Photosynthesis and metabolism interact duringacclimation of Arabidopsis thaliana to high irradianceand sulphur depletionpce_2199 1974..1988

CRISTIÁN WULFF-ZOTTELE1,2, NICOLE GATZKE1, JOACHIM KOPKA1, ARIEL ORELLANA3, RAINER HOEFGEN1,JOACHIM FISAHN1 & HOLGER HESSE1

1Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany, 2Departamento de Agroindustria,Universidad de La Frontera, Casilla 54-D, Temuco, Chile and 3Nucleo Milenio en Biotecnología Celular Vegetal, Centro deBiotecnología Vegetal, Universidad Andrés Bello, Santiago, Chile

ABSTRACT

Arabidopsis plants were exposed to high light or sulphurdepletion alone or in combination for 6 d, and changes ofphotosynthetic parameters and metabolite abundanceswere quantified. Photosynthetic electron transport rates(ETRs) of plants exposed to sulphur depletion and highlight decreased strongly at day 2 of the acclimation period.After 3 d of treatment, the photosynthetic capacityrecovered in plants exposed to the combined stresses,indicating a short recovery time for re-adjustment ofphotosynthesis. However, at metabolic level, the stresscombination had a profound effect on central metabolicpathways such as the tricarboxylic acid (TCA) cycle, gly-colysis, pentose phosphate cycle and large parts of aminoacid metabolism. Under these conditions, central metabo-lites, such as sugars and their phosphates, increased, whilesulphur-containing compounds were decreased. Furtherdifferential responses were found for the stress indicatorproline accumulating already at day 1 of the high-lightregime, but in combination with sulphur depletion firstdeclined and after a recovery phase reached a delayedelevated level. Other metabolites such as raffinose andputrescine seem to replace proline during the early com-binatorial stress response and may act as alternative pro-tectants. Our findings support the notion that plantsintegrate the selectively sensed stress factors in centralmetabolism.

Key-words: Arabidopsis; high light; metabolic profiling;sulphur metabolism.

Abbreviations: GC/MS, gas chromatography coupled tomass spectrometry; PPFD, photosynthetic photon fluxdensity; PVPP, polyvinylpyrrolidone; ROS, reactive oxygenspecies.

INTRODUCTION

Photosynthesis and cellular metabolism are tidily linked tooptimize plant performance. Nutrient depletion severelyaffects metabolism and ultimately causes severe stresssymptoms that inhibit growth. Exposure of plants to exces-sive irradiance activates plant defence mechanisms andmight cause damage to the photosynthetic reaction centres.High light and sulphur depletion represent an excellentexample of two different abiotic stresses that occur in thefield simultaneously (e.g. Buri, Masunaga & Wakatsuki2000). Experimentally, combining sulphur depletion withhigh irradiance enables to challenge the plant antioxidantdefence system related to sulphur-containing compounds,and enforces the induction of alternative defence mecha-nisms (Resurreccion et al. 2002; Lunde et al. 2008). Eventhough different light conditions (Broeckling et al. 2005;Gibon et al. 2006; Nakamura et al. 2007) and sulphur deple-tion have been extensively studied as isolated stress factors(Hirai et al. 2003, 2004, 2005; Maruyama-Nakashita et al.2003; Nikiforova et al. 2003, 2005a,b), little is known aboutthe impact of the single and combined application of highirradiance and S limitation to plants in a time-courseexperiment.

Photosynthetic carbon fixation provides the carbon skel-etons for nutrient metabolism and structural components.S-containing compounds and proteins mediate the defenceagainst ROS that are produced in the Mehler reaction andenhanced by high light (Hesse et al. 2004; Foyer et al. 2009).Specifically, glutathione catalyses the recycling of the anti-oxidant ascorbate from dehydroascorbate, a reaction whichis localized directly in chloroplasts (Grene 2002; Tausz,Šircelj & Grill 2004). Levels of glutathione depend on theavailability of cysteine and the sulphate reduction pathway,respectively (Harms et al. 2000; Hesse et al. 2004). There-fore, this pathway significantly determines the fitness of theplant in the defence against ROS. The level of glutathionedecreases under sulphur-limiting conditions. Moreover,genes involved in the antioxidant defence system wereinduced upon sulphur depletion (Hirai et al. 2003, 2004,2005; Nikiforova et al. 2003, 2005a,b).

Correspondence: H. Hesse. Fax: +49 (0) 331-567898247; e-mail:hesse@mpimp-golm.mpg.de

Plant, Cell and Environment (2010) 33, 1974–1988 doi: 10.1111/j.1365-3040.2010.02199.x

© 2010 Blackwell Publishing Ltd1974

Adaptation of plants to high light has been extensivelystudied in relation to photosynthetic performance at thelevel of the thylakoid membranes (Li et al. 2009). However,only few studies have been performed to phenotype thephotosynthetic response to excess light in combination withmetabolic adjustment. These studies focus on primarymetabolites involved in carbon metabolism, amino acidmetabolism and secondary metabolites. For example,tomato plants grown 7 d under low- or high-light conditionsexhibited characteristic metabolic adjustments of aminoacid, organic acid and carbohydrate pools (Urbanczyk-Wochniak & Fernie 2005). In particular, a dramatic increaseof the lysine pool was detected after prolonged (21 d) expo-sure to high light. As can be expected, the responses ofcarbohydrate levels to altered light conditions were ingeneral larger than the changes induced by altered nitrateavailability (Urbanczyk-Wochniak & Fernie 2005). Using aredox signalling system that preferentially excites eitherphotosystem I or II, the time-dependent impact of redoxsignals on the transcriptome and metabolome of Arabidop-sis thaliana was investigated (Bräutigam et al. 2009). Rapidand dynamic changes emerged in nuclear transcript accu-mulation, resulting in differential and specific expressionpatterns for genes associated with photosynthesis andmetabolism. Metabolite pools also exhibited dynamicchanges and indicate re-adjustments between distinctmetabolic states depending on the respective illumination(Bräutigam et al. 2009). These states reflect re-allocation ofenergy resources in a defined and reversible manner, indi-cating that structural changes in the photosynthetic appa-ratus during long-term acclimation are additionallysupported at the level of metabolism.

To investigate the interactions between acclimation inphotosynthesis and cellular metabolism, we exposed Arabi-dopsis plants to single and combined treatments of sulphurdepletion and high irradiance in a time-course experiment.Investigation of photosynthesis rates demonstrated a shortacclimation time to recover photosynthetic performance. Inparticular, we provide a first catalogue of metabolic path-ways that are affected during early acclimation to high lightand sulphur depletion up to 6 d of treatment. This impliedthe identification of characteristic metabolic response pat-terns in a time-resolved manner during acclimation. More-over,we attempted to identify specific metabolic compoundsthat aid in maintenance of photosynthetic performanceduring acclimation to changed environments. Time-courseexperiments of Arabidopsis plants upon high-irradianceexposure and sulphur depletion provided temporal resolu-tion between permanently present protective compoundsand de novo synthesized alternative photoprotectivemetabolites. Based on chlorophyll fluorescence imaging andGC/TOFMS, we aimed at identification of metabolic statesthat support acclimation of the photosynthetic apparatus tosingle and combined stresses of sulphur depletion and highlight. We specifically addressed the question, if acclimationof photosynthetic performance to high light and sulphurdepletion is supported by clear metabolic re-adjustmentduring the first 6 d of stress exposure.

MATERIALS AND METHODS

Plant material and growth conditions

Arabidopsis thaliana (Col-0, g-1) plants were grown inhydroponic cultures in the greenhouse using Hoagland’smedium (50 mm [Fe-EDTA] SO4, 1.5 mm Ca(NO3)2, 0.5 mmKH2PO4, 0.75 mm MgSO4, 1.25 mm KNO3 and trace ele-ments) at ambient temperature 20–25 °C. The plants weregrown for 3 weeks prior to the start of each experimentinitiated by changing the culture medium. Controlplants remained in regular Hoagland’s medium solution.For sulphate depletion experiments, the complete mediumwas changed to a medium in which [Fe-EDTA] nitrate andchloride salts substituted sulphate, respectively. To studythe influence of light and sulphate depletion on photosyn-thetic parameter, the population was divided in subsets ofcontrol and depleted plants, and cultivated under high-lightand greenhouse light conditions. Experiments with plantsunder changing PFD during growth were carried out in agrowth chamber adjusted to 20 °C, 60–70% relative humid-ity, 16 h of light at 700–1.000 mmol m-2 s-1, while controlplants were grown in the greenhouse adjusted to 21 °C,60% relative humidity, 16 h of light at 140 mmol m-2 s-1

PFD generated by metal halogen lamps (HPI-T 400W-645;Philips, Hamburg, Germany). Leaf material was harvestedfrom plants daily up to 6 d after start of the different treat-ments from control and treated Arabidopsis plants. Allplant material was sampled at 1100 h in the morning andimmediately frozen in liquid nitrogen before storage at–80 °C. Experiments were performed with three to fourbiological replicas.

Chlorophyll fluorescence imaging and leafsample harvesting

Chlorophyll fluorescence imaging was performed by anIMAGING-PAM chlorophyll fluorometer (Heinz WalzGmbH, Effeltrich, Germany). At the start of each measure-ment, a plant was dark adapted for 20 min for determina-tion of Fo and Fm (Bilger et al. 1995; Fisahn et al. 1995).Then,the first level of PFD (4 mmol m-2 s-1) was applied and a setof values was measured. The same procedure was repeatedfor each PFD level (10, 50, 100, 225, 365, 585 and955 mmol m-2 s-1). All experiments were performed in trip-licates and repeated at least two times (Büssis et al. 2006).FII, photochemical quenching (qP) and electron transportrate (ETR) provide measures of the reduction state of theprimary electron acceptor of photosystem II and the appar-ent electron transfer within the thylakoid membrane(Bilger et al. 1995; Baker 2008).

In parallel to the chlorophyll fluorescence measurements,young leaves of a set of 30 plants were harvested with theaim to compare the biochemical status of leaves under thecombined conditions of sulphur depletion and differentlight regimes. Once, the leaf tissues were frozen in liquidnitrogen; all tissues were ground in a mortar, divided andused for GC/MS metabolite profiling, chlorophyll and thiol

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© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1974–1988

content analyses. The respective data sets were achievedfrom three to four biological replicates.

Quantification of total chlorophyll and totalcarotenoids in leaf tissues

One hundred milligrams of leaf tissues was ground to a finepowder and extracted with 80% acetone at 4 °C in darknessfor 1 h. The samples were centrifuged 15 min at 15 777 g,4 °C, and the supernatant was collected. Then, 50 mL ofsupernatant was diluted in 950 mL of 80% acetone, and usedfor chlorophyll measurements following the protocoldescribed by Lichtenthaler (1987).

Extraction and analysis of solublethiol compounds

Individual soluble thiols were determined as the sum oftheir reduced and oxidized forms. Fifty milligrams of frozenground leaf tissue was added to 25.0 mg PVPP (previouslywashed with 0.1 m HCl) and 500 mL of 0.1 m HCl. Thesamples were shaken for 60 min at room temperature.Aftercentrifugation (15 min at 15.777 g, 4 °C), the supernatantswere frozen at –20.0 °C until reduction/derivatization. Thelevels of glutathione and cysteine were determined byhigh-performance liquid chromatography (HPLC)-basedmethod after subsequent reduction and derivatization withmonobromobimane, as described by Kreft, Höfgen & Hesse(2003).

Metabolite profiling using GC/MS

For GC/MS analysis, polar metabolite fractions were pre-pared from 50 mg frozen leaf tissue, which was ground to afine powder and then extracted with methanol/CHCl3. Themetabolite extraction and the preparation of the fractionof polar metabolites by liquid partitioning into water wereas described earlier (Roessner et al. 2000; Wagner, Sefkow& Kopka 2003). Metabolite samples were derivatizedby methoxyamination, using a 20 mg mL-1 solution ofmethoxyamine hydrochloride in pyridine, followed by asubsequent trimethylsilylation step, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (Fiehn et al. 2000; Roess-ner et al. 2000). A C12, C15, C19, C22, C28, C32 and C36n-alkane mixture was used for the determination of reten-tion time indices and chromatographic alignment (Wagneret al. 2003). Ribitol, isoascorbate and deuterated alaninewere added for internal standardization (Kaplan et al.2004). Samples were injected using splitless mode (1 mLsample) and analysed using a quadrupole-type GC/MSsystem (MD800; ThermoQuest, Manchester, UK). Thechromatograms and mass spectra were evaluated using theMASSLAB software (ThermoQuest), and data were pro-cessed using the software TagFinder (Luedemann et al.2008). Following the procedure of Desbrosses, Kopka &Udvardi (2005), peak areas, X, were defined to representthe fragment responses (Xi, of fragment i). Fragment

responses were normalized to fresh weight, and response ofthe internal standard ribitol using fragment m/z = 319, atRI = 1733.7. The response of ascorbic acid was normalizedto the chromatographically separated isoascorbate peak,mass fragment m/z = 332, which was shown to be absentfrom non-spiked plant samples. This procedure corrects forthe slightly varying sample amount, as well as pipetting andother volume processing errors of all monitored metabo-lites, and in the case of ascorbic acid corrects additionallyfor the variable recovery of this metabolite. Isoascorbicacid contains a contamination of dehydroascorbate whichwas not chromatographically separated from the silylationproduct of endogenous dehydroascorbate. Endogenousdehydroascorbate was determined after background sub-traction of internal standard contributions using non-sample control experiments. Subsequently, response ratios(C) were calculated using the normalized fragmentresponses of each metabolite. The ratios were built relativeto the mean normalized response of the control samplegroup (i.e. the cultivation under sulphate-sufficient andregular light conditions at the initial time-point of stressexposure). Changes in the relative concentration of thesemetabolites caused by stress acclimation were analysedby calculating the response ratio (%C). %C denotes theaverage fold change of metabolite pool sizes expressed aspercentage relative to the concentration measured usingcontrol plants. Both the treated and the reference sampleswere replicated (n = 5) pools of three plants, respectively.

Statistical analysis

Heat map presentation was performed on the data setsobtained from metabolite profiling with the softwarepackage TMEV (Saeed et al. 2003). The data were log2

transformed before analysis.The term ‘significant’ is used inthe text only when the change in question had been con-firmed to be significant (P < 0.05). The t-tests were per-formed using the two-sided algorithm embedded intoMicrosoft Excel.

Principal component analysis (PCA) was performedusing the software STATISTICA 6.0 (StatSoft, Inc., Tulsa,OK, USA). Prior to PCA, we log10 transformed the relativeresponses of each metabolite, log10 (Ri). In the case of iden-tified metabolites, missing values were corrected by manualpeak extraction or defined as the average value of therespective sample group after log10 transformation. If noresponse value was retrievable for any of the samplesof specific organ, log10 (Ri) = 0 was substituted for PCAanalysis.

RESULTS

Phenotypic characterization of Arabidopsisplants under sulphate depletion and high PFDs

During a time-course experiment of six consecutive days,no significant differences in the macroscopic phenotypewere detected between control and sulphate-deprived

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plants under low light intensities (Fig. 1a). However, plantsexposed to high light showed large phenotypical changesthat were severely more pronounced than leaf morphologi-cal symptoms induced by sulphur depletion (Fig. 1a).Young

leaves subjected to high light in the presence of sulphateturned dark green during a 4 d period. Subsequently, theseleaves acquired a green-reddish colouration up to day 6.A similar pattern of acclimation was detected in

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Figure 1. Six-day adaptation kinetics of Arabidopsis plants exposed to different PFDs and sulphate depletion were characterized byvisible phenotype and photosynthetic performance using chlorophyll fluorescence imaging. Visual phenotypes observed in control andsulphate-depleted Arabidopsis thaliana plants grown in the greenhouse and transferred to high-light (HL) conditions with PPFD of750 mmol (m2s) after 1, 2, 3 and 6 d of sulphate depletion (a). (b) Images of the yield (FII) of chlorophyll fluorescence detected at PPFDof 365 mmol (m2s) from A. thaliana leaves of control and sulphate-depleted plants at different light regimes. The yield of chlorophyllfluorescence was mapped to the colour scheme shown in (c).

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sulphur-depleted plants. However, at day 2, the leaves ofsulphur-depleted plants exhibited a paler phenotype incomparison to high light and presence of sulphur (Fig. 1a).

Chlorophyll fluorescence imaging of youngleaves under sulphate depletion andhigh-light regimes

At day 1 of the time-course, young leaves of control plantsunder high light displayed an increase in the yield of fluo-rescence (FII; Fig. 1b), qP and ETR (data not shown) incomparison to greenhouse controls. In contrast, youngleaves of sulphate-depleted plants exhibited a reduction ofthe photosynthetic parameters before the onset of chloroticsymptoms (Fig. 1b). The overall yield of chlorophyll fluo-rescence of the leaves declined by local reductions (Fig. 1b).After 2 d under these conditions, marked decreases of FII,ETR and qP were observed when compared to the respec-tive sulphur-treated plants (Figs 1b & 2a). Intriguingly, after3 d under high-light conditions, a recovery of FII, ETR andqP emerged in young leaves of sulphate-depleted plants(Fig. 1b). However, the performance did not reach thelevels of the control plants. In contrast, leaves of sulphatedepleted and leaves of plants grown under low-light inten-sities of the respective control groups showed no significantchanges in parameters of photosynthetic performanceduring the time-course of six consecutive days exposed toeither or combined stress (Figs 1b & 2c,d).

The non-photochemical quenching (qN) of chlorophyllfluorescence was determined for young leaves from plantsgrown under the above described conditions. A significantincrease of qN was detected in young leaves of sulphate-depleted plants after 2 d of exposure to high light (Fig. 2b).

From day 3 on, qN decreased in young leaves of sulphate-depleted plants under high light (data not shown). In agree-ment with the above observations, plants grown underlow-light regimes in the greenhouse with or withoutsulphate displayed no significant difference in their rates ofqN (Fig. 2d).

Recent studies have shown that sulphate depletioncauses a reduction in chlorophyll content (Nikiforova et al.2005a). To study the effect of different light regimes incombination with sulphate availability on chlorophylllevels, we extracted total chlorophyll content from youngleaves. For all plant subsets, the total chlorophyll contentremained relatively constant during the time-course(Fig. 3a,b). However, an increase of the total chlorophyllcontent emerged in young leaves of control plants after 3 dunder high light exposure which decreased towards day 6approaching the original content of days 0–2 (Fig. 3b, blackbars). Furthermore, the chlorophyll content tends todecrease, but not significantly, decreased under sulphatedepletion combined with high-light intensities (Fig. 3b,white bars).

Plots of the relative ETR against the total amount ofchlorophyll (Supporting Information Fig. S1a,b) demon-strated that under low-light conditions, sulphate depletionderived data clustered indistinguishably with data derivedfrom untreated plants (Supporting Information Fig. S1a).However, the relationship between ETR and total chloro-phyll content diverged in young leaves of sulphate-depletedplants under high-light conditions starting at day 2 of thetime-course (Supporting Information Fig. S1b). Because ofthe higher chlorophyll content in control plants at day 3, thecorrelation deviates significantly compared to day 2 (Sup-porting Information Fig. S1b, black squares). Subsequently,

Figure 2. Chlorophyll fluorescenceparameters of Arabidopsis thaliana undersulphate deficiency and different lightconditions after 2 d of the adaptationperiod. Apparent photosynthetic electrontransport rates (ETRs) (a, c) andnon-photochemical quenching (qN) ofchlorophyll fluorescence (b, d) wereobtained during stepwise increase ofPFD (see Materials and methods) incontrol ( ) and sulphate-deprived plants(�). (a, b) Plants were exposed to PFDsof 750 mmol (m2s) at day 0 or maintainedin the greenhouse (c, d). Data wereobtained from the same set of plants asin Fig. 1. Each data point represents theaverage of three to five individualplants � SE.

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a recovery in the relationship between ETR and the totalamount of chlorophyll was observed towards initial ratios.

Metabolic profiling of Arabidopsis plantsexposed to combined S depletion and lightstress

Typical metabolite profiles of samples harvested fromplants grown under different cultivation conditions clearlyshow that the metabolite composition varies depending onthe type of treatment (Supporting Information Fig. S2).Metabolites with differences in their respective contentsare indicated with an arrow. Additionally, thiol-containingcompounds were measured by HPLC (Kreft et al. 2003),and photosynthetic pigments by photometry.Altogether, 84compounds of known chemical structure were assessed withrespect to their metabolic response to the selected stressconditions. Results were catalogued onto plant biosyntheticpathways according to the metabolic pathway schemes ofAraCyc (http://www.arabidopsis.org/tools/aracyc/; Zhanget al. 2005). Response ratios %C was log2 transformed andvisualized as a heat map in Fig. 4 (see detailed informationin Supporting Information Table S1). Statistical significanceof the differences in relative concentrations was analysedby t-test comparing to reference samples. Differences wereconsidered to be significant with P < 0.05 and C > 2.0 or<0.5 in at least one of the time-points monitored for assess-ing the tested conditions. Thus, the filtered metabolicresponses represented a clear metabolic phenotype as isvisualized in the heat map (Fig. 4; for details, SupportingInformation Table S1). In particular, the heat map demon-strated a far reaching effect of the stress treatments on mostof the monitored metabolites (Fig. 4). High light alone andin combination with sulphur depletion had a considerableimpact on primary metabolism. Two major clusters ofsamples are formed, separating the single sulphate deple-tion treatment under ambient light from the sulphur-sufficient high-light condition and the combined stress ofsulphate depletion and high light.

Metabolite profiling of Arabidopsis plantssubjected to low light and sulphur depletion

Under low light, sulphur depletion resulted in large changesin metabolite pools. At day 1 (DS day 1, Fig. 4), the systemrevealed a significant perturbation response from the initialmetabolic equilibrium at day 0 (control, Fig. 4). In general,a slight increase in metabolite levels could be observed.However, already at day 2 of sulphur depletion, most of themetabolites re-equilibrated to their initial metabolic state.Only carotenoids, glutamine, tryptophan, proline, serineand glucose-6-phosphate went through a concentrationminimum at day 2 after onset of sulphate depletion, whileraffinose reached a minimum at day 3. After 6 d of sulphurdeprivation, the metabolic equilibrium closely resemblesthe control(for details, Supporting Information Table S1).

Metabolite profiling of Arabidopsis plantssubjected to high irradiance in the presenceof sulphur

In contrast to sulphur depletion at reference light, themetabolite response to high-light conditions exhibited amore pronounced deviation from the initial equilibrium.Cluster analysis identified three groups of metabolites: thefirst cluster exhibits dramatic changes in the pool sizes, thesecond is characterized by significant modifications, whilethe third cluster contains metabolites that are not affectedby high light (Fig. 4).A subset of metabolites that displayeda pronounced response at day 1 of high light (HL day 1)re-equilibrated towards its initial control state at day 2 (HLday 2). However, at day 6 under high-light conditions, mostof these metabolites reached a new acclimation state.Glutamine, raffinose and glutamate increase in contentapproaching a new equilibrium at a higher level.

Consistent with the observed increased photosyntheticactivity caused by high-light exposure, major increases inmetabolite levels occurred already at day 1 of the time-course experiment (Fig. 4). For intermediates of glycolysis,we observed significant increases in hexose phosphates,

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Figure 3. Correlation between photosynthetic electron transport rate (ETR) obtained by chlorophyll fluorescence imaging and totalamount of chlorophyll during the 6 d adaptation period. Total chlorophyll contents of young leaves of control (black bars) andsulphate-deprived (white bars) Arabidopsis plants grown in the greenhouse (a) and high-light conditions (b). Errors bars denote � SE(n = 10). Student’s t-test was significant (P < 0.05) at time points marked with an asterisk (*).

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Figure 4. Heat map visualization and cluster tree representations of relative change of metabolite pools in response to the time-courseof sulphur depletion and differential light treatments. Plants grown under greenhouse and high-light conditions in the presence or absenceof sulphate for 6 d (cf. compare Fig. 1). The heat map was generated by using log base 2-transformed fold changes (response ratiosrelative to sulphur-sufficient and regular-light conditions). Each metabolite is represented by a single row. Each treatment is shown as asingle column obtained by averaging the replicate measurements of each condition. Red indicates increased metabolite content, whereasblue indicates decreased contents of metabolites. The underlying numerical data are presented in Supporting Information Table S1.Sulphur depletion is indicated by DS (delta S), high light by HL.

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glucose 6-phosphate and fructose 6-phosphate, and sub-sequently in sucrose and trehalose. Nearly all elevatedmetabolic pool sizes persisted throughout the 6 d oftreatment. Elevation in serine and glycine (linked to3-phosphoglycerate) was measured together with accumu-lation of shikimate and tyrosine (linked to phospho-enolpyruvate), alanine, leucine and valine (linked topyruvate), the aspartate branch of amino acids linked tooxalacetate (asparagine, b-alanine, lysine, homoserine,threonine) and the glutamate branch linked to 2-oxoglutarate (glutamate, glutamine, proline).

The third cluster compiles metabolites which did notrespond notably to high light; methionine pools stayed con-stant as levels of aspartate, citrate and the fatty acids palmi-tate and stearate. Intriguingly, glutathione levels exhibited aslight increase at day 1 of high-light exposure which duringthe subsequent days relaxed to its initial equilibriumconcentration.

Metabolite profiling of Arabidopsis plantssubjected to a combination of high light andsulphur depletion

Cluster analysis suggests that high light dominates as stressover sulphate depletion throughout the full acclimationperiod. However, the combination with sulphate depletionaffected several individual metabolites at specific time-points of the treatment. At day 1, most metabolites exhib-ited similar changes upon exposure to high light in theabsence of sulphur in comparison to the presence of suffi-cient nutrient availability (Fig. 4). However, depletion ofsulphur induced significant reduction in the levels ofglucose and fructose, while isocitrate and citrate increased.Contents of glutamine, raffinose, arginine and homoserinechanged slightly. At day 2 of the combined stress regime,major differences emerged. Glutathione and proline levelsdropped dramatically in sulphur-depleted plants (Fig. 4).Glucose, fructose and raffinose levels also declined signifi-cantly, but were still at elevated levels compared to thecontrol. This is also the case for ascorbate (as was deter-mined by specific internal standardization of GC/MS pro-files using isoascorbate, cf. Materials and methods) andmore pronounced for glutathione under sulphate depletion.Day 3 of high-light treatment displays more similaritybetween metabolite concentrations when sulphur defi-ciency and availability are compared. Raffinose levels havesignificantly increased in accordance with the glucose andfructose behaviour. As expected, glutathione levels weresignificantly reduced at day 3, which is also the case forcysteine and methionine, while the precursor of cysteineO-acetylserine accumulated. Metabolites such as dehy-droascorbate and ascorbate declined under sulphur-depleted conditions, indicating that ascorbate may beinvolved in a fast response to oxidative stress, while glu-tathione levels decrease under the combined stress. A pro-nounced accumulation of threonate, a breakdown productof ascorbate could be detected indicating a failure to recycleall of the oxidized ascorbate via the ascorbate–glutathione

cycle. Generally, contents of cysteine and glutathionedecreased, while a marker for S starvation O-acetylserineincreased.

General metabolic response patterns duringequilibration to high light and sulphurdepletion

Overall, three characteristic classes of metabolite acclima-tion kinetics, classes A, B and C, emerged upon perturba-tion within the investigated time-course (Fig. 5a,b). Thedisaccharide raffinose representing metabolite class Aexhibited an oscillatory type of adaptation upon perturba-tion by high light (Fig. 5a,c). In sulphate-saturated plants,the amount of raffinose started to increase at the first dayof high-light stress. Sulphate-deprived plants exhibitedincreased levels of raffinose. After 2 d of high-light expo-sure, a marked inhibition in the pool size of raffinose wasobserved in leaves of sulphate-depleted plants. Finally, astrong increase in the level of raffinose was detected afterthe third day of high light in young leaves of sulphate-depleted plants.

An example for a metabolite B strongly increasing insulphate-depleted plants during the first day under high-light intensities and subsequently decreasing is maltose(Fig. 5b). Allantoin belongs to the group of late-responsivemetabolites C (Fig. 5b,e,f) that strongly increase in contentafter 3 d in sulphate-depleted plants exposed to high light.

PCA reveals specific metabolites thatdistinguish the response of plants to sulphatedeficiency and high light

To reduce the complexity provided by the heat map to thenumber of metabolic pattern delimited by the major vari-ances of the data set, PCA was performed on 54 samplesharvested from plants subjected to the complete set ofexperimental conditions (Fig. 6). PCA evaluates the majorvariances in a data set rather than statistical significance ofobserved changes.Thus, we used PCA to independently testthe deductions summarized in the previous paragraphs.Indeed, classification by PCA confirmed most of our obser-vations on sample classes and behaviour which were madeby a manual and hypothesis-driven survey of the data. Indetail, PCA revealed that the four highest-ranking compo-nents accounted for 80.55% of the total variance within thedata set comprising the 20 most influential metaboliteswhich were deduced from the loading values of the compo-nents 1, 2 and 3 (Fig. 4; Supporting Information Tables S1 &S3). The assessment of three of these components allowedconsistent classification of all treatments and time-pointsduring the acclimation periods to single and combinedstress regimes. Component 1 accounted for 55.39% of thevariance, indicating a strong differential response of plantsgrown in the greenhouse and transferred to high-light con-ditions. In addition, samples of young leaves harvested after2 d of combined sulphate deficiency and high-light stress

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cluster together in an independent group. This group ofsamples is defined by high loading values of metabolites,such as arabinose, threonate (ascorbate metabolism),b-alanine (pyrimidine metabolism), quinate (phenylpro-panoid biosynthesis), glutathione (sulphate metabolism),

a-oxoglutarate [tricarboxylic acid (TCA) cycle], raffinose(environmental stress indicator), phosphate (e.g. energy,phosphorylation of proteins), benzoate (involved in thesalicylic acid biosynthesis) and glutamine (nitrogenmetabolism) (Supporting Information Table S2).

(a) (b)

(c) (d)

(e) (f)

Figure 5. Time-course of metabolites detected during a period of 6 d. (a, b) Model patterns of metabolic adaptation kinetics which wererepeatedly observed. Exemplary response patterns of the pool size changes of raffinose (c), maltose (d) and putrescine (e, f) as obtainedby GC/MS metabolite profiling are shown; control plants (black bars), plants subjected to sulphur deficiency (dark grey bars), high light(blue bars) or combined sulphur deficiency and high-light stress (red bars). (e) Representative zoom-in of the 26.5–27.5 minchromatogram region putrescine signal (grey arrow) which exhibits a dramatic differential response after 3 d of different treatments. (f)Putrescine mean � SE (n = 5) at all experimental conditions. RR, relative response of metabolite.

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1 The second component encompassing 15.85% of the vari-ance enables to distinguish between leaf tissues of controlplants and sulphate-deprived plants after 6 d of treat-ment with high-light conditions.The component had highcontributions by metabolites involved in lipid biosynthe-sis such as glycerol-1-P, glycerol, palmitate and stearate;purine and pyrimidine metabolism (uric acid, thymine,uracil, allantoin); cysteine biosynthesis (O-acetylserine);aspartate pathway (aspartate, homoserine); nitrogenmetabolism (aspartate, glutamine, ornithine, arginine);and starch degradation (maltose) (Supporting Informa-tion Table S2).

2 The third component encompasses 5.52% of the vari-ance, and it resolved the time series of high light andcombined sulphate deficiency, and high-light stressresponses. Component 3 was defined by metabolitesinvolved in photorespiration (glycine), photosynthesis(chlorophyll a and glyceric-3-P), nitrogen assimilation(glutamine), involved in radical scavenging (l-ascorbicand glutathione), aromatic amino acid biosynthesis(tryptophan) and stress-related response metabolites(g-amino-butyrate; GABA) when the plants are in con-ditions of high light and combined sulphate deficiency,and high-light conditions.

High light

Day 2 (–S/HL)

Low light

–15 –10 –5 0 5 10–10

–5

0

5

10

Com

ponent 2 (

15.8

5%

of variance)

Component 1 (55.39% of variance)

Day 6 –S/HL

Day 1 –S/HL

Day 3 –S/HL

Day 2 –S/HL

Day 6 HL

Day 3 HL

Day 1 HLDay 2 HL

Low light

C/low light

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–15 –10 –5 0 5 10

–10

–5

0

5

10

Com

ponent 3 (

5.2

9%

of variance)

Component 1 (55.39% of variance)

Figure 6. Principal component analysis (PCA) of metabolites during the 6 d response period to single- or double-stress exposure.Samples representing control greenhouse-grown plants and plants exposed to high-light intensities were clustered according to thetreatment and time of stress exposure. Components 1, 2 and 3 depict 73.30% of the total metabolite variance of the data set, and clearlyseparate the treatments according to the metabolic phenotype inherent of each treatment. In detail, plants grown in the greenhouse (lowlight) were distinguished from plants exposed to high-light conditions (high light or HL), and sulphate-depleted plants after 2 d oftreatment with high-light intensities (upper panel, component 1 versus component 2). Note that the score plots of components 1 and 3demonstrate the time-course of plants exposed to high light and sulphur depletion in a time-dependent manner (lower panel).

Acclimation to high light and sulphur depletion in Arabidopsis 1983

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1974–1988

DISCUSSION

Photosynthetic and metabolic changes underlow light and sulphur depletion

The onset of sulphur depletion had no consequence for thephotosynthetic properties of the investigated system at day1 of the time-course experiment. However, on metaboliclevel, a significant but transient elevation of severalmetabolite pools could be observed at day 1 (Fig. 4; Sup-porting Information Figs S3 & S4). Sulphur depletion at lowlight induced modulation in concentrations of centralmetabolites belonging to glycolysis and oxidative pentosephosphate pathway (OPPP) such as glucose and fructose,and their hexose phosphates glucose-6-phosphate andfructose-6-phosphate, but also gluconate-6-phosphate,respectively. Furthermore, an increase of glycine and serinelevels was observed, indicating a changed photorespirationrate (Foyer et al. 2009). Taking these data into account, wesuggest that sulphur depletion has been sensed by leavesalready at day 1 and the immediate response results in ashift of pool sizes within major metabolic pathways. Modu-lation of fluxes through these pathways could be affectedthrough thioredoxin-mediated activation of major enzymesin these pathways (Bräutigam et al. 2009).Together, we con-clude that initial responses to sulphur depletion are delim-ited to changes of the internal sulphate pool(s) which maybe sensed by the availability of thioredoxins. These modu-lations in levels of thioredoxin might also induce differ-ences in the redox state of the cell. Detection of metabolicmisbalance at day 1 of sulphur depletion is further mani-fested in the elevated levels of raffinose and proline that areclassical indicators of stress (Fig. 4; Sharma & Dietz 2006;Nishizawa, Yabuta & Shigeoka 2008).

At the second day of the adaptation period, light satura-tion curves of the ETR indicated an increase at saturatingPFDs in sulphur-depleted plants at low light (Fig. 2c). Thisacclimation in the electron transport capacity might becaused by the metabolic acclimation at day 1 of sulphurdepletion. Re-adjustment of metabolite concentrations toinitial pre-acclimation pool sizes was completed 3 d aftersystem perturbation by S limitation. The aforementionedobservation supports the notion that under the conditionschosen in this study, external sulphur limitation leads to are-arrangement of internal sulphur resources and ulti-mately to a slow S depletion from the plant system. In short,at days 1–2 of our experiments, external limitation of Ssupply can be sensed, but re-arrangement of internal Sresources did not yet reach S depletion or even more severeS depletion.

Correlation between the chlorophyll content and theETR formed a single cluster during the 6 d adaptationperiod (Fig. 3a; Supporting Information Fig. S1a). There-fore, non-invasive measurements of the chlorophyll fluores-cence imaging parameters reflect the results that wereobtained by the biochemical analysis of chlorophyll concen-trations. Taken together, sulphur depletion at ambient lightconditions induces characteristic adaptation in metabolismand photosynthetic performance. In particular, changes in

metabolite levels emerge already at day 1 which in a bidi-rectional feedback/feed-forward interaction might affectphotosynthetic capacity at subsequent days.

Photosynthetic and metabolic changes underhigh irradiance

Generally, metabolite levels of plants exposed to high lightalone or in combination with sulphur depletion show animmediate response, changing most of the investigatedmetabolite levels. Approximately 75% of the metabolitesincreased in their content while the remaining 25% stayedon the level or decreased at later time-points. The lattergroup comprises chlorophyll, glutathione, methionine,palmitate, stearate and glycerol which are partially in accor-dance with metabolites decreasing under sulphur depletiondescribed in a recent publication (Nikiforova et al. 2005a).

Generally, most of the metabolites of the glycolysis, TCAand OPPP are altered in their content, indicating that plantsexposed to high light undergo a metabolic shift andenhance the Calvin cycle to fix carbon. For example, expo-sure of plants to elevated light induces increasing photosyn-thetic activity (Schlüter et al. 2003; Couée et al. 2006). Theseelevations in ETR may be reflected by increased metabolicpool sizes in the presence and absence of sulphur supply(Fig. 4). In particular, elevations in glucose, fructose andglycine a product of photorespiration emerged upon high-light treatment (Fig. 4). Elevated pool sizes of amino acidsand organic acids are caused by increased availability ofprecursors derived from photosynthesis.

Interestingly, almost all investigated metabolites revertedtheir pool sizes at day 2 of high light (Fig. 4). Only fewmetabolites such as alanine, homoserine and threonateremained constant, while O-acetylserine slightly increasedin content. This observation has not been described before,and the reason for this behaviour is speculative and has tobe addressed in further investigations. However, the generaldecline of metabolites, but still on a high level with respectto control conditions, seems to be a general phenomenon ofhigh-light treatment.The obtained results are in accordancewith general observations that plants exposed to stressreduce their respiration (Atkin & Macherel 2009; Kornaset al. 2009) and respiratory enzymes (Miszalski et al. 2001),and thus affecting central metabolite pathways such as gly-colysis, TCA and OPPP. Another explanation could be theavailability of resources which were used up at day 1 underhigh light. The acclimation process is not advanced enoughto cope with the new condition. For the acclimation process,a ‘biphasic response’ could be observed, which is displayedby a fast response to the perturbation and re-adjustment tothe initial metabolic state (days 1 and 2). This might be partof the typical response to natural fluctuations in lightconditions. This first phase of transient responses may befollowed by a slower acclimation process that becomesapparent at days 3–6, and is part of the long-term acclima-tion process to continued/persistent high-light stress(Fig. 4).Therefore, the adaptation kinetics of many metabo-lites exhibits an oscillation during the first three days of

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increased illumination. Mostly all metabolites stayed ona high content level until the remaining time of theexperiment.

Chlorophyll content increased transiently upon exposureto high light (Fig. 3b). As a consequence, the correlationbetween the relative ETR and the chlorophyll content(Supporting Information Fig. S1a) exhibited an increasedscatter in comparison to ambient conditions (SupportingInformation Fig. S1a). This is in contrast to potato plantswhich were exposed to increasing PFDs. These plantsdecreased their chlorophyll content upon exposure to highlight (Bilger et al. 1995). However, chlorophyll contentof potato plants was significantly modulated by Calvincycle activity. Up-regulation of Calvin cycle activity gaverise to elevated chlorophyll content (Bilger et al. 1995).Clearly, exposure of Arabidopsis to high light results in anup-regulation of the Calvin cycle activity that could give riseto the elevation in chlorophyll content (Schlüter et al. 2003).Thus, we conclude that the acclimation of the photosyn-thetic apparatus to high light is plant type specific andexhibits a characteristic time-course in chlorophyll equili-bration in A. thaliana.

Photosynthetic and metabolic responsesto high light in combination withsulphur depletion

High light in the absence of sulphate gave rise to similarmetabolic pool modifications as high light exclusively.However, several metabolites displayed significant differ-ences between the two treatments (e.g. glucose, fructose,raffinose and glutamine; Fig. 4). Again, these deviationsindicate the onset of sulphur stress sensing and the regula-tory impact of thioredoxin-mediated activation of centralmetabolic pathways. Metabolites that exhibited differentialacclimation with and without sulphate are comparable tothose that shifted their equilibrium under low light andsulphur deprival. PCA supports the metabolic differentia-tion at day 1 with and without sulphur (Fig. 6).

Parallel to the strong decline in photosynthetic activity(Figs 1 & 2), sulphur depletion caused significant deviationsin the metabolite pools at the second day of the treatment.In particular, combination of high light and sulphur deple-tion induced pronounced damage to the photosyntheticapparatus after 2 d (Fig. 2a). Light saturation curves of theETR indicated severe down-regulation of photosystem II inleaves exposed to multiple stresses. In contrast, the photo-protective mechanisms were still able to dissipate the exces-sive light absorbed by the light-harvesting complex uponexposure to high light exclusively (Fig. 2c).This finding indi-cates that the presence and effective performance of severalphotoprotective reactions are significantly affected by theavailability of sulphur.

Exposure of plants to high light induces the production ofROS and intermediates that damage key biomolecules suchas proteins, lipids and DNA (Halliwell 2006). Organismshave evolved several defence mechanisms including thewell-established ascorbate–glutathione system or the

plasma membrane-located NADPH-dependent oxidase.The decline of glutathione is thus not unexpected becauseof its important role in defence against various stressesincluding high light (Payton et al. 2001) and sulphur stress(Nikiforova et al. 2005a). In addition, metabolites involvedin the antioxidant stress response and their degradationproducts, such as threonic acid (Green & Fry 2005) anddehydroascorbate, accumulate in both sets of experimentsthat apply high light with and without sulphate depletion(Fig. 4; Supporting Information Table S1, days 2, 3 and 6).These results suggest that ascorbate and glutathione areinvolved in regulating the acclimation responses to highlight in plants treated with limited sulphate nutrient supply.The observed changes in the amount of glutathione, ascor-bate and threonic acid suggest the expression of genesinvolved in the up-regulation and resource saving of ascor-bate, which then allows the provision of low-molecular anti-oxidants employed to maintain several functions, such asphotosynthesis and direct quenching of ROS species.Results derived from the ascorbate-deficient mutant vtc-2support the key role of ascorbate in high-light acclimationmechanisms (Müller-Moulé, Golan & Niyogi 2004), and thedirect effect of light intensity on regulation of genesinvolved in the ascorbate biosynthesis (Yabuta et al. 2007).

Activation of alternative defence compoundsduring high-light exposure (ROS)

In addition to the discussed metabolites, raffinose,polyamines and glutamate are increased in their contentssupporting the hypothesis that alternative defence mecha-nisms are activated. During the time-course of the experi-ment, we found that the changes in the amount of raffinosecorrelated with the adaptation kinetics of the photosyn-thetic capacity observed in young leaves of sulphate-depleted plants treated with high-light stress (Fig. 5c).Raffinose levels were at the detection limit at the secondday of the adaptation period in plants exposed to sulphurdepletion and high light. In parallel, the yield of chlorophyllfluorescence (Fig. 1b) decreased strongly at this time.

Recovery of the photosynthetic performance at day 3(Fig. 1b) correlated with an increase in raffinose (Fig. 6c).Furthermore, presence of detectable levels of raffinose atday 2 of the adaptation period might have been in favour ofmaintaining continuous photosynthetic performance underhigh light in the presence of sulphur (Figs 5c & 1b). Severalrecent studies suggest that raffinose family oligosaccha-rides, such as raffinose, stachyose and verbatose, can func-tion as intracellular antioxidant under several types ofstress as response to ROS production (Hannah et al. 2006;Kaplan et al. 2007; Nishizawa et al. 2008; Wienkoop et al.2008; Widodo et al. 2009). Thus, raffinose might partiallycompensate the reduction in glutathione content because ofsulphur depletion.

The level of qN, which provides estimation for dissipationof absorbed light energy as heat, was higher in the absenceof sulphur (Fig. 2b). Therefore, we suggest that sulphurdepletion activates xanthophyll cycle-mediated excess light

Acclimation to high light and sulphur depletion in Arabidopsis 1985

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1974–1988

dissipation (Fig. 2b), whereas antioxidant defence systemsare down-regulated at day 2 of the adaptation period. Unex-pectedly, photosynthetic performance recovered at day 3 ofhigh light and sulphur depletion (Fig. 1b).This recovery wasassociated with pronounced modifications in metaboliccomposition (Fig. 4). In particular, the concentration ofraffinose increased at day 3.

Conclusion

This paper presents a detailed and comprehensive analysisof a time-course experiment of sulphur depletion, high lightand the combination of both stresses, a novel aspect toinvestigate the dynamic behaviour of the photosyntheticand metabolic adjustments of Arabidopsis plants. Based onthis analysis, we suggest that metabolism and photosyn-thetic adaptation undergo a complex regulatory circuitduring a 6 d acclimation period. The obtained resultssuggest that in a coordinated manner, a systemic responseto high light and sulphur depletion is initiated. Our resultssuggest that already a slightly reduced sulphur level issensed at day 1 of sulphate depletion to initiate a biochemi-cal response via modulation of the activity of the Calvincycle and glycolysis probably via thioredoxin. These accli-mation processes are independent of light availability.Exposure to high light alone elevates pool sizes of a greatnumber of central metabolites involved in glycolysis, TCAand OPPP pathways. Metabolite acclimation kinetics athigh light or the combination of high light and sulphurdepletion display similar characteristics at early stages, indi-cating dominance over the latter treatment. Advancementin time and sulphur depletion emphasized the effect of highlight in an additive process, resulting in unbalanced defencemetabolites causing drastic metabolic alterations. High lightalone and in combination with sulphur depletion inducedROS formation, which requires detoxification by ascorbate/gluthatione especially at later time-points. A dramaticreduction in photosynthetic performance is observed at day2 in the absence of sulphate which results in a transientdecline of metabolite levels. To survive under combinedstress conditions of high light and sulphur depletion, plantsactivate several alternative defence mechanisms thatinclude the synthesis of raffinose, trehalose and putrescine.

ACKNOWLEDGMENTS

We wish to thank Dr Bertrand Gakiere and Dr VictoriaNikiforova for fruitful discussions.This work was supportedby funding from the German Service of AcademicExchange (DAAD), Germany (C.W-Z.), the MECESUPgrants of the Faculty of Science of the University of Chile(PCB P06-065-F and PFB-16) and the Max Planck Society.

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Received 19 April 2010; accepted for publication 7 June 2010

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Relative electron transport rate (ETR) versustotal amount of chlorophyll in young leaves of control ( )and sulphate-deprived (�) plants grown in the greenhouse(a) and high-light conditions, 750 mmol m-2 s-1 (b).Figure S2. Representative GC/MS-based metabolite profileof control and sulphate-depleted plants after 3 d of expo-sure to different light intensities. At the 22.00–32.0 minregion of the GC/MS chromatograms: peak identification:(a), pentadecane; (b), octadecane; (c), nonadecane; Rib,ribitol; 1, malate; 2, putrescine 3 TMS; 3, threonate; 4,glutamine; 5, putrescine 4 TMS; 6, shikimate; 7, citrate; dehy-droascorbate; 9, tyrosine. Yet, non-identified compoundsdetected in these chromatogram regions are not indicated.A, B and C are internal standards used for retention index(RI) calculation and chromatogram alignment.Figure S3. Heat map visualization and cluster tree repre-sentations as zoom-in of relative change of metabolite poolsin response to the time-course of sulphur depletion at days0, 1 and 2 of reference light (cf. compare Fig. 4). The heatmap was generated by using log base 2-transformed foldchanges (response ratios relative to sulphur-sufficient andregular-light conditions). Each metabolite is represented bya single row. Each treatment is shown as a single columnobtained by averaging the replicate measurements of eachcondition. Red indicates increased metabolite content,whereas blue indicates decreased contents of metabolites.

The underlying numerical data are presented in SupportingInformation Table S1. Sulphur depletion is indicated by DS(delta S), high light by HL.Figure S4. Principal component analysis (PCA) of metabo-lites during the first two days response period to sulphurdepletion at reference light. Samples representing controlgreenhouse-grown plants under control and sulphur deple-tion. PCA based on data of Fig. 4, and Supporting Informa-tion Fig. S3 and Table S1.Table S1. Metabolites determined in extracts of Arabidop-sis leaves during the time-course in the greenhouse with orwithout sulphur depletion, in high-light conditions or acombination of sulphate deficiency and high-light stress.%C ratio represents the average of the relative concentra-tion at the stress condition to the average of relativeconcentration in control plants. The results observed inArabidopsis leaves subjected to sulphate deficiency in thegreenhouse were not regarded in this table. Differences inmetabolites levels that were significant are indicated in boldformat. MX, methoxyaminated derivate.Table S2. The most influential metabolites, which contrib-ute to the components 1, 2 and 3 derived from the principalcomponent analysis (PCA). Values in bold are metabolitesinvolved in response to sulphate deprivation (Nikiforovaet al. 2005a). (1) Thiol compounds detected by high-performance liquid chromatography (HPLC); and (2)GC/MS analysis. MX, methoxyaminated derivate.Table S3. Averaged yield of chlorophyll fluorescence atPPFD of 365 mmol (m2s) at days 1, 2, 3 and 4 of the time-course experiment. Numerical values correspond toFig. 1b.

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1988 C. Wulff-Zottele et al.

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