportion and absolute amounts of AGPase monomer (Fig. 4B-C) is explained by the decrease in the total amount of AGPase protein as the enzyme was activated. A similar tendency has been observed in previous studies [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF]36], and indicates that there are changes in AGPase expression and/or turnover over this time period, in addition to changes in redox activation.

Changes in Tre6P levels during dark-light transitions in wild type

Arabidopsis thaliana Col-0 and the starchless pgm mutant

A second set of experiments was carried out to investigate whether endogenous changes in sugar levels during light-dark transitions are accompanied by changes in Tre6P, the activation state of AGPase and the rate of starch synthesis. Experiments were done with wild-type plants under normal conditions or after a prolonged dark treatment, and with the starchless pgm mutant. In the latter two systems there are even larger changes in the levels of sugars than under normal conditions in wild-type.

Wild-type Col-0 plants growing in a standard 12 h/12 h light-dark diurnal cycle retain substantial levels of sugars and glycolytic intermediates in their leaves at the end of the night (Table 1). When they are re-illuminated there is a small but marked increase in Copyright 2006 Biochemical Society very low levels by the end of the dark period. Re-illumination after an extended night leads to a larger increase in sugars, and a stronger stimulation of starch synthesis than normal (Table 1) [36]. An even more extreme situation occurs in the pgm mutant, which is unable to synthesize starch because it lacks plastidial phosphoglucomutase [START_REF] Caspar | Alterations in growth, photosynthesis and respiration in a starch deficient mutant of Arabidopsis thaliana (L.) Heynh. deficient in chloroplast phosphoglucomutase[END_REF]. Sugar levels in the pgm mutant are very low at the end of the night, but increase dramatically after illumination (Table 1), as reported previously [START_REF] Caspar | Alterations in growth, photosynthesis and respiration in a starch deficient mutant of Arabidopsis thaliana (L.) Heynh. deficient in chloroplast phosphoglucomutase[END_REF]36]. In the pgm mutant, or after an extension of the night in wild type, the rapid accumulation of sugars in the light occurs because the preceding period of low carbon leads to a temporary inhibition of carbohydrate utilization when the plants are re-illuminated [36]. Fig. 5A shows the accompanying changes in Tre6P. In wild-type Col-0, Tre6P is present at low levels at the end of the night, and increases 2-fold during the first 4 h of the light period. Extension of the night for 6 h leads to a 3-fold decrease in Tre6P. When the plants are re-illuminated for 4 h there is a 12-fold increase in Tre6P, which reaches levels that are 2-fold higher than after illumination at the end of the normal night. In the starchless pgm mutant Tre6P is very low at the end of the night, and increases 20-fold after 4 h illumination, reaching levels that are more than 3-fold higher than in wild type plants in the light (Fig. 5A).

In wild-type Col-0, AGPase is almost completely inactivated in the dark (Fig. 5B-C), and is only moderately activated after 4 h of illumination. Re-illumination after a 6-h extension of the normal night leads to a much greater activation of AGPase. An even more pronounced effect is observed in the pgm mutant, where AGPase is almost fully activated in the light. These changes in the activation state of AGPase show a strikingly similar pattern to those observed in the levels of Tre6P (Fig. 5A-C), and are consistent with differences in the accumulation of starch in the wild-type plants (Fig. 5D).

DISCUSSION

Measurement of Tre6P by LC/MS-Q3 compared to other methods

We have developed a highly sensitive and specific assay for measurement of Tre6P in plant extracts, based on separation of anionic compounds by LC coupled to detection with a triple quadrupole mass spectrometer. Using this method we can reliably measure Tre6P in the femto-picomole range, allowing measurements from as little as 5 mg of plant tissue. The reliability of the extraction and analytic procedures was confirmed by recovery experiments, which showed that >80% of Tre6P added to plant tissues before extraction was recovered and measured in the final extract. The specificity of the assay was clearly demonstrated by incubation of plant extracts with TPP and SPP to selectively remove Tre6P and Suc6P, respectively (Fig. 1C-D). Although we have only reported LC/MS-Q3 data for Tre6P, Suc6P and ADPGlc, this procedure has wider application and can be used to measure many other anionic metabolites simultaneously.

With a detection limit in the femtomole range, the LC/MS-Q3 method is much more sensitive (>100-fold) than the hexokinase inhibition and enzymatic assay methods, which have a detection limit of between 50-100 pmol Tre6P [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF]14]. The relatively simple sample preparation is an added advantage, and allows higher throughput. The LC/MS-Q3 method is also much less susceptible to interference from other compounds in the plant extracts. The HPLC method described in [START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF] avoids some of these problems, but still involves pre-treatment of samples with strong alkali, and is less sensitive than our LC/MS-Q3 method, requiring 20-times more plant tissue (100 mg) for reliable measurements.

In wild type A. thaliana Col-0 rosette leaves, we found Tre6P levels ranging from 23 to 298 pmol g -1 FW, with values of up to 380 pmol g -1 FW in the pgm mutant.

Previously reported values for plants of similar age grown in soil (16 h light/8 h dark) were much higher -up to 12 nmol g -1 FW [START_REF] Schluepmann | Trehalose 6-phosphate is indispensible for carbohydrate utilization and growth in Arabidopsis thaliana[END_REF]. These values were obtained using the hexokinase inhibition assay, and one possible explanation for this discrepancy is that inhibition of the hexokinase by other compounds in the plant extract gave rise to an overestimation of Tre6P levels. Another possibility is that plants developing under long day conditions have higher levels of Tre6P than our plants, which were grown with a 12 h photoperiod. In 9-d-old seedlings grown in sterile liquid culture, we found levels of Tre6P ranging from 18 pmol g -1 FW in sugar-starved plants to 482 pmol g -1 FW after sucrose re-addition. These levels are also lower than previously reported values from 7-day-old seedlings grown in liquid culture (2-11 nmol g -1 FW [14]), or in 7-day-old seedlings grown on solid media (1.8 nmol g -1 FW [START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF]). The former values were obtained using the enzymatic assay, and the latter by HPLC. All of these previously reported values would have been calibrated with Tre6P standards, but none of the reports describes verification of the Tre6P standards, as we did using the two independent enzymatic assays. Our data suggest that the standards used for these other studies were likely to contain less Tre6P than expected, and this could have led to significant overestimation of Tre6P in the plant extracts. At present, we are unable to fully account for the significantly higher levels of Tre6P reported from these previous studies. However, it is worth noting that none of the previous studies reported use of TPP to specifically remove the Tre6P from plant extracts to check the specificity of the assay. We believe this is an essential test for the reliability of any measurement of Tre6P in plant extracts.

In spinach leaves, mesophyll cells occupy 58% of the total leaf volume and the cytosol comprises 3.4 % of the total volume of mesophyll cells, or about 2% of total leaf volume [START_REF] Winter | Subcellular volumes and metabolite concentrations in spinach leaves[END_REF]. If we assume that most of the Tre6P in A. thaliana leaves is present in the cytosol of mesophyll cells, and that the leaves of this species have a similar cellular composition to spinach, we estimate that the intracellular concentration of Tre6P ranges from about 1 to 15 µM in wild type A. thaliana Col-0 leaves. These estimates are somewhat lower than the lowest concentration (100 µM) of Tre6P supplied to isolated pea leaf chloroplasts [START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF], therefore, it will be important to repeat the isolated chloroplast experiments with lower concentrations of Tre6P, to see if they also lead to redox activation of AGPase.

Changes in sugar levels lead to large and rapid changes in Tre6P

Two complementary approaches were used to alter sugar levels in A. thaliana -depletion and re-addition of exogenous sucrose to seedlings, and perturbation of endogenous sugar levels in leaves of older plants by changing the duration of the diurnal cycle, or use of a starchless mutant. In both experimental systems, Tre6P levels were low when sugars were low, and rose when sugars increased, with the relative changes in Tre6P being about 10-fold greater than those of the phosphorylated glycolytic intermediates. The large and dynamic fluctuations in the level of Tre6P that parallel the changes in sugars strongly support the hypothesis that Tre6P acts as a signal of sugar status in plants.

Inspection of the temporal dynamics reveals that Tre6P correlates better with sucrose than it does with glucose or fructose (Figs. 2, 3 and5; Table 1). This could mean that Tre6P is either a sucrose-specific signal or that its concentration reflects only cytosolic sugar levels, because most of the free hexoses are probably sequestered in the vacuole [START_REF] Heineke | Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or apoplasm[END_REF]. The levels of Tre6P and sucrose remained above those found in the control seedlings even 8 h after sucrose readdition, whereas the hexose-phosphates, UDPGlc and ATP were close to control levels by 3 h, and changed relatively little thereafter (Figs 2 and3). The increases in UDPGlc and Glc6P following sucrose resupply to starving tissue might have contributed to the increase in Tre6P, by stimulating TPS activity. However, the relative increase in Tre6P was much greater than the changes in UDPGlc and Glc6P, suggesting that other factors, such as sucrose-induced changes in expression or phosphorylation of TPS and TPP [15][START_REF] Avonce | The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signalling[END_REF][17][START_REF] Glinski | Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana -a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis[END_REF], also contributed to the increase in Tre6P.

Athough further work is needed to understand in detail the control of Tre6P levels, our results provide clear evidence that Tre6P acts as a signal of sugar status in plants.

Changes in Tre6P are correlated with changes in redox activation of

AGPase and the rate of starch synthesis

In both of our experimental systems, increasing levels of sugars and Tre6P were accompanied by an increase in the redox activation state of AGPase, higher levels of ADPGlc and a stimulation of starch synthesis. These observations are consistent with the proposal that sucrose acts via changes in the level of Tre6P to influence redox activation of AGPase and the rate of starch synthesis. Within 3 h of feeding sucrose to starved seedlings, glycolytic intermediates and ATP rose to levels similar to those found in control seedlings but then remained more or less constant (Fig. 2). This indicates that central metabolism was restored to a full-nutrition-like state during the first 3 h of sucrose resupply. In contrast, sucrose and Tre6P were both higher than in control seedlings at Copyright 2006 Biochemical Society and 8 h after sucrose resupply (Figs 2 and3). The high Tre6P might account for the maintenance of AGPase in the activated state (Fig. 4), and could be acting as a signal to divert some of the excess sucrose towards starch synthesis. Some of the excess sucrose also appears to be hydrolysed to free hexoses (Fig. 2), probably by acid invertase in the vacuole, but at present there is no evidence of a role for Tre6P in this process.

Transitory starch reserves in leaves buffer plants against diurnal fluctuations in their carbon balance, and both the rate and amount of starch accumulation are adjusted to the prevailing growth conditions [START_REF] Thimm | MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes[END_REF]27,36]. Current models for the regulation of photosynthate partitioning explain how starch and sucrose synthesis are coordinated to avoid withdrawing fixed carbon too rapidly from the Calvin cycle. They also provide a framework to understand how sucrose synthesis is decreased, and starch synthesis increased, when sugars accumulate in the leaf. Feedback inhibition or inactivation of sucrose-phosphate synthase leads to higher levels of fructose-2,6-bisphosphate in the cytosol, which inhibit the cytosolic fructose-1,6-bisphosphatase and restrict the export of triose-phosphates from the chloroplast. This leads to an increase in the ratio of 3PGA/Pi in the stroma, activating AGPase and increasing the rate of starch synthesis [39]. The recently discovered redox regulation of AGPase provides a second and complementary way to regulate starch synthesis in response to light [START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF] and the accumulation of sugars [START_REF] Kolbe | Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase[END_REF][START_REF] Hendriks | ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species[END_REF], and may help to explain how starch synthesis is coordinated with carbon utilisation over the 24-h diurnal cycle, or when unfavourable conditions decrease the amount of carbon fixed in a diurnal cycle. Even a few hours of low sugar levels towards the end of the night period lead to massive changes in gene expression, and an inhibition of metabolism and growth [START_REF] Thimm | MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes[END_REF]36]. These effects are only gradually reversed when plants are re-illuminated, so sugars accumulate in the leaves and roots when photosynthesis starts up again (Table 1) [36]. This is followed by an increase in Tre6P levels in the leaves, redox activation of AGPase and a stimulation of starch synthesis (Fig. 5). From Copyright 2006 Biochemical Society

Materials

  Tre6P and other biochemicals were obtained from Sigma-Aldrich Chemie GmbH, (Taufkirchen, Germany). Enzymes were from Roche Diagnostics GmbH (Mannheim, Germany) unless indicated otherwise. Deuterated Tre6P -[6,6-2 H]Tre6P -was synthesized enzymatically from [6,6-2 H]D-glucose. The reaction contained: 785 nmol [6,6-2 H]D-glucose, 1.2 µmol ATP, 1.2 µmol UDPGlc, 10 U hexokinase and 5 U TPS

  2 ms. The Q1 and Q3 peak 10 Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083 Copyright 2006 Biochemical Society widths were 0.5 and 0.7, respectively. Tre6P and Suc6P were selected using a parent ion of 421.1 m/z in the first quadrupole, a collision energy of 46 eV, and a product ion of 79 m/z [33] in the third quadrupole. Metabolites was quantified by comparison of the integrated MS-Q3 signal peak area with a calibration curve obtained using authentic standards. The standard addition method [34] was used to correct for matrix effects in the LC/MS-Q3 analysis.

RESULTS 11 Biochemical

 11 Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083 Copyright 2006 Biochemical Society Assay of Tre6P using anion-exchange liquid chromatography linked to triple quadrupole mass spectrometry Three techniques have been used previously to measure Tre6P in plants: a yeast

  Tre6P was quantified by comparison with authentic standard solutions, whose concentrations were verified by two independent enzymatic tests using either phosphotrehalase or TPP (see Experimental). Solutions with a nominal Tre6P concentration of 1 mM were prepared from three separate batches of Tre6P. The actual concentrations of Tre6P measured with phosphotrehalase or TPP (in brackets) were: 0.65 mM (0.66 mM), 0.68 mM (not determined) and 0.77 mM (0.82 mM). All measurements of Tre6P in plant extracts were calibrated with standard solutions whose concentration 13 Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083 Copyright 2006 Biochemical Society had been independently verified in this way. A typical calibration curve from the LC/MS-Q3 is shown in Fig. 1E, demonstrating the linear response from 0.18 to 3.4 pmol of Tre6P.

  2, 2.4 or 6.0 pmol of authenticated Tre6P standard to samples (22-30 mg) of the frozen plant material (containing 2.3-3.2 pmol Tre6P) before extraction. Recoveries of the added 1.2, 2.4 and 6.0 pmol of Tre6P were 85 ± 20%, 82 ± 11% and 86 ± 11% (mean ± SD, n = 4), respectively, demonstrating that even the smallest amounts of Tre6P were quantitatively recovered. For routine measurements, another way to assess recoveries is to spike samples with isotopically labelled Tre6P. For this purpose, Tre6P containing two deuterium atoms on C 6 of the phosphorylated glucose moiety -[6,6-2 H]Tre6P -was synthesised enzymatically, by incubation of [6,6-2 H]D-glucose with a slight stoichiometric excess of ATP and UDPGlc, plus hexokinase and TPS. The reaction resulted in over 93% conversion of glucose to Tre6P. The deuterated Tre6P was shown to have the same retention time in the LC step as unlabelled Tre6P, but the parent ion was 2 atomic mass units heavier as expected. Recovery experiments using the deuterated Tre6P gave comparable results to those described previously (data not shown). Changes in Tre6P levels after addition of sucrose to sugar-starved Arabidopsis thaliana seedlings The development and validation of the LC/MS-Q3 assay for Tre6P allowed us to investigate how the level of Tre6P in A. thaliana responds to sugar starvation and sucrose 14 Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083

  monomeric form at each time point, from three biological replicates. The proportion of 15 Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083 Copyright 2006 Biochemical Society

  sucrose and reducing sugars, and starch begins to accumulate. When wild-type plants are subjected to an extension of the night, sugars and glycolytic intermediates are depleted to 16 Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083

3 h 20 Biochemical

 20 Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083

  these results, we propose that sugar-induced changes in Tre6P play a central role in coordinating carbohydrate utilisation and storage in plants. Further studies are needed to reveal exactly how sugars affect Tre6P levels, and how Tre6P acts within the plastid to promote redox activation of AGPase, as well as to identify other processes and targets that are regulated by Tre6P in plants. Gotsche, S. and Dahl, M.K. (1995) Purification and characterization of the phospho-(1,1)glucosidase (TreA) of Bacillus subtilis 168. J. Bacteriol.

Figure 1 .

 1 Figure 1. Assay of trehalose 6-phosphate (Tre6P) using liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS-Q3). (A) Standards containing 3.4 pmol Tre6P and 10 pmol Suc6P. (B-D) Aqueousmethanol extract (100 µl) from seedlings grown in liquid culture with full nutrition. (B) untreated; (C) pre-incubated with 0.25 U sucrose-phosphatase; (D) preincubated with 0.37 U trehalose-phosphatase. (E) Calibration curve for Tre6P, values are mean ± SD, n = 3. RT, retention time; AA, area.

Figure 2 .

 2 Figure 2. Effects of carbon starvation and sucrose resupply on glycolytic intermediates, ATP, sugars and starch in Arabidopsis thaliana seedlings. Seedlings were grown axenically for 7 days under continuous light in sucrosecontaining liquid medium, then starved for 2 days in sucrose-free medium. At time = 0 h, sucrose (15 mM final concentration) was added to the medium, and seedlings were harvested at 0.2, 0.5, 1.25, 3 and 8 h after sucrose resupply. Control samples were grown with full nutrition for 9 days, and harvested at the time of sucrose re-addition to the starved samples. Values are mean ± SD, n = 2 (n = 4 for ADPGlc).

Figure 3 .

 3 Figure 3. Sucrose starvation and resupply affect trehalose 6-phosphate levels in Arabidopsis thaliana seedlings. For experimental details see legend to Figure 2. Values are mean ± SD, n = 3-4.

Figure 4 .

 4 Figure 4. Sucrose starvation and resupply affect redox regulation of ADPglucose pyrophosphorylase in Arabidopsis thaliana seedlings. Seedlings were starved and then resupplied with sucrose as described in the legend to Figure 2. (A) Representative immunoblots showing the oxidised AGPase small subunit dimer (100 kDa) and reduced monomer (50 kDa), separated by non-reducing SDS-PAGE. (B) The degree of activation of

Figure 5 .Figure 2

 52 Figure 5. Effects of light and extended darkness on Tre6P and redox regulation of ADPglucose pyrophosphorylase in rosette leaves from Arabidopsis thaliana Col-0 (WT) and the starchless pgm mutant. WT and pgm plants were grown for 5 weeks in soil with a 12h/12h light-dark cycle, and rosette leaves were harvested at the end of the night (EN) and after 4 h illumination. WT plants were also subjected to a single 6-h extension of the night (WT XN), and leaves were harvested at the end of this extension and 4 h after re-illumination. (A) Tre6P levels, values are mean ± SD, n = 4. (B) Representative immunoblots showing the 100-kDa dimeric (inactive) and 50-kDa monomeric (active) forms of the ADPglucose pyrophosphorylase small subunit. (C) Degree of activation of ADPglucose pyrophosphorylase, values are mean ± SD, n = 5. (D) Starch content (µmol hexose equivalents g -1 FW), values are mean ± SD, n = 4-5.

Table 1 . Effects of light and extended darkness on sugars, starch and Glc6P levels in Arabidopsis thaliana rosette leaves.

 1 Arabidopsis thaliana ecotype Col-0 and starchless pgm mutant plants were grown in soil with a 12h/12h light-dark cycle for 5 weeks. Rosette leaves were harvested at the end of the dark period (EN) and after 4 h illumination for measurement of carbohydrates and Glc6P. WT plants were also subjected to a single 6-h extension of the night before re-illumination, and rosette leaves were harvested at the end of the extended night (EN) and 4 h after re-illumination. Values are mean ± SD, n = 4-5 independent biological samples, each containing leaves from 3 plants.

	177, 2721-2726

Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083

Schluepmann, H., van Dijken, A., Aghdasi, M., Wobbes, B., Paul, M. and Smeekens, S. (2004) Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation. Plant Physiol. 135, 879-890

Biochemical Journal Immediate Publication. Published on 21 Mar 2006 as manuscript BJ20060083 Copyright 2006 Biochemical Society

Acknowledgements

This research was supported by the Max Planck Society and by the BMBF funded project GABI Verbund Arabidopsis III "Gauntlets, Carbon and Nutrient Signaling: Test Systems, and Metabolite and Transcript Profiles" (0312277A). R.M. was supported by a Ramón y Cajal research contract. We thank Melanie Höhne for excellent technical assistance. We also thank Dr Michael Dahl (University of Konstanz, Germany) and Dr Carlos Gancedo (Instituto de Investigaciones Biomedicas C.S.I.C., Madrid, Spain) for the generous gifts of the pSG2 plasmid and Yarrowia lipolytica HXK1 over-expressing yeast strain, respectively.