Abstract

Glutathione, NAD, and NADP are key nonprotein redox couples in the aqueous phase of virtually all cells, whereas in plant cellsascorbate also plays an important role in redox homeostasis. This work presents the development and validation of plate reader assaysthat allow rapid analysis of these four redox couples in extracts of Arabidopsis leaves. Analytical methods were adapted and validated forspeciWc measurement of oxidized and reduced forms. Oxidized and reduced forms of glutathione and ascorbate, as well as NAD+ andNADP+, were measured in HCl extracts, NADH, and NADPH in parallel alkaline extracts. Both standards and extracts gave linear assayresponses, and recovery quotients of added metabolites through the extraction procedure were generally high. The plate reader methodwas validated against more conventional spectrophotometric assays and also, for glutathione, by HPLC analysis. The method was shownto yield quantitative data for six independent extracts with a total sample preparation and analysis time of 4 h. Analysis of the four redoxcouples throughout Arabidopsis rosette development showed that redox states were relatively constant but that total pools of NAD, glu-tathione, and ascorbate were signiWcantly modiWed by day length and developmental stage. 2007 Elsevier Inc. All rights reserved.

Keywords: Pyridine nucleotides; GSH; GSSG; Dehydroascorbate; Redox state; Redox metabolites; Oxidative stress

ModiWcations in redox state play important roles inmediating or modulating signaling linked to developmentalprocesses and interactions with the environment in bothplants and animals. Key players in determining cellularredox status are the thioldisulWde buVer, glutathione,1 andpyridine nucleotides, which are central to redox metabolismand maintenance of intracellular redox status [1,2]. Ascor-bate (vitamin C) also plays an important role as a generalantioxidant and, in plants, as the main nonprotein reduc-tant for H2O2-detoxifying peroxidases [3]. Recent work has

established that the content and/or reduction status of bothascorbate and glutathione play roles in the control of plantdevelopment as well as in responses to symbionts and path-ogenic organisms [412]. Enrichment of both ascorbate andglutathione contents in plant tissues has been the focus ofstudies seeking to manipulate plant stress resistance andnutritional value [1315].

Key players in maintaining the redox status of ascorbateand glutathione are NAD(P)H pools. Thus, there is closeinterplay among NAD(P), ascorbate, and glutathione inplant metabolism and environmental responses [16]. More-over, total NAD pools may show considerable plasticity inAnalytical Biochemistry 3

A plate reader method for the mglutathione, and ascorbate in tiss

proWling during Arabido

Guillaume Queval,

Institut de Biotechnologie des Plantes, UMR CNRS 8

Received 18 SeAvailable online 0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2007.01.005

* Corresponding author. Fax: +33 169153424.E-mail address: graham.noctor@u-psud.fr (G. Noctor).

1 Ascorbate, glutathione, NAD, and NADP are used here as genericterms that denote oxidized and reduced forms of each redox couple with-out distinction. SpeciWc reduced forms are denoted ASC, GSH, NADH,and NADPH, whereas respective oxidized forms are termed DHA, GSSG,NAD+, and NADP+.ANALYTICALBIOCHEMISTRY

63 (2007) 5869

www.elsevier.com/locate/yabio

easurement of NAD, NADP, ue extracts: Application to redox psis rosette development

Graham Noctor

618, Universit Paris XI, 91405 Orsay cedex, France

ptember 200610 January 2007plants in response to factors such as H2O2 and oxidativestress [17,18]. Such changes are potentially important indetermining metabolic integration and stress resistance inplants [19,20]. Indeed, NAD synthesis and contents arereceiving renewed attention as roles of NAD in signalingprocesses are becoming apparent [2123].

Plate reader method for redox proWling / G. Queva

Despite the importance of interplay among these redoxcouples in governing the physiological outcome of stressexposure, and despite the large body of data generated forglutathione and ascorbate in various stress conditions, veryfew studies have generated data that provide an integratedproWle of the four compounds. It is still unclear to whatextent ascorbate, glutathione, NAD, and NADP areaVected in a coordinated manner and to what degree theirstatus changes independently.

To facilitate the analysis of these questions, we havedeveloped plate reader assays that produce quantitativedata on ascorbate, glutathione, NAD+, and NADP+ byrapid analysis of acid extracts. Parallel analysis of alkalineextracts from the same tissue allows determination ofNADH and NADPH. The method allows convenient andaccurate measurement of pool sizes and reduction statesand has been applied to establish how the status of thesefour key redox couples changes throughout development inleaves of the model plant, Arabidopsis thaliana.

Materials and methods

Unless stated otherwise, all reagents were obtained fromSigma (Saint Quentin Fallavier, France). Glutathione disul-Wde (GSSG),2 glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) were obtained fromRoche Diagnostics (Meylan, France). Assays were devel-oped on a Multiskan Spectrum variable wavelength platereader (Thermo Labsystems, Cergy Pontoise, France) usingCorning 96-well UV-transparent plates and a Wnal assayvolume of 0.2 ml in all cases. Where indicated, assays werealso performed on a Cary 50 UVVis spectrophotometer(Varian, Les Ulis, France). All solutions used MilliporeWltered water. HPLC analysis was performed on a WatersAlliance instrument (Saint Quentin en Yvelines, France)with a Waters 2475 multiwavelength Xuorescence detectorset at ex D 380 and em D 480. The reverse-phase columnwas a Waters Symmetry C18 (150 4.6 mm i.d., 3.5m)with a Sentry guard column (10 2.1 mm i.d.).

Plant material and sampling

Seeds of A. thaliana (ecotype Columbia) were sown insoil in a controlled environment growth chamber at 8 hphotoperiod and irradiance of 200 mol.m2 s1 at the leafsurface. Other conditions were 20/18 C day/night tempera-ture and 60% humidity. Following germination, seedlingswere transferred to individual pots of 7 cm diameter andwatered with nutrient solution twice per week. At 37 days

2 Abbreviations used: GSSG, glutathione disulWde; GR, glutathione re-ductase; G6PDH, glucose-6-phosphate dehydrogenase; FW, fresh weight;AO, ascorbate oxidase; ASC, reduced ascorbate; DHA, dehydroascorbate;DTT, dithiothreitol; GSH, reduced glutathione; DTNB, 5,5-dithiobis(2-nitro-benzoic acid); VPD, 2-vinylpyridine; PMS, phenazine methosulfate;

DCPIP, dichlorophenolindophenol; ADH, alcohol dehydrogenase; RSD,relative standard deviation; SE, standard error; NEM, N-ethylmaleimide.l, G. Noctor / Anal. Biochem. 363 (2007) 5869 59

after sowing, the photoperiod was increased to 16 h. Sam-ples of approximately 100 mg fresh weight (FW) rosette leafmaterial were taken at the developmental stages indicated,taking care to exclude material from the stem and hypo-cotyl. For young plants, samples consisted of severalrosettes suYcient to obtain 100 mg FW. For older plants,100-mg samples consisted of mixed material from at leasttwo diVerent leaves. Following weighing, samples wereintroduced into 2-ml Eppendorf tubes, rapidly frozen in liq-uid nitrogen, and stored at 80 C until extraction.

Extractions

All extraction steps were performed at 4 C or belowusing an extraction medium/FW ratio of 1 ml/100 mg. Sam-ples were ground in liquid nitrogen and then extracted into1 ml of 0.2 N HCl. The homogenate was transferred toEppendorf tubes, aliquots were withdrawn for the chloro-phyll assay as described below, and the remainder was cen-trifuged at 16,000g for 10 min at 4 C. Two aliquots of thesupernatant were taken and neutralized independently. TheWrst aliquot, for the assays of NAD+ and NADP+, con-sisted of 0.2 ml that was incubated in boiling water for1 min, rapidly cooled, and neutralized as follows. First, 20lof 0.2 M NaH2PO4 (pH 5.6) was added, followed by thestepwise addition of aliquots of 0.2 M NaOH. The samplewas vortexed after each addition, and the pH was veriWedwith pH indicator paper. The Wnal pH of all samples wasbetween 5 and 6, requiring approximately 0.16 ml of 0.2 MNaOH. To assay ascorbate and glutathione, a secondsupernatant aliquot of 0.5 ml supernatant was neutralizedas above, without heating, with approximately 0.4 ml of 0.2M NaOH in the presence of 50l of 0.2 M NaH2PO4 (pH5.6). The Wnal pH of the neutralized acid extracts wasbetween 5 and 6. To measure NADH and NADPH, parallelleaf samples were extracted as for NAD+ and NADP+

except that the extraction medium was 0.2 M NaOH andthe heated supernatant aliquot was neutralized with 0.2 NHCl to a Wnal pH of between 7 and 8.

Plate reader assays

For all assays, the reaction mix was homogenized byprogrammed shaking. Following initiation of each reactionas stated below, the mix was shaken twice and then read-ings were taken at the appropriate wavelength every 23 swith programmed mixing by shaking between each reading.For the endpoint assay of ascorbate, the Wnal absorbancewas measured 5 min after the initiation of ascorbate oxida-tion. For other compounds, the absorbance was linear overat least 3 min. Rates were calculated by linear regression ofcurves generated for the Wrst Wve points ( Wrst 90 s).

AscorbateAscorbate was measured by a method adapted fromRefs. [24] and [25]. This assay measures the A265 that is

60 Plate reader method for redox proWling / G. Queva

speciWcally removable by ascorbate oxidase (AO), whichconverts reduced ascorbate (ASC) to nonabsorbing oxi-dized forms. ASC is measured without pretreatment ofextracts; ASC and the relatively stable oxidized form, dehy-droascorbate (DHA), are measured together as totalascorbate after conversion of DHA to ASC by incubationwith thiols such as dithiothreitol (DTT). AO was dissolvedin 0.2 M NaH2PO4 (pH 5.6) at 40 U.ml

1, divided into ali-quots of 0.2 ml, and stored at 20 C. Each day, an aliquotwas freshly thawed and unused enzyme was discarded atthe end of the experiment. To assay ASC, triplicate aliquotsof 20l neutralized supernatant (unless stated otherwise)were introduced into plate wells containing 0.1 ml of 0.2MNaH2PO4 (pH 5.6) and 75l water. The solutions weremixed twice by programmed shaking, and then A265 wasrecorded and 5l AO was added. Solutions were remixedby shaking, and the decrease in A265 value was monitoredIn general, a stable value was reached within 1 to 2 min.Values were taken 5 min after the addition of AO. To assaytotal ascorbate, 0.1 ml neutralized supernatant was Wrstadded to 0.14 ml of 0.12 M NaH2PO4 (pH 7.5) and 10l of25 mM DTT, and solutions were incubated for 30 min atroom temperature unless stated otherwise (see Fig. 2 later).Triplicate aliquots of this solution were then assayed asdescribed for ASC.

Glutathione

Glutathione was measured by the recycling assayinitially described by Tietze [26] by adapting methods inRef. [27]. The method relies on the GR-dependent reduc-tion of 5,5-dithiobis(2-nitro-benzoic acid) (DTNB, Ell-mans reagent), monitored at 412 nm. Withoutpretreatment of extracts, the method measures total gluta-thione, that is, reduced glutathione (GSH) plus GSSG.SpeciWc measurement of GSSG was achieved by pretreat-ment of extract aliquots with 2-vinylpyridine (VPD), asdescribed by GriYth [28]. GR was freshly prepared eachday by centrifugation of an (NH4)2SO4 suspension andresuspension of the pellet to 20 U.ml1 in 0.2 M NaH2PO4(pH 7.5) and 10 mM EDTA. To measure total glutathione,triplicate aliquots of 10l neutralized extract (unless statedotherwise) were added to plate wells containing 0.1 ml of 0.2M NaH2PO4 (pH 7.5), 10 mM EDTA, 10l of 10 mMNADPH, 10l of 12 mM DTNB, and 60l of water. Thereaction was started by the addition of 10l GR. Afterautomatic mixing by shaking, the increase in A412 was mon-itored for 5 min. Standards were run concurrently in thesame plates as triplicate assays of 0 to 1 nmol GSH in thewell. Rates generally were calculated over the Wrst 90 s andin all cases were corrected for GSH-independent reductionof DTNB by subtraction of the mean value of triplicateblank assays (0 GSH). GSSG was measured by the sameprinciple after incubation of 0.2 ml neutralized extract with1l VPD for 30 min at room temperature to complex GSH.

To remove excess VPD, the derivatized solution was centri-fuged twice and triplicate 20-l aliquots (unless statedl, G. Noctor / Anal. Biochem. 363 (2007) 5869

otherwise) of the Wnal supernatant were assayed asdescribed above. GSSG standards run concurrently weresubjected to the same VPD derivatization as the extracts,and amounts in the plate well ranged from 0 to 80 pmol.Rates were calculated as for total glutathione and correctedby subtraction of the blank (0 GSSG).

Total thiols

Total nonprotein thiols were assayed in neutralized acidextracts as DTNB-reactive thiols using GSH as standard.For plate reader assays, each well contained 0.1 ml of 0.2 MNaH2PO4 (pH 7.5), 10 mM EDTA, 10l of 12 mM DTNB,and 90l extract. For standards, extract was replaced by 0,10, 20, and 50 nmol GSH (total volume 0.2 ml). Assays byspectrophotometer used 0.7 ml of 0.12 M NaH2PO4 (pH7.5), 6 mM EDTA, 0.1 ml of 6 mM DTNB, and 0.2 mlextract. For standards, extract was replaced by 0, 10, 20, 50,and 100 nmol GSH (total volume 1 ml). In all cases, A412was measured 5 min after the addition of standard orextract. GSH standards gave A412 close to that predicted bythe established extinction coeYcient for both plate readerand spectrophotometer (13,600 M1 cm1). Assays ofextracts were corrected for A412 in both the absence ofDTNB (plate well or cuvette with extract but no DTNB)and the basal absorbance of DTNB (plate well or cuvettewith DTNB but no extract or standard), which were mea-sured in parallel.

Thiol analysis by HPLC

Individual thiols were analyzed by a method adaptedfrom Ref. [27]. Following neutralization, 0.2ml extract super-natant was added to 0.1 ml of 0.5 M Ches (pH 8.5) and 20lof 10 mM DTT. After incubation at room temperature for30 min to reduce disulWdes, thiols were derivatized by theaddition of 20l of 30 mM monobromobimane. The mixturewas incubated in the dark at room temperature for 15 min,and then the reaction was stopped by the addition of 0.66 mlof 10% (v/v) acetic acid. The derivatized mix was centrifugedat 10,000g for 10 min, and 0.9 ml supernatant was Wlteredthrough 0.2m mesh into autosampler vials. Vials wereloaded into the HPLC, and 50l was injected onto the col-umn. Bimane derivatives were separated by isocratic elutionwith 10% methanol and 0.25% acetic acid (pH 4.3) at a Xowrate of 0.8 ml/min and a column temperature of 40 C. Peakswere identiWed by reference to authentic standards and werequantiWed according to mixed standard solutions using qua-dratic curve Wtting. Thiolbimane derivatives eluted at6.5 min (Cys), 7.1min (Cys-Gly), 10 min (-Glu-Cys), and12.2 min (glutathione).

Pyridine nucleotides

Pyridine nucleotides were assayed by adapting methods

described by Monger and coworkers [29]. The assayinvolves the phenazine methosulfate (PMS)-catalyzed

Plate reader method for redox proWling / G. Queva

reduction of dichlorophenolindophenol (DCPIP) in thepresence of ethanol and alcohol dehydrogenase (ADH) (forNAD+ and NADH) or glucose 6-phosphate and G6PDH(for NADP+ and NADPH). Reduced and oxidized formsare distinguished by preferential destruction in acid or base.To assay NAD+ and NADH, ADH was freshly dissolved in0.1 M Hepes (pH 7.5) and 2 mM EDTA to a concentrationof 2500 U.ml1. Unless stated otherwise, triplicate aliquotsof 20l neutralized supernatant were introduced into platewells containing 0.1 ml of 0.1 M Hepes (pH 7.5), 2 mMEDTA, 20l of 1.2 mM DCPIP, 10l of 20 mM PMS, 25lwater, and 10l ADH. The reaction was started by theaddition of 15l absolute ethanol. Following automaticmixing by shaking, the decrease in A600 was monitored for5 min. Contents were calculated by reference to standardsrun concurrently (040 pmol NAD+ or NADH in the well),and unknowns and standards were corrected for absor-bance decreases measured for triplicate blank assays(0 NAD+ or NADH). To assay NADP+ and NADPH,G6PDH was freshly prepared each day by centrifugation ofan (NH4)2SO4 suspension and resuspension of the pellet to200 U.ml1 in 0.1M Hepes (pH 7.5) and 2 mM EDTA.Triplicate aliquots of 30l neutralized supernatant (unlessstated otherwise) were introduced into plate wells contain-ing 0.1 ml of 0.1 M Hepes (pH 7.5), 2 mM EDTA, 20l of1.2 mM DCPIP, 10l of 20 mM PMS, 10l of 10 mM glu-cose 6-phosphate, and 30l water. The reaction was startedby the addition of 10l G6PDH. Following automatic mix-ing by shaking, the decrease in A600 was monitored for5 min and rates were calculated over the Wrst 2 min usingrelevant standards and blank correction, as describedabove for NAD+.

Recoveries of added standards through the extraction procedure

Recovery quotients of known amounts of added metab-olites were examined as follows. Except for NADH andNADPH, approximately 1 g of Arabidopsis leaf materialwas ground to a homogeneous powder in liquid nitrogen.For each metabolite, triplicate aliquots of approximately100 mg powder were transferred to precooled mortars andextracted into either 0.2 N HCl or 0.2 N HCl spiked with aknown amount of the metabolite in question. In parallel,triplicate aliquots of the spiked HCl solution were takenthrough the sample preparation procedure in the absenceof leaf material, that is, neutralized and heated (for NAD+

and NADP+). Exact contents of each aliquot of homoge-neous powder were estimated as chlorophyll, and the recov-ery of metabolites was estimated by subtraction of themean leaf contribution to the spiked samples The sameprocedure was used to analyze recoveries of NADH andNADPH except that extraction was into either 0.2 MNaOH or 0.2 M NaOH plus NADH or NADPH and sam-ples of chopped and homogenized leaf material were pre-

weighed to enable calculation of leaf contents on an FWbasis. To separate the inXuence of the leaf sample from thatl, G. Noctor / Anal. Biochem. 363 (2007) 5869 61

of the neutralization/heating step on recoveries of pyridinenucleotides, recovery quotients were calculated both withreference to external standard curves and with reference tostandards subjected to the sample preparation steps.

Chlorophyll

Chlorophyll was estimated in acid extracts as pheophy-tin (stoichiometric conversion occurs by acid-induced lossof magnesium from chlorophyll). After the addition of acidto leaf powder, duplicate aliquots of the homogenate (50l)were withdrawn and added to 1 ml acetone and 200lwater. The acetone extract was vortexed, stored at 4 Cuntil completion of the above assays (generally 90 min), andcentrifuged to remove insoluble material, and the clearsupernatant was assayed at 666 and 655 nm. Pheophytinconcentration was calculated using established extinctioncoeYcients according to the following equation: Pheophy-tin (g.ml1) D (A666 6.75) + (A655 26.03).

Data analysis

Absorbance changes were calculated automatically byplate reader or spectrophotometer software and were pro-cessed in Microsoft Excel. Variability was expressed as rela-tive standard deviation (RSD) for percentage deviation orstandard error (SE) for absolute deviation. Line Wtting wasperformed by the regression analysis tool in SigmaPlot. Tocalculate glutathione redox potential, the glutathione mid-point redox potential (E0) at pH 7 and 25 C was taken as 230 mV and the highest glutathione concentration (GSH +2 GSSG) measured in wild-type leaves was taken as 5 mM.This concentration value is close to the values derived fromlabeling of glutathione in Arabidopsis suspension cells [30]and to concentrations that can be inferred from Arabidopsisleaf glutathione contents, assuming that glutathione con-centrations are very low in the leaf cell vacuole. For a halfcell involving a two-electron transfer under standard condi-tions, the Nernst equation for calculation of the actualredox potential can be simpliWed to ED E0 (29.6log10[Reduced Form] / [Oxidized Form]). For the glutathi-one half cell (2 GSH ! GSSG + 2e + 2H+), this equationbecomes ED 230 mV (29.6 log10([GSH]2 / [GSSG]).

Results

Typical standard curves for plate reader assays of ascor-bate, glutathione, NAD, and NADP are shown in Fig. 1.Unlike the enzymatic methods used for glutathione andpyridine nucleotides, which rely on kinetics analysis, themethod for ascorbate is an endpoint assay and quantitiesusually are calculated according to the commonly usedextinction coeYcient of 14,000 M1 cm1 [31]. Fig. 1A com-pares A265 for equivalent concentrations of ascorbate in thespectrophotometer with a conventional 1-cm path (white

circles) and plate reader (black circles), where we calculatedfrom well dimensions that an assay volume of 0.2 ml

62 Plate reader method for redox proWling / G. Queva

represented an optical path length of 5 mm. Using standardsolutions of ascorbate, the response of the spectrophotome-ter was close to the value predicted from the extinctioncoeYcient, with 50M giving A265 of approximately 0.7(Fig. 1A). The plate reader response was very close to 50%of this value (Fig. 1A). Therefore, all of the plate readerdata below for ascorbate were analyzed with the conversionfactor of 0.1 mM D A412 of 0.7, that is, 7000 M1 For othercompounds, unknown quantities always were calculatedaccording to known standards run concurrently. Figs. 1B toD show that a linear response to standards was observedfor each compound. Glutathione as GSH gave a linearresponse up to 1 nmol in the plate well (Fig. 1B). When theglutathione assay response was tested against GSSG, theresponse was also linear up to at least 0.2 nmol (data notshown), although (as predicted) an approximately twofoldgreater response was observed on a molar basis relative toGSH (see Fig. 3 later). Oxidized and reduced forms ofNAD gave a very similar response (Fig. 1C), as did bothforms of NADP (Fig. 1D).

A key factor in the assay of redox couples is distinguish-ing between reduced and oxidized forms. For this, weadapted conventional techniques and veriWed their applica-bility to the plate reader assays. In the case of ascorbate, theassay measures only the reduced form (ASC), and detection

Fig. 1. Typical standard curves for plate reader assays of ascorbate, gluta-thione, NAD, and NADP. (A) Absorbance at 265 nm versus ascorbateconcentration. White symbols represent spectrophotometric assay withstandard 1-cm cuvette, and black symbols represent plate reader assay.(B) Rates of absorbance change at 412 nm versus amount of GSH in theplate reader well. (C) Rates of absorbance change at 600 nm versusamount of NAD+ (black circles) and NADH (white circles) in the platewell. (D) Rates of absorbance change at 600 nm versus amount of NADP+

(black circles) and NADPH (white circles) in the plate well.

[Ascorbate] (M)0 20 40 60

A26

5

0.0

0.4

0.8

1.2

Glutathione (nmol)0.0 0.4 0.8 1.2

A41

2.m

in-1

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1.5

2.0

NAD (pmol) )0 10 20 30 40 50

A60

0.m

in-1

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NADP (pmol0 10 20 30 40 50

A60

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in-1

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A B

C Dof DHA requires prior conversion to this form, for whichDTT was used. The eYcacy of DHA conversion to ASCl, G. Noctor / Anal. Biochem. 363 (2007) 5869

was dependent on both the incubation time and the DTTconcentration (Fig. 2). Using a DHA concentration in theincubation (400M) that is at the upper limit of that foundin extracts, optimal results were obtained with 1 mM DTTand 30 min incubation (Fig. 2A). Whereas 5 mM DTT didnot allow full conversion of DHA to ASC, even after60 min incubation (Fig. 2B), incubation with 20 mM DTTcaused even less eVective reduction (Fig. 2C). In the lattercase, failure to reduce DHA was accompanied by decreasesin ASC, suggesting that high DTT concentrations eithercause destruction of ASC during the incubation (e.g.,through free radical-catalyzed reactions) or interfere withthe assay due to carryover. To measure total ascorbate in

Fig. 2. EVects of varying DTT concentrations and incubation times onascorbate and DHA yield. Ascorbate (white circles) and DHA (black cir-cles) were incubated at a concentration of 400 M with DTT at 1 mM (A),5 mM (B), and 20 mM (C). The Wnal concentration of ascorbate or DHAin the plate well was 40 M, and predicted A265 after the addition ofAO D 0.28. Values at time 0 are in the absence of DTT. At each time point,data are duplicate points obtained in two separate experiments. Wheretwo points are not apparent, this is due to symbol overlap.

A

B

C

Time (min)0 20 40 60 80

A26

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0.2

0.3

0.4

Time (min)0 20 40 60 80

A26

5

0.0

0.1

0.2

0.3

0.4tissue extracts, therefore, we used 1 mM DTT and an incu-bation period of 30 min.

Plate reader method for redox proWling / G. Queva

Methods to distinguish between GSH and GSSG in theGRDTNB assay include pretreatment of extracts to com-plex GSH using thiol reagents such as N-ethylmaleimide(NEM) and VPD. VPD was chosen because of its eYciencyat slightly acidic pH and its lack of eVect on GR after carry-over into the assay [28]. Preliminary assays using standardsshowed that VPD was able to rapidly block glutathione sulf-hydryl groups (Fig. 3A), whereas its presence did not greatlyaVect the assay response to GSSG (Fig. 3B). Under our con-ditions, therefore, the inhibitory eVect of VPD was relativelyspeciWc for the assay of GSH. Although VPD has limited sol-ubility in water and most can be removed by centrifugationof VPD-treated solutions prior to assay [28], other controlexperiments indicated that carryover of VPD can have aslight inhibitory eVect on the GRDTNB method (data notshown). Because VPD does not inhibit GR appreciably [28],this eVect probably is observed because any VPD that is car-ried over competes with DTNB for GSH that is formed fromGSSG in the assay. Thus, to account for possible assay arti-

Fig. 3. Selective measurement of GSSG by derivatization of GSH withVPD. (A) Time course of GSH removal by VPD at 1:200 dilution (v/v,black squares) and 1:40 dilution (v/v, white squares). GSH was present inthe incubation at 4 M, and the quantity transferred to the assay well was80 pmol. (B) EVect of VPD on the response of glutathione solutions withdiVerent GSH/GSSG values. White circles represent no VPD treatment,and black symbols represent treatment with VPD for 30 min at VPD dilu-tions of 1:200 (circles) and 1:40 (triangles). The amount of GSH equiva-lents transferred to the assay well was 80 pmol throughout. For panel A, atypical experiment is shown. Similar results were obtained in other experi-ments. For panel B, values are means of two independent experiments,and error bars indicate actual values.

A

Time (min)0 20 40 60 80

A41

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in-1

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% glutathione as GSSG0 20 40 60 80 100

A41

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0.2facts, standard curves for GSSG always were generated usingVPD-treated standards.l, G. Noctor / Anal. Biochem. 363 (2007) 5869 63

A spectrophotometric assay has been described forassay of NADP+ and NADPH by extraction into neutralbuVer followed by assay of NADPH at 340 nm with orwithout pretreatment to interconvert the two forms [32].However, this method measures NADP(H) in the micro-molar to millimolar range [32], whereas the recycling assayusing DCPIP reduction is sensitive down to nanomolarconcentrations (standard curves for pyridine nucleotides inFig. 1 represent 50200 nM in the plate well). Using theDCPIP reduction method, speciWc assay of reduced andoxidized pyridine nucleotides can be achieved by preferen-tial destruction in acid and alkali. Oxidized forms aredestroyed in alkaline conditions, and reduced forms aredestroyed by acid [29]. This procedure was Wrst checkedusing standard solutions. In our conditions, heating wasfound to be necessary for eYcient removal of forms tar-geted for destruction. When extracts were not heated, gen-erally more than 10% of the targeted forms remained (datanot shown). However, when acid extracts were heatedbrieXy, negligible NADPH and no NADH were detected(Fig. 4). Heating of alkaline extracts caused complete lossof NAD+ and left only trace amounts of NADP+ (Fig. 4).Destruction of pyridine nucleotides was not 100% speciWc,and heating in acid or base caused small but signiWcantloss of nontargeted forms. Control experiments with stan-dards showed that the mean losses of NAD+ and NADP+

through heating in acid were 8 and 13%, respectively,whereas losses of NADH and NADPH after heating inNaOH were 12 and 18%. Other experiments revealed thatthe brief heating of acid extracts to remove NADH andNADPH had a slight but signiWcant eVect on measuredascorbate and glutathione values, in particular causingsome decrease in the reduction state. Because of this, HClextracts were split into two aliquots. One aliquot was neu-tralized directly for measurement of ascorbate and gluta-thione, and the other aliquot was heated prior toneutralization for assay of NAD+ and NADP+ (for furtherdetails, see Materials and methods).

Fig. 4. Selective plate reader assay of reduced and oxidized pyridine nucle-otides by preheating in acid and base. Standard solutions were dilutedinto 0.2 N HCl or 0.2 M NaOH, heated 1 min at 95 C, neutralized, andassayed as described in Materials and methods. Black bars represent oxi-

HCl NaOH

NAD

+

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NAD

HN

ADP+

NAD

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+

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NAD

PHdized forms, and white bars represent reduced forms. Where no bar isapparent, the compound was undetectable.

64 Plate reader method for redox proWling / G. Queva

Preliminary assays of NAD and NADP in leaf extractsused 5 U ADH and 0.4 U G6PDH in the assay well. Toimprove reproducibility and sensitivity of NAD(H) andNADP(H) assays in plant extracts, enzyme concentrationswere increased to 25 U ADH and 2 U G6PDH, respec-tively, and amounts were calculated according to standardcurves run in parallel using the same enzyme concentration.Under these conditions, the response of the assays was lin-ear with respect to volume of leaf extract, and the Wttedlines passed close to the origin (Fig. 5). DiVerences in thetwo slopes for certain compounds reXect diVerences in FWbetween the two extracts. For all data reported below,extract volumes used were intermediate within the rangesshown in Fig. 5.

Recoveries of metabolites through the extraction proce-dure in the presence of leaf sample were on the order of 75to 90% except for DHA, where recovery was somewhat var-iable (Table 1). Recovery of DHA was dependent on reduc-tion with DTT and was negligible when assayed withoutthis pretreatment (data not shown). Less than 10% of the

Fig. 5. Linearity of the assays with increasing amounts of extract in theplate well. Leaf material was extracted as described in the text, and threediVerent volumes of the Wnal sample extract were assayed. Data are shownfor two independent alkaline extracts (NADH and NADPH) or acidextracts (other assays). Black circles represent extract 1, and white circlesrepresent extract 2. Where data points are not visible, they are hidden by

pmol

det

ecte

d

GSSG

sample volume (l) sample volume (l)0 10 20 30 40

nm

ol d

etec

ted

0

200

400

GLUTATHIONE

0

500

1000

ASC + DHA

0

2

ASC

0

2

4

6

NADPH

0 10 20 30 400

10

20

NADP+

0

10

20

NADH

0

3

6

NAD+

0

15

30

45

pmol

det

ecte

d

pmol

det

ecte

dthe corresponding data points for the other extracts. Regressions are lin-ear without constraint through the origin.l, G. Noctor / Anal. Biochem. 363 (2007) 5869

Table 1Recovery quotients of metabolites through the extraction procedure

Note. Known amounts of metabolite standards were added to 0.2 MNaOH (for NADH and NADPH) or 0.2 N HCl (for other compounds),and the spiked media were used to extract triplicate aliquots of homoge-nized leaf powder. Recoveries were calculated relative to triplicate parallelextracts performed into unspiked media. For pyridine nucleotides, data inparentheses show percentage recoveries adjusted for extract-independentloss of metabolites, calculated against standards treated exactly asextracts. For further details, see Materials and methods.

Redox couple Recovery (%)

Reduced form Oxidized form

ASC/DHA 92.1 1.4 58.4 25.0GSH/GSSG 76.7 0.5 93.7 8.1NADH/NAD+ 76.6 2.1 (87.1 2.4) 79.6 13.5 (86.1 14.7)NADPH/NADP 82.1 5.4 (100.9 6.6) 72.9 1.2 (84.1 1.4)

Table 2Comparison of leaf contents assayed by spectrophotometer and platereader

Note. Data are from young Arabidopsis plants sampled at 15 to 31days after sowing. The two methods were used for assay of the sameextracts treated identically. For plate reader assays, extract volumes inthe plate well were 20 l except for total glutathione, where 10 l wasassayed. For spectrophotometer assay, volumes were 20 l (pyridinenucleotides, glutathione, and GSSG) and 100 l (ASC and ASC +DHA). Units are in mol.g1 FW for ascorbate and DHA and are innmol.g1 FW for other compounds. All values are means SE of fourindependent leaf extracts except for GSSG (three independentextracts).

Metabolite Leaf contents

Plate reader Spectrophotometer

ASC 2.51 0.11 2.42 0.12ASC + DHA 3.11 0.13 2.83 0.15Total glutathione 493 45 535 34GSSG 28.4 7.1 25.2 6.3NADH 2.8 0.4 3.9 1.5NAD+ 13.1 1.3 14.0 0.8NADPH 9.9 0.1 7.6 0.3NADP+ 9.7 0.6 9.9 1.3

Table 3Assay-to-assay variability and sample-to-sample variability for redoxmetabolites measured by plate reader assay

Note. Between-assay variability shows the mean RSD values among tripli-cate assays, averaged for six independent extracts (n D 6). Variability dueto the extraction and condition-independent biological diVerence (rightcolumn) is expressed as the means of RSD values among three extracts

Metabolite Variability (RSD, %)

Between assays Between plants

ASC 3.7 0.4 18.4 4.3ASC + DHA 4.2 1.1 17.4 4.9Total glutathione 2.7 0.7 15.6 2.5GSSG 6.0 1.3 35.0 6.2NAD+ 8.1 2.3 16.5 2.7NADH 19.1 7.7 22.5 6.2NADP+ 6.0 1.8 14.0 3.0NADPH 4.4 1.1 20.2 4.2from diVerent plants sampled in the same growth conditions, obtained insix independent experiments (n D 6).

Fig. 6. Plate reader assays of total contents and reduction states of ascorbate,Total contents are in nmol.g1 FW except for ascorbate, where values are in

0

Days after 0 20 40 60

NAD

P

0

10

20

30GSH equivalents; that is, GSSG values are multiplied by 2 before subtraction extracts of diVerent plants. glutathione, NAD, and NADP throughout Arabidopsis shoot development.mol.g1 FW. Percentage reductions of the glutathione pool are calculated in

20 40 60 80

DP %

NADPH

0

20

40

60

80

sowingPlate reader method for redox proWling / G. Queval, G. Noctor / Anal. Biochem. 363 (2007) 5869 65

added GSH was detected when extracts were treated withVPD (data not shown). Loss of pyridine nucleotides was onthe order of 20% (Table 1). Recoveries were improved whenthey were calculated relative to standards taken throughthe extraction procedure in the absence of leaf extract(Table 1, values in parentheses). This shows that loss of pyr-idine nucleotides was due partly to factors present in theleaf tissue and partly to the extraction and heating proce-dure itself. In the case of NADPH, loss was independent ofleaf extracts given that recoveries were 100% when related

to standards taken through the extraction and heating step(Table 1).

To further validate the assay, metabolites were extractedfrom young leaves and assayed in parallel by plate readerand spectrophotometer. Table 2 shows that very similarvalues were obtained by both methods. For example, in thecase of GSSG, which usually is the minority form of gluta-thione, plate reader analysis gave values that were0.93 0.17 of values measured in the same extracts byspectrophotometric assay (n D 14 independent leaf extracts

Total leaf contents % Reduction

NA

NAD

% N

ADH

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20

40

60

80

NAD

0

20

40

Glutathione %

GSH

0

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40

60

80

100

Glu

tath

ione

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250

500

750

0

20

40

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120

Asco

rbat

e

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4

6

8 Ascorbate % ASC

Photoperiod8 h 16 h

Flowering

Photoperiod8 h 16 h

FloweringVegetative growth Vegetative growthfrom the total glutathione values. Data are means SE of three independent

66 Plate reader method for redox proWling / G. Queva

in Wve independent experiments). An advantage of the 96-well plate reader method is the ease of replicate assays ofextracts. Table 3 shows that the mean RSD between assayswas less than 10% for all compounds except NADH, whichwas the least abundant form of pyridine nucleotide (Table 2and Fig. 6). We also tested variability between individualplants growing in identical controlled conditions (Table 3,right column). The experimental design involved indepen-dent analysis of single extracts from three diVerent plants,with the value for each metabolite in each extract beingobtained from the mean of the triplicate assay. Therefore,these RSD values represent biological variation as well asvariation due to the extraction and sample preparation, andthey were between 14 and 35% and relatively constant oversix independent experiments (Table 3).

The assay was applied to the analysis of how the redoxmetabolites change over the course of Arabidopsis shootdevelopment. At the vegetative (leaf-producing) stage, theArabidopsis shoot consists of a low-lying rosette. When theXowering program is initiated, the main stem elongatesgreatly and produces multiple Xowers, and this eventually isaccompanied by leaf senescence. Most known Arabidopsisecotypes, including Columbia, are long-day plants; thatis, Xowering is accelerated by long photoperiods. We grewplants for 5 weeks in short days (8-h photoperiod) and thentransferred them to long-day conditions (16-h photope-riod). Redox metabolites were measured throughout thisdevelopmental program by assay of triplicate leaf samplesfrom diVerent plants at each stage (Fig. 6). The initial stageof vegetative growth involved an increase in total contentsof all four redox couples, most notably those of NAD,ascorbate, and glutathione (Fig. 6, left panels). Ascorbateand NAD showed a further increase after the photoperiodwas lengthened, and both decreased markedly after the ini-tiation of the Xowering program. In contrast, glutathioneshowed an increase in the oldest leaves, that is, when theXower stem was well developed and leaves were beginningto undergo senescence (Fig. 6, left panels). Changes inredox states generally were less marked (Fig. 6, right pan-els). Ascorbate and glutathione remained more than 80%reduced at all stages, although a gradual trend of the gluta-thione pool toward increased oxidation was observed dur-ing development (Fig. 6, right panels). Unlike the highlyreduced ascorbate and glutathione pools, NAD remainedmore than 80% oxidized at all stages. Small changes in theNAD reduction state were observed, and these largelyreXected changes in NAD+ given that NADH contents gen-erally were very constant. Of the four redox couples, theleast variable was NADP, for which no marked trends wereobserved for either total contents or redox state, with thelatter remaining at 50% reduced throughout development(Fig. 6).

The speciWc enzymatic assay of glutathione was furthercompared with total acid-extractable thiols measured byreaction with DTNB and measurement of individual thiols

by precolumn monobromobimane labeling and separationon reverse-phase HPLC with Xuorescence detection. Thel, G. Noctor / Anal. Biochem. 363 (2007) 5869

data in Table 4 show reasonable agreement between valuesfor glutathione, whether measured by HPLC or enzymati-cally on the plate reader or the spectrophotometer. Valueswere close to those found in young Arabidopsis leaves inFig. 6. GSH accounted for 60 to 66% of acid-soluble thiolsextractable from young leaves, whether these were mea-sured by spectrophotometer or plate reader (Table 4).HPLC proWles of bimane-labeled thiols showed that theonly other signiWcant detectable peaks were (in order ofelution) Cys, Cys-Gly, -Glu-Cys, and homoCys. AlthoughhomoCys could not be accurately quantiWed, the summedleaf content of the other three thiols was 17 nmol.g1 FW(Table 4).

Discussion

A wealth of literature data exists on the responses ofascorbate and glutathione to stress in plants [13], and gluta-thione status is considered to be an important oxidativestress marker in most cells [1,2]. Although ascorbate andglutathione have key functions as antioxidants, both com-pounds also play important roles in other aspects of metab-olism. Inversely, although most of the focus on NAD andNADP has occurred within the context of studies of meta-bolic regulation, emerging data point to new roles, particu-larly for NAD, in the regulation of gene expression andresponses to stress in diverse groups of organisms[21,22,33]. Despite the physiological overlap and biochemi-cal interplay among the four redox couples, few studieshave taken an integrated approach to their analysis. Forthis, a rapid and convenient extraction and assay method isrequired. Methods have been described for proWling of

Table 4Comparison of leaf contents of glutathione and total thiols in youngleaves of A. thaliana

Note. Independent acid extractions of three diVerent samples of Arabidop-sis rosettes 15 days after sowing were performed. Following neutraliza-tion, aliquots of extract supernatant were used for HPLC analysis ofthiols after monobromobimane derivatization, enzymatic assay of totalglutathione and GSSG by spectrophotometer and plate reader, and analy-sis of total DTNB-reactive thiols by spectrophotometer and plate reader.For spectrophotometer and plate reader assays, GSH was calculated bysubtraction of GSSG from total glutathione, taking into account that 1GSSG is equivalent to 2 GSH. All values except percentages are innmol.g1 FW. For further details, see Materials and methods.

Compound Leaf contents

HPLC Spectrophotometer Plate reader

Total glutathione 291 6 275 9 242 13GSH 257 6 227 10GSSG 9 2 7 2GSH/Glutathione (%) 93 1 94 1Total thiols 426 16 343 6GSH/Total thiols (%) 60 1 66 1-Glu-Cys 4.2 0.2 Cys-Gly 0.7 0.1 Cysteine 11.8 0.1 redox metabolites in biological samples by HPLC with cou-lometric or diode array detection [34,35]. These methods

Plate reader method for redox proWling / G. Queva

require more specialized equipment or extraction proce-dures than do those used in the current study, where wehave validated a method of general applicability that is ableto proWle the chief nonprotein components of redox statusin biological tissues.

Assays by plate reader yielded values similar to thoseobtained on a conventional spectrophotometer. However,the plate reader assay has several advantages. It is poten-tially more sensitive in terms of sample required. Althoughthe shorter optical path length in the plate reader approxi-mately halves the sensitivity on a concentration basis, this ismore than oVset by the smaller assay volume, meaning thatless sample is required than in a conventional spectropho-tometer assay. The accuracy of the current method is good,with acceptably low between-assay RSD for most com-pounds, and the triplicate assay improves the robustness ofthe method. The principal advantage of the current methodis high sample throughput without the need for expensiveor specialized equipment. Theoretically, on a 96-well platereader, it is possible to adapt the method to measure in trip-licate up to 28 diVerent extracts together with four knownstandard concentrations. Although sample preparation andassay time would increase accordingly, the gain over a con-ventional spectrophotometric assay would be very substan-tial. Here we routinely used a triplicate assay of 6 extractsand four standards. This protocol involves simultaneoususe of up to 30 wells and allows a threefold gain in assaytime, even when compared with a spectrophotometer with acell changer facility. Total analysis time for acid extracts of6 independent samples was 2 h 30 min (1 h sample prepara-tion and 1 h 30 min analysis). For alkaline extracts, the sam-ple preparation was similar but analysis time was less than30 min. We applied the method to Arabidopsis, a genus thathas become the model for functional genomics studies inplants. Thus, comparison of triplicate samples of two geno-types is possible in slightly more than 4 h, and further gainsin time would come from preparation and assay of acid andalkaline extractions in parallel. Total analysis time is animportant consideration in view of the limited stability ofsome of the metabolites and the necessity to minimizepostextraction artifacts due to, for example, gradual oxida-tion of reduced forms of ascorbate and glutathione. Weestimate that the method could be readily adapted for trip-licate analysis of up to 12 tissue extracts without loss ofrobustness due to excessive delays during postextractionassay preparation.

The extraction method was based on procedures com-monly used for assay of pyridine nucleotides and was val-idated by the generally acceptable recoveries. DHA wasthe most problematic compound, with relatively low andsomewhat variable recoveries that could be related toincomplete reduction to ASC (Fig. 2). This compound,however, rarely exceeds 10% of the total tissue ascorbatepool in plants, and so incomplete recovery does notgreatly aVect total ascorbate contents even though it

could cause overestimation of the extent of tissue ascor-bate reduction state. For pyridine nucleotides, wherel, G. Noctor / Anal. Biochem. 363 (2007) 5869 67

reduced and oxidized forms are measured independently,parallel extracts with internal standards could be used toimprove precision, but this would necessarily increaseanalysis time, and we found that although recoveries wereless than 100%, this was due to small systematic loss, asreXected in the reproducible recovery quotients. The cur-rent data on leaf contents and redox states of NAD,ascorbate, and glutathione are similar to values obtainedin studies where these compounds have been measuredindividually in Arabidopsis [25,36,37].

Analysis of changes in redox couples during develop-ment of Arabidopsis revealed that total contents were morevariable than redox states. Standard errors were greater forthe reduction states of NAD and NADP than for those ofascorbate and glutathione. This can be explained partly byan additional source of experimental variation because thereduction state is calculated from two independent extractsfor pyridine nucleotides, whereas in the case of ascorbateand glutathione it is calculated from assays performed onaliquots of the same extracts. Changes in redox states of allfour redox couples, either in whole plant tissue or in speciWcsubcellular compartments, can occur in response toenvironmental stress and/or abrupt changes in irradiance[3842]. The current data show that, during Arabidopsisrosette development in controlled conditions, only limitedchange in redox state is observed in the form of slight varia-tion in NAD redox status and a general trend towardincreased oxidation of the glutathione pool (Fig. 6). Gluta-thione is unique among the four redox couples examined inthat its redox potential is dependent on total concentration(because the concentration term of the Nernst equationrelates to [GSH]2 / GSSG). Calculations from the data ofFig. 6 reveal that although the GSH/GSSG ratio changesduring Arabidopsis leaf development, the estimated gluta-thione redox potential is remarkably stable at around 180mV (Fig. 7). Thus, it appears that adjustments in total glu-tathione concentration act to oVset the changes in GSH/GSSG and that the global redox potentials of all four redox

Fig. 7. Calculated values for GSH/GSSG (white circles) and glutathioneredox potential (black circles) throughout Arabidopsis rosette develop-ment. Values are derived from the glutathione data shown in Fig. 6.

Days after sowing0 20 40 60 80

GSH

/GSS

G

0

10

20

30 -220

-180

-140

-100

Redox potential (mV)

Photoperiod8 h 16 h

FloweringVegetative growthcouples are very stable throughout Arabidopsis shootdevelopment.

68 Plate reader method for redox proWling / G. Queva

In contrast to the stability of tissue redox potentials,the total contents of ascorbate, glutathione, and NAD allshowed clear age- and photoperiod-related trends.Recent studies, notably of Arabidopsis mutants, havehighlighted the importance of total ascorbate andglutathione concentrations in processes such as plantpathogen and plantsymbiont interactions, meristemdevelopment, and light signaling [412,36]. Emerging evi-dence, particularly from work on yeast and mammaliancells, points to roles for NAD in modulating develop-ment and gene expression [4345]. Other studies in plantshave shown that photoperiod and/or growth irradiancecauses adjustments in some components of leaf redoxstate [4648]. The current study shows that both NADand ascorbate increased signiWcantly in response to thetransfer of plants to long days, although the transfer hadlittle immediate eVect on glutathione contents (Fig. 6).Whereas NAD and ascorbate both decreased at the onsetof Xowering, glutathione behaved in an opposing mannerand contents were highest in the oldest leaves. A compar-ison of glutathione contents with total thiols shows thatthe glutathione contents of the oldest leaves (Fig. 6) weresigniWcantly higher than the total soluble thiol contentsof the youngest leaves (Table 4). Thus, the increase in leafglutathione with plant age is not simply the result of itsdecreased use, for example, in the synthesis of phytochel-atins, which if present should be detected by the DTNBassay for total acid-soluble thiols [49]. Interestingly, arole in the control of Xowering recently has been ascribedto changes in both glutathione and ascorbate concentra-tions [8,50]. Ascorbate concentration has also been linkedto growth rate, cell diVerentiation, and pathogen resis-tance in Arabidopsis [6,10,51], whereas enhanced stressresistance is observed in tobacco mutants with high NADcontents [19,20]. Decreases in leaf pyridine nucleotidecontents following Xowering in spinach have beenreported [52]. It remains to be seen whether the generalincreases in NAD and ascorbate that occur throughoutthe vegetative phase of the Arabidopsis life cycle have anyrole in controlling growth or determining age-relateddiVerences in susceptibility to stress.

Acknowledgments

We thank Dorothe Thominet for excellent technicalassistance and Myroslawa Miginiac-Maslow for helpfuladvice on measuring pyridine nucleotides. This work wasfunded partly by the French ANR-Gnoplante programGNP0508, Redoxome.

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A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: Application to redox profiling during Arabidopsis rosette developmentMaterials and methodsPlant material and samplingExtractionsPlate reader assaysAscorbateGlutathioneTotal thiolsThiol analysis by HPLCPyridine nucleotidesRecoveries of added standards through the extraction procedureChlorophyllData analysis

ResultsDiscussionAcknowledgmentsReferences