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ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 363 (2007) 5869
www.elsevier.com/locate/yabio

A plate reader method for the measurement of NAD, NADP,


glutathione, and ascorbate in tissue extracts: Application to redox
proWling during Arabidopsis rosette development
Guillaume Queval, Graham Noctor
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Universit Paris XI, 91405 Orsay cedex, France
Received 18 September 2006
Available online 10 January 2007

Abstract
Glutathione, NAD, and NADP are key nonprotein redox couples in the aqueous phase of virtually all cells, whereas in plant cells
ascorbate also plays an important role in redox homeostasis. This work presents the development and validation of plate reader assays
that allow rapid analysis of these four redox couples in extracts of Arabidopsis leaves. Analytical methods were adapted and validated for
speciWc measurement of oxidized and reduced forms. Oxidized and reduced forms of glutathione and ascorbate, as well as NAD+ and
NADP+, were measured in HCl extracts, NADH, and NADPH in parallel alkaline extracts. Both standards and extracts gave linear assay
responses, and recovery quotients of added metabolites through the extraction procedure were generally high. The plate reader method
was validated against more conventional spectrophotometric assays and also, for glutathione, by HPLC analysis. The method was shown
to yield quantitative data for six independent extracts with a total sample preparation and analysis time of 4 h. Analysis of the four redox
couples throughout Arabidopsis rosette development showed that redox states were relatively constant but that total pools of NAD, glutathione, 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 in


mediating or modulating signaling linked to developmental
processes and interactions with the environment in both
plants and animals. Key players in determining cellular
redox status are the thioldisulWde buVer, glutathione,1 and
pyridine nucleotides, which are central to redox metabolism
and maintenance of intracellular redox status [1,2]. Ascorbate (vitamin C) also plays an important role as a general
antioxidant and, in plants, as the main nonprotein reductant for H2O2-detoxifying peroxidases [3]. Recent work has

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 generic
terms that denote oxidized and reduced forms of each redox couple without distinction. SpeciWc reduced forms are denoted ASC, GSH, NADH,
and NADPH, whereas respective oxidized forms are termed DHA, GSSG,
NAD+, and NADP+.
0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2007.01.005

established that the content and/or reduction status of both


ascorbate and glutathione play roles in the control of plant
development as well as in responses to symbionts and pathogenic organisms [412]. Enrichment of both ascorbate and
glutathione contents in plant tissues has been the focus of
studies seeking to manipulate plant stress resistance and
nutritional value [1315].
Key players in maintaining the redox status of ascorbate
and glutathione are NAD(P)H pools. Thus, there is close
interplay among NAD(P), ascorbate, and glutathione in
plant metabolism and environmental responses [16]. Moreover, total NAD pools may show considerable plasticity in
plants in response to factors such as H2O2 and oxidative
stress [17,18]. Such changes are potentially important in
determining metabolic integration and stress resistance in
plants [19,20]. Indeed, NAD synthesis and contents are
receiving renewed attention as roles of NAD in signaling
processes are becoming apparent [2123].

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

Despite the importance of interplay among these redox


couples in governing the physiological outcome of stress
exposure, and despite the large body of data generated for
glutathione and ascorbate in various stress conditions, very
few studies have generated data that provide an integrated
proWle of the four compounds. It is still unclear to what
extent ascorbate, glutathione, NAD, and NADP are
aVected in a coordinated manner and to what degree their
status changes independently.
To facilitate the analysis of these questions, we have
developed plate reader assays that produce quantitative
data on ascorbate, glutathione, NAD+, and NADP+ by
rapid analysis of acid extracts. Parallel analysis of alkaline
extracts from the same tissue allows determination of
NADH and NADPH. The method allows convenient and
accurate measurement of pool sizes and reduction states
and has been applied to establish how the status of these
four key redox couples changes throughout development in
leaves of the model plant, Arabidopsis thaliana.

59

after sowing, the photoperiod was increased to 16 h. Samples of approximately 100 mg fresh weight (FW) rosette leaf
material were taken at the developmental stages indicated,
taking care to exclude material from the stem and hypocotyl. For young plants, samples consisted of several
rosettes suYcient to obtain 100 mg FW. For older plants,
100-mg samples consisted of mixed material from at least
two diVerent leaves. Following weighing, samples were
introduced into 2-ml Eppendorf tubes, rapidly frozen in liquid nitrogen, and stored at 80 C until extraction.
Extractions

Unless stated otherwise, all reagents were obtained from


Sigma (Saint Quentin Fallavier, France). Glutathione disulWde (GSSG),2 glutathione reductase (GR), and glucose-6phosphate dehydrogenase (G6PDH) were obtained from
Roche Diagnostics (Meylan, France). Assays were developed on a Multiskan Spectrum variable wavelength plate
reader (Thermo Labsystems, Cergy Pontoise, France) using
Corning 96-well UV-transparent plates and a Wnal assay
volume of 0.2 ml in all cases. Where indicated, assays were
also performed on a Cary 50 UVVis spectrophotometer
(Varian, Les Ulis, France). All solutions used Millipore
Wltered water. HPLC analysis was performed on a Waters
Alliance instrument (Saint Quentin en Yvelines, France)
with a Waters 2475 multiwavelength Xuorescence detector
set at ex D 380 and em D 480. The reverse-phase column
was a Waters Symmetry C18 (150 4.6 mm i.d., 3.5 m)
with a Sentry guard column (10 2.1 mm i.d.).

All extraction steps were performed at 4 C or below


using an extraction medium/FW ratio of 1 ml/100 mg. Samples were ground in liquid nitrogen and then extracted into
1 ml of 0.2 N HCl. The homogenate was transferred to
Eppendorf tubes, aliquots were withdrawn for the chlorophyll assay as described below, and the remainder was centrifuged at 16,000g for 10 min at 4 C. Two aliquots of the
supernatant were taken and neutralized independently. The
Wrst aliquot, for the assays of NAD+ and NADP+, consisted of 0.2 ml that was incubated in boiling water for
1 min, rapidly cooled, and neutralized as follows. First, 20 l
of 0.2 M NaH2PO4 (pH 5.6) was added, followed by the
stepwise addition of aliquots of 0.2 M NaOH. The sample
was vortexed after each addition, and the pH was veriWed
with pH indicator paper. The Wnal pH of all samples was
between 5 and 6, requiring approximately 0.16 ml of 0.2 M
NaOH. To assay ascorbate and glutathione, a second
supernatant aliquot of 0.5 ml supernatant was neutralized
as above, without heating, with approximately 0.4 ml of 0.2
M NaOH in the presence of 50 l of 0.2 M NaH2PO4 (pH
5.6). The Wnal pH of the neutralized acid extracts was
between 5 and 6. To measure NADH and NADPH, parallel
leaf samples were extracted as for NAD+ and NADP+
except that the extraction medium was 0.2 M NaOH and
the heated supernatant aliquot was neutralized with 0.2 N
HCl to a Wnal pH of between 7 and 8.

Plant material and sampling

Plate reader assays

Seeds of A. thaliana (ecotype Columbia) were sown in


soil in a controlled environment growth chamber at 8 h
photoperiod and irradiance of 200 mol.m2 s1 at the leaf
surface. Other conditions were 20/18 C day/night temperature and 60% humidity. Following germination, seedlings
were transferred to individual pots of 7 cm diameter and
watered with nutrient solution twice per week. At 37 days

For all assays, the reaction mix was homogenized by


programmed shaking. Following initiation of each reaction
as stated below, the mix was shaken twice and then readings were taken at the appropriate wavelength every 23 s
with programmed mixing by shaking between each reading.
For the endpoint assay of ascorbate, the Wnal absorbance
was measured 5 min after the initiation of ascorbate oxidation. For other compounds, the absorbance was linear over
at least 3 min. Rates were calculated by linear regression of
curves generated for the Wrst Wve points ( Wrst 90 s).

Materials and methods

2
Abbreviations used: GSSG, glutathione disulWde; GR, glutathione reductase; 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(2nitro-benzoic acid); VPD, 2-vinylpyridine; PMS, phenazine methosulfate;
DCPIP, dichlorophenolindophenol; ADH, alcohol dehydrogenase; RSD,
relative standard deviation; SE, standard error; NEM, N-ethylmaleimide.

Ascorbate
Ascorbate was measured by a method adapted from
Refs. [24] and [25]. This assay measures the A265 that is

60

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

speciWcally removable by ascorbate oxidase (AO), which


converts reduced ascorbate (ASC) to nonabsorbing oxidized forms. ASC is measured without pretreatment of
extracts; ASC and the relatively stable oxidized form, dehydroascorbate (DHA), are measured together as total
ascorbate after conversion of DHA to ASC by incubation
with thiols such as dithiothreitol (DTT). AO was dissolved
in 0.2 M NaH2PO4 (pH 5.6) at 40 U.ml1, divided into aliquots of 0.2 ml, and stored at 20 C. Each day, an aliquot
was freshly thawed and unused enzyme was discarded at
the end of the experiment. To assay ASC, triplicate aliquots
of 20 l neutralized supernatant (unless stated otherwise)
were introduced into plate wells containing 0.1 ml of 0.2M
NaH2PO4 (pH 5.6) and 75 l water. The solutions were
mixed twice by programmed shaking, and then A265 was
recorded and 5 l AO was added. Solutions were remixed
by shaking, and the decrease in A265 value was monitored
In general, a stable value was reached within 1 to 2 min.
Values were taken 5 min after the addition of AO. To assay
total ascorbate, 0.1 ml neutralized supernatant was Wrst
added to 0.14 ml of 0.12 M NaH2PO4 (pH 7.5) and 10 l of
25 mM DTT, and solutions were incubated for 30 min at
room temperature unless stated otherwise (see Fig. 2 later).
Triplicate aliquots of this solution were then assayed as
described for ASC.
Glutathione
Glutathione was measured by the recycling assay
initially described by Tietze [26] by adapting methods in
Ref. [27]. The method relies on the GR-dependent reduction of 5,5-dithiobis(2-nitro-benzoic acid) (DTNB, Ellmans reagent), monitored at 412 nm. Without
pretreatment of extracts, the method measures total glutathione, that is, reduced glutathione (GSH) plus GSSG.
SpeciWc measurement of GSSG was achieved by pretreatment of extract aliquots with 2-vinylpyridine (VPD), as
described by GriYth [28]. GR was freshly prepared each
day by centrifugation of an (NH4)2SO4 suspension and
resuspension 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 10 l neutralized extract (unless stated
otherwise) were added to plate wells containing 0.1 ml of 0.2
M NaH2PO4 (pH 7.5), 10 mM EDTA, 10 l of 10 mM
NADPH, 10 l of 12 mM DTNB, and 60 l of water. The
reaction was started by the addition of 10 l GR. After
automatic mixing by shaking, the increase in A412 was monitored for 5 min. Standards were run concurrently in the
same plates as triplicate assays of 0 to 1 nmol GSH in the
well. Rates generally were calculated over the Wrst 90 s and
in all cases were corrected for GSH-independent reduction
of DTNB by subtraction of the mean value of triplicate
blank assays (0 GSH). GSSG was measured by the same
principle after incubation of 0.2 ml neutralized extract with
1 l VPD for 30 min at room temperature to complex GSH.
To remove excess VPD, the derivatized solution was centrifuged twice and triplicate 20-l aliquots (unless stated

otherwise) of the Wnal supernatant were assayed as


described above. GSSG standards run concurrently were
subjected 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 corrected
by subtraction of the blank (0 GSSG).
Total thiols
Total nonprotein thiols were assayed in neutralized acid
extracts as DTNB-reactive thiols using GSH as standard.
For plate reader assays, each well contained 0.1 ml of 0.2 M
NaH2PO4 (pH 7.5), 10 mM EDTA, 10 l of 12 mM DTNB,
and 90 l extract. For standards, extract was replaced by 0,
10, 20, and 50 nmol GSH (total volume 0.2 ml). Assays by
spectrophotometer used 0.7 ml of 0.12 M NaH2PO4 (pH
7.5), 6 mM EDTA, 0.1 ml of 6 mM DTNB, and 0.2 ml
extract. For standards, extract was replaced by 0, 10, 20, 50,
and 100 nmol GSH (total volume 1 ml). In all cases, A412
was measured 5 min after the addition of standard or
extract. GSH standards gave A412 close to that predicted by
the established extinction coeYcient for both plate reader
and spectrophotometer (13,600 M1 cm1). Assays of
extracts were corrected for A412 in both the absence of
DTNB (plate well or cuvette with extract but no DTNB)
and the basal absorbance of DTNB (plate well or cuvette
with DTNB but no extract or standard), which were measured in parallel.
Thiol analysis by HPLC
Individual thiols were analyzed by a method adapted
from Ref. [27]. Following neutralization, 0.2 ml extract supernatant was added to 0.1 ml of 0.5 M Ches (pH 8.5) and 20 l
of 10 mM DTT. After incubation at room temperature for
30 min to reduce disulWdes, thiols were derivatized by the
addition of 20 l of 30 mM monobromobimane. The mixture
was incubated in the dark at room temperature for 15 min,
and then the reaction was stopped by the addition of 0.66 ml
of 10% (v/v) acetic acid. The derivatized mix was centrifuged
at 10,000g for 10 min, and 0.9 ml supernatant was Wltered
through 0.2 m mesh into autosampler vials. Vials were
loaded into the HPLC, and 50 l was injected onto the column. Bimane derivatives were separated by isocratic elution
with 10% methanol and 0.25% acetic acid (pH 4.3) at a Xow
rate of 0.8 ml/min and a column temperature of 40 C. Peaks
were identiWed by reference to authentic standards and were
quantiWed according to mixed standard solutions using quadratic curve Wtting. Thiolbimane derivatives eluted at
6.5 min (Cys), 7.1 min (Cys-Gly), 10 min (-Glu-Cys), and
12.2 min (glutathione).
Pyridine nucleotides
Pyridine nucleotides were assayed by adapting methods
described by Monger and coworkers [29]. The assay
involves the phenazine methosulfate (PMS)-catalyzed

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

reduction of dichlorophenolindophenol (DCPIP) in the


presence of ethanol and alcohol dehydrogenase (ADH) (for
NAD+ and NADH) or glucose 6-phosphate and G6PDH
(for NADP+ and NADPH). Reduced and oxidized forms
are distinguished by preferential destruction in acid or base.
To assay NAD+ and NADH, ADH was freshly dissolved in
0.1 M Hepes (pH 7.5) and 2 mM EDTA to a concentration
of 2500 U.ml1. Unless stated otherwise, triplicate aliquots
of 20 l neutralized supernatant were introduced into plate
wells containing 0.1 ml of 0.1 M Hepes (pH 7.5), 2 mM
EDTA, 20 l of 1.2 mM DCPIP, 10 l of 20 mM PMS, 25 l
water, and 10 l ADH. The reaction was started by the
addition of 15 l absolute ethanol. Following automatic
mixing by shaking, the decrease in A600 was monitored for
5 min. Contents were calculated by reference to standards
run concurrently (040 pmol NAD+ or NADH in the well),
and unknowns and standards were corrected for absorbance decreases measured for triplicate blank assays
(0 NAD+ or NADH). To assay NADP+ and NADPH,
G6PDH was freshly prepared each day by centrifugation of
an (NH4)2SO4 suspension and resuspension of the pellet to
200 U.ml1 in 0.1M Hepes (pH 7.5) and 2 mM EDTA.
Triplicate aliquots of 30 l neutralized supernatant (unless
stated otherwise) were introduced into plate wells containing 0.1 ml of 0.1 M Hepes (pH 7.5), 2 mM EDTA, 20 l of
1.2 mM DCPIP, 10 l of 20 mM PMS, 10 l of 10 mM glucose 6-phosphate, and 30 l water. The reaction was started
by the addition of 10 l G6PDH. Following automatic mixing by shaking, the decrease in A600 was monitored for
5 min and rates were calculated over the Wrst 2 min using
relevant standards and blank correction, as described
above for NAD+.
Recoveries of added standards through the extraction
procedure
Recovery quotients of known amounts of added metabolites were examined as follows. Except for NADH and
NADPH, approximately 1 g of Arabidopsis leaf material
was ground to a homogeneous powder in liquid nitrogen.
For each metabolite, triplicate aliquots of approximately
100 mg powder were transferred to precooled mortars and
extracted into either 0.2 N HCl or 0.2 N HCl spiked with a
known amount of the metabolite in question. In parallel,
triplicate aliquots of the spiked HCl solution were taken
through the sample preparation procedure in the absence
of leaf material, that is, neutralized and heated (for NAD+
and NADP+). Exact contents of each aliquot of homogeneous powder were estimated as chlorophyll, and the recovery of metabolites was estimated by subtraction of the
mean leaf contribution to the spiked samples The same
procedure was used to analyze recoveries of NADH and
NADPH except that extraction was into either 0.2 M
NaOH or 0.2 M NaOH plus NADH or NADPH and samples of chopped and homogenized leaf material were preweighed to enable calculation of leaf contents on an FW
basis. To separate the inXuence of the leaf sample from that

61

of the neutralization/heating step on recoveries of pyridine


nucleotides, recovery quotients were calculated both with
reference to external standard curves and with reference to
standards subjected to the sample preparation steps.
Chlorophyll
Chlorophyll was estimated in acid extracts as pheophytin (stoichiometric conversion occurs by acid-induced loss
of magnesium from chlorophyll). After the addition of acid
to leaf powder, duplicate aliquots of the homogenate (50 l)
were withdrawn and added to 1 ml acetone and 200 l
water. The acetone extract was vortexed, stored at 4 C
until completion of the above assays (generally 90 min), and
centrifuged to remove insoluble material, and the clear
supernatant was assayed at 666 and 655 nm. Pheophytin
concentration was calculated using established extinction
coeYcients according to the following equation: Pheophytin (g.ml1) D (A666 6.75) + (A655 26.03).
Data analysis
Absorbance changes were calculated automatically by
plate reader or spectrophotometer software and were processed in Microsoft Excel. Variability was expressed as relative standard deviation (RSD) for percentage deviation or
standard error (SE) for absolute deviation. Line Wtting was
performed by the regression analysis tool in SigmaPlot. To
calculate glutathione redox potential, the glutathione midpoint 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 from
labeling of glutathione in Arabidopsis suspension cells [30]
and to concentrations that can be inferred from Arabidopsis
leaf glutathione contents, assuming that glutathione concentrations are very low in the leaf cell vacuole. For a half
cell involving a two-electron transfer under standard conditions, the Nernst equation for calculation of the actual
redox potential can be simpliWed to E D E0 (29.6
log10[Reduced Form] / [Oxidized Form]). For the glutathione half cell (2 GSH ! GSSG + 2e + 2H+), this equation
becomes E D 230 mV (29.6 log10([GSH]2 / [GSSG]).
Results
Typical standard curves for plate reader assays of ascorbate, glutathione, NAD, and NADP are shown in Fig. 1.
Unlike the enzymatic methods used for glutathione and
pyridine nucleotides, which rely on kinetics analysis, the
method for ascorbate is an endpoint assay and quantities
usually are calculated according to the commonly used
extinction coeYcient of 14,000 M1 cm1 [31]. Fig. 1A compares A265 for equivalent concentrations of ascorbate in the
spectrophotometer with a conventional 1-cm path (white
circles) and plate reader (black circles), where we calculated
from well dimensions that an assay volume of 0.2 ml

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

1.2

A265

0.8

0.4

2.0

1.5

1.0

0.5

0.0
0

20

40

0.2
0.1

0.0
0.0

60

0.4
0.3

A265

A412.min-1

62

0.4

0.8

0.0

1.2

Glutathione (nmol)

[Ascorbate] (M)

20

40

60

80

60

80

60

80

0.10

0.05

0.20

0.15

0.05

0.00
10

20

30

NAD (pmol)

40

50

0.2
0.1

0.00
0

0.4
0.3

0.10

A265

A600.min-1

0.15

A600.min-1

Time (min)

10

20

30

40

50

0.0

NADP (pmol )

20

40

Time (min)

0.4
0.3

A265

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

0.2
0.1

represented an optical path length of 5 mm. Using standard


solutions of ascorbate, the response of the spectrophotometer was close to the value predicted from the extinction
coeYcient, with 50 M 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 reader
data below for ascorbate were analyzed with the conversion
factor of 0.1 mM D A412 of 0.7, that is, 7000 M1 For other
compounds, unknown quantities always were calculated
according to known standards run concurrently. Figs. 1B to
D show that a linear response to standards was observed
for each compound. Glutathione as GSH gave a linear
response up to 1 nmol in the plate well (Fig. 1B). When the
glutathione assay response was tested against GSSG, the
response was also linear up to at least 0.2 nmol (data not
shown), although (as predicted) an approximately twofold
greater response was observed on a molar basis relative to
GSH (see Fig. 3 later). Oxidized and reduced forms of
NAD gave a very similar response (Fig. 1C), as did both
forms of NADP (Fig. 1D).
A key factor in the assay of redox couples is distinguishing between reduced and oxidized forms. For this, we
adapted conventional techniques and veriWed their applicability to the plate reader assays. In the case of ascorbate, the
assay measures only the reduced form (ASC), and detection
of DHA requires prior conversion to this form, for which
DTT was used. The eYcacy of DHA conversion to ASC

0.0
0

20

40

Time (min)
Fig. 2. EVects of varying DTT concentrations and incubation times on
ascorbate and DHA yield. Ascorbate (white circles) and DHA (black circles) 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 DHA
in the plate well was 40 M, and predicted  A265 after the addition of
AO 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. Where
two points are not apparent, this is due to symbol overlap.

was dependent on both the incubation time and the DTT


concentration (Fig. 2). Using a DHA concentration in the
incubation (400 M) that is at the upper limit of that found
in extracts, optimal results were obtained with 1 mM DTT
and 30 min incubation (Fig. 2A). Whereas 5 mM DTT did
not allow full conversion of DHA to ASC, even after
60 min incubation (Fig. 2B), incubation with 20 mM DTT
caused even less eVective reduction (Fig. 2C). In the latter
case, failure to reduce DHA was accompanied by decreases
in ASC, suggesting that high DTT concentrations either
cause destruction of ASC during the incubation (e.g.,
through free radical-catalyzed reactions) or interfere with
the assay due to carryover. To measure total ascorbate in
tissue extracts, therefore, we used 1 mM DTT and an incubation period of 30 min.

0.0
0

20

40

60

80

0.2

A412.min-1

Time (min)

0.1

0.0
0

20

40

60

80

100

% glutathione as GSSG
Fig. 3. Selective measurement of GSSG by derivatization of GSH with
VPD. (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 in
the incubation at 4 M, and the quantity transferred to the assay well was
80 pmol. (B) EVect of VPD on the response of glutathione solutions with
diVerent GSH/GSSG values. White circles represent no VPD treatment,
and black symbols represent treatment with VPD for 30 min at VPD dilutions of 1:200 (circles) and 1:40 (triangles). The amount of GSH equivalents transferred to the assay well was 80 pmol throughout. For panel A, a
typical experiment is shown. Similar results were obtained in other experiments. For panel B, values are means of two independent experiments,
and error bars indicate actual values.

Methods to distinguish between GSH and GSSG in the


GRDTNB assay include pretreatment of extracts to complex GSH using thiol reagents such as N-ethylmaleimide
(NEM) and VPD. VPD was chosen because of its eYciency
at slightly acidic pH and its lack of eVect on GR after carryover into the assay [28]. Preliminary assays using standards
showed that VPD was able to rapidly block glutathione sulfhydryl groups (Fig. 3A), whereas its presence did not greatly
aVect the assay response to GSSG (Fig. 3B). Under our conditions, therefore, the inhibitory eVect of VPD was relatively
speciWc for the assay of GSH. Although VPD has limited solubility in water and most can be removed by centrifugation
of VPD-treated solutions prior to assay [28], other control
experiments indicated that carryover of VPD can have a
slight inhibitory eVect on the GRDTNB method (data not
shown). Because VPD does not inhibit GR appreciably [28],
this eVect probably is observed because any VPD that is carried over competes with DTNB for GSH that is formed from
GSSG in the assay. Thus, to account for possible assay artifacts, standard curves for GSSG always were generated using
VPD-treated standards.

A spectrophotometric assay has been described for


assay of NADP+ and NADPH by extraction into neutral
buVer followed by assay of NADPH at 340 nm with or
without pretreatment to interconvert the two forms [32].
However, this method measures NADP(H) in the micromolar to millimolar range [32], whereas the recycling assay
using DCPIP reduction is sensitive down to nanomolar
concentrations (standard curves for pyridine nucleotides in
Fig. 1 represent 50200 nM in the plate well). Using the
DCPIP reduction method, speciWc assay of reduced and
oxidized pyridine nucleotides can be achieved by preferential destruction in acid and alkali. Oxidized forms are
destroyed in alkaline conditions, and reduced forms are
destroyed by acid [29]. This procedure was Wrst checked
using standard solutions. In our conditions, heating was
found to be necessary for eYcient removal of forms targeted for destruction. When extracts were not heated, generally more than 10% of the targeted forms remained (data
not shown). However, when acid extracts were heated
brieXy, negligible NADPH and no NADH were detected
(Fig. 4). Heating of alkaline extracts caused complete loss
of 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 signiWcant
loss of nontargeted forms. Control experiments with standards 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 in
NaOH were 12 and 18%. Other experiments revealed that
the brief heating of acid extracts to remove NADH and
NADPH had a slight but signiWcant eVect on measured
ascorbate and glutathione values, in particular causing
some decrease in the reduction state. Because of this, HCl
extracts were split into two aliquots. One aliquot was neutralized directly for measurement of ascorbate and glutathione, and the other aliquot was heated prior to
neutralization for assay of NAD+ and NADP+ (for further
details, see Materials and methods).
0.06

0.04

0.02

0.00

NAD+
NADH
NADP+
NADPH

0.1

63

NAD+
NADH
NADP+
NADPH

0.2

A600.min-1

A
A412.min-1

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

HCl

NaOH

Fig. 4. Selective plate reader assay of reduced and oxidized pyridine nucleotides by preheating in acid and base. Standard solutions were diluted
into 0.2 N HCl or 0.2 M NaOH, heated 1 min at 95 C, neutralized, and
assayed as described in Materials and methods. Black bars represent oxidized forms, and white bars represent reduced forms. Where no bar is
apparent, the compound was undetectable.

64

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

Preliminary assays of NAD and NADP in leaf extracts


used 5 U ADH and 0.4 U G6PDH in the assay well. To
improve reproducibility and sensitivity of NAD(H) and
NADP(H) assays in plant extracts, enzyme concentrations
were increased to 25 U ADH and 2 U G6PDH, respectively, and amounts were calculated according to standard
curves run in parallel using the same enzyme concentration.
Under these conditions, the response of the assays was linear with respect to volume of leaf extract, and the Wtted
lines passed close to the origin (Fig. 5). DiVerences in the
two slopes for certain compounds reXect diVerences in FW
between the two extracts. For all data reported below,
extract volumes used were intermediate within the ranges
shown in Fig. 5.
Recoveries of metabolites through the extraction procedure in the presence of leaf sample were on the order of 75
to 90% except for DHA, where recovery was somewhat variable (Table 1). Recovery of DHA was dependent on reduction with DTT and was negligible when assayed without
this pretreatment (data not shown). Less than 10% of the
+

ASC

ASC/DHA
GSH/GSSG
NADH/NAD+
NADPH/NADP

30

15

ASC + DHA

NADH

NADP

GLUTATHIONE

20

pmol detected

1000

500

GSSG
400

Reduced form

Oxidized form

92.1 1.4
76.7 0.5
76.6 2.1 (87.1 2.4)
82.1 5.4 (100.9 6.6)

58.4 25.0
93.7 8.1
79.6 13.5 (86.1 14.7)
72.9 1.2 (84.1 1.4)

Table 2
Comparison of leaf contents assayed by spectrophotometer and plate
reader
Metabolite

Leaf contents
Plate reader

Spectrophotometer

2.51 0.11
3.11 0.13
493 45
28.4 7.1
2.8 0.4
13.1 1.3
9.9 0.1
9.7 0.6

2.42 0.12
2.83 0.15
535 34
25.2 6.3
3.9 1.5
14.0 0.8
7.6 0.3
9.9 1.3

Note. Data are from young Arabidopsis plants sampled at 15 to 31


days after sowing. The two methods were used for assay of the same
extracts treated identically. For plate reader assays, extract volumes in
the plate well were 20 l except for total glutathione, where 10 l was
assayed. For spectrophotometer assay, volumes were 20 l (pyridine
nucleotides, glutathione, and GSSG) and 100 l (ASC and ASC +
DHA). Units are in mol.g1 FW for ascorbate and DHA and are in
nmol.g1 FW for other compounds. All values are means SE of four
independent leaf extracts except for GSSG (three independent
extracts).

Recovery (%)

Note. Known amounts of metabolite standards were added to 0.2 M


NaOH (for NADH and NADPH) or 0.2 N HCl (for other compounds),
and the spiked media were used to extract triplicate aliquots of homogenized leaf powder. Recoveries were calculated relative to triplicate parallel
extracts performed into unspiked media. For pyridine nucleotides, data in
parentheses show percentage recoveries adjusted for extract-independent
loss of metabolites, calculated against standards treated exactly as
extracts. For further details, see Materials and methods.

ASC
ASC + DHA
Total glutathione
GSSG
NADH
NAD+
NADPH
NADP+

NAD

pmol detected

nmol detected

Redox couple

45

pmol detected

Table 1
Recovery quotients of metabolites through the extraction procedure

10

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

NADPH
20

Metabolite

Variability (RSD, %)
Between assays

10

200

0
0

10

20

30

40

sample volume (l)

10

20

30

40

sample volume (l)

Fig. 5. Linearity of the assays with increasing amounts of extract in the


plate well. Leaf material was extracted as described in the text, and three
diVerent volumes of the Wnal sample extract were assayed. Data are shown
for two independent alkaline extracts (NADH and NADPH) or acid
extracts (other assays). Black circles represent extract 1, and white circles
represent extract 2. Where data points are not visible, they are hidden by
the corresponding data points for the other extracts. Regressions are linear without constraint through the origin.

ASC
ASC + DHA
Total glutathione
GSSG
NAD+
NADH
NADP+
NADPH

3.7 0.4
4.2 1.1
2.7 0.7
6.0 1.3
8.1 2.3
19.1 7.7
6.0 1.8
4.4 1.1

Between plants
18.4 4.3
17.4 4.9
15.6 2.5
35.0 6.2
16.5 2.7
22.5 6.2
14.0 3.0
20.2 4.2

Note. Between-assay variability shows the mean RSD values among triplicate assays, averaged for six independent extracts (n D 6). Variability due
to the extraction and condition-independent biological diVerence (right
column) is expressed as the means of RSD values among three extracts
from diVerent plants sampled in the same growth conditions, obtained in
six independent experiments (n D 6).

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

to standards taken through the extraction and heating step


(Table 1).
To further validate the assay, metabolites were extracted
from young leaves and assayed in parallel by plate reader
and spectrophotometer. Table 2 shows that very similar
values were obtained by both methods. For example, in the
case of GSSG, which usually is the minority form of glutathione, plate reader analysis gave values that were
0.93 0.17 of values measured in the same extracts by
spectrophotometric assay (n D 14 independent leaf extracts

added GSH was detected when extracts were treated with


VPD (data not shown). Loss of pyridine nucleotides was on
the order of 20% (Table 1). Recoveries were improved when
they were calculated relative to standards taken through
the extraction procedure in the absence of leaf extract
(Table 1, values in parentheses). This shows that loss of pyridine nucleotides was due partly to factors present in the
leaf tissue and partly to the extraction and heating procedure itself. In the case of NADPH, loss was independent of
leaf extracts given that recoveries were 100% when related

Total leaf contents

% Reduction

Photoperiod

Photoperiod

8h

8h

16 h

16 h

Vegetative growth Flowering Vegetative growth

Flowering

120

80
4

60
40

Ascorbate % ASC

100
6

Ascorbate

65

20
0

Glutathione

80
500

60
40

250
20
0

Glutathione % GSH

100

750

NAD

60
40
20

NAD % NADH

80
40

20
0

NADP

60
20
40
10

20

NADP % NADPH

80

30

0
0

20

40

60

20

40

60

80

Days after sowing


Fig. 6. Plate reader assays of total contents and reduction states of ascorbate, glutathione, NAD, and NADP throughout Arabidopsis shoot development.
Total contents are in nmol.g1 FW except for ascorbate, where values are in mol.g1 FW. Percentage reductions of the glutathione pool are calculated in
GSH equivalents; that is, GSSG values are multiplied by 2 before subtraction from the total glutathione values. Data are means SE of three independent
extracts of diVerent plants.

66

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

in Wve independent experiments). An advantage of the 96well plate reader method is the ease of replicate assays of
extracts. Table 3 shows that the mean RSD between assays
was less than 10% for all compounds except NADH, which
was the least abundant form of pyridine nucleotide (Table 2
and Fig. 6). We also tested variability between individual
plants growing in identical controlled conditions (Table 3,
right column). The experimental design involved independent analysis of single extracts from three diVerent plants,
with the value for each metabolite in each extract being
obtained from the mean of the triplicate assay. Therefore,
these RSD values represent biological variation as well as
variation due to the extraction and sample preparation, and
they were between 14 and 35% and relatively constant over
six independent experiments (Table 3).
The assay was applied to the analysis of how the redox
metabolites change over the course of Arabidopsis shoot
development. At the vegetative (leaf-producing) stage, the
Arabidopsis shoot consists of a low-lying rosette. When the
Xowering program is initiated, the main stem elongates
greatly and produces multiple Xowers, and this eventually is
accompanied by leaf senescence. Most known Arabidopsis
ecotypes, including Columbia, are long-day plants; that
is, Xowering is accelerated by long photoperiods. We grew
plants for 5 weeks in short days (8-h photoperiod) and then
transferred them to long-day conditions (16-h photoperiod). Redox metabolites were measured throughout this
developmental program by assay of triplicate leaf samples
from diVerent plants at each stage (Fig. 6). The initial stage
of vegetative growth involved an increase in total contents
of all four redox couples, most notably those of NAD,
ascorbate, and glutathione (Fig. 6, left panels). Ascorbate
and NAD showed a further increase after the photoperiod
was lengthened, and both decreased markedly after the initiation of the Xowering program. In contrast, glutathione
showed an increase in the oldest leaves, that is, when the
Xower stem was well developed and leaves were beginning
to undergo senescence (Fig. 6, left panels). Changes in
redox states generally were less marked (Fig. 6, right panels). Ascorbate and glutathione remained more than 80%
reduced at all stages, although a gradual trend of the glutathione pool toward increased oxidation was observed during development (Fig. 6, right panels). Unlike the highly
reduced ascorbate and glutathione pools, NAD remained
more than 80% oxidized at all stages. Small changes in the
NAD reduction state were observed, and these largely
reXected changes in NAD+ given that NADH contents generally were very constant. Of the four redox couples, the
least variable was NADP, for which no marked trends were
observed for either total contents or redox state, with the
latter remaining at 50% reduced throughout development
(Fig. 6).
The speciWc enzymatic assay of glutathione was further
compared with total acid-extractable thiols measured by
reaction with DTNB and measurement of individual thiols
by precolumn monobromobimane labeling and separation
on reverse-phase HPLC with Xuorescence detection. The

Table 4
Comparison of leaf contents of glutathione and total thiols in young
leaves of A. thaliana
Compound

Total glutathione
GSH
GSSG
GSH/Glutathione (%)
Total thiols
GSH/Total thiols (%)
-Glu-Cys
Cys-Gly
Cysteine

Leaf contents
HPLC

Spectrophotometer

Plate reader

291 6

4.2 0.2
0.7 0.1
11.8 0.1

275 9
257 6
92
93 1
426 16
60 1

242 13
227 10
72
94 1
343 6
66 1

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

data in Table 4 show reasonable agreement between values


for glutathione, whether measured by HPLC or enzymatically on the plate reader or the spectrophotometer. Values
were close to those found in young Arabidopsis leaves in
Fig. 6. GSH accounted for 60 to 66% of acid-soluble thiols
extractable from young leaves, whether these were measured by spectrophotometer or plate reader (Table 4).
HPLC proWles of bimane-labeled thiols showed that the
only other signiWcant detectable peaks were (in order of
elution) Cys, Cys-Gly, -Glu-Cys, and homoCys. Although
homoCys could not be accurately quantiWed, the summed
leaf content of the other three thiols was 17 nmol.g1 FW
(Table 4).
Discussion
A wealth of literature data exists on the responses of
ascorbate and glutathione to stress in plants [13], and glutathione status is considered to be an important oxidative
stress marker in most cells [1,2]. Although ascorbate and
glutathione have key functions as antioxidants, both compounds also play important roles in other aspects of metabolism. Inversely, although most of the focus on NAD and
NADP has occurred within the context of studies of metabolic regulation, emerging data point to new roles, particularly for NAD, in the regulation of gene expression and
responses to stress in diverse groups of organisms
[21,22,33]. Despite the physiological overlap and biochemical interplay among the four redox couples, few studies
have taken an integrated approach to their analysis. For
this, a rapid and convenient extraction and assay method is
required. Methods have been described for proWling of
redox metabolites in biological samples by HPLC with coulometric or diode array detection [34,35]. These methods

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

Photoperiod
8h

Vegetative growth Flowering

30

GSH/GSSG

16 h
-220

20

-180

10

-140

-100

0
0

20

40

60

Redox potential (mV)

require more specialized equipment or extraction procedures than do those used in the current study, where we
have validated a method of general applicability that is able
to proWle the chief nonprotein components of redox status
in biological tissues.
Assays by plate reader yielded values similar to those
obtained on a conventional spectrophotometer. However,
the plate reader assay has several advantages. It is potentially more sensitive in terms of sample required. Although
the shorter optical path length in the plate reader approximately halves the sensitivity on a concentration basis, this is
more than oVset by the smaller assay volume, meaning that
less sample is required than in a conventional spectrophotometer assay. The accuracy of the current method is good,
with acceptably low between-assay RSD for most compounds, and the triplicate assay improves the robustness of
the method. The principal advantage of the current method
is high sample throughput without the need for expensive
or specialized equipment. Theoretically, on a 96-well plate
reader, it is possible to adapt the method to measure in triplicate up to 28 diVerent extracts together with four known
standard concentrations. Although sample preparation and
assay time would increase accordingly, the gain over a conventional spectrophotometric assay would be very substantial. Here we routinely used a triplicate assay of 6 extracts
and four standards. This protocol involves simultaneous
use of up to 30 wells and allows a threefold gain in assay
time, even when compared with a spectrophotometer with a
cell changer facility. Total analysis time for acid extracts of
6 independent samples was 2 h 30 min (1 h sample preparation and 1 h 30 min analysis). For alkaline extracts, the sample preparation was similar but analysis time was less than
30 min. We applied the method to Arabidopsis, a genus that
has become the model for functional genomics studies in
plants. Thus, comparison of triplicate samples of two genotypes is possible in slightly more than 4 h, and further gains
in time would come from preparation and assay of acid and
alkaline extractions in parallel. Total analysis time is an
important consideration in view of the limited stability of
some of the metabolites and the necessity to minimize
postextraction artifacts due to, for example, gradual oxidation of reduced forms of ascorbate and glutathione. We
estimate that the method could be readily adapted for triplicate analysis of up to 12 tissue extracts without loss of
robustness due to excessive delays during postextraction
assay preparation.
The extraction method was based on procedures commonly used for assay of pyridine nucleotides and was validated by the generally acceptable recoveries. DHA was
the most problematic compound, with relatively low and
somewhat variable recoveries that could be related to
incomplete reduction to ASC (Fig. 2). This compound,
however, rarely exceeds 10% of the total tissue ascorbate
pool in plants, and so incomplete recovery does not
greatly aVect total ascorbate contents even though it
could cause overestimation of the extent of tissue ascorbate reduction state. For pyridine nucleotides, where

67

80

Days after sowing


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

reduced and oxidized forms are measured independently,


parallel extracts with internal standards could be used to
improve precision, but this would necessarily increase
analysis time, and we found that although recoveries were
less than 100%, this was due to small systematic loss, as
reXected in the reproducible recovery quotients. The current data on leaf contents and redox states of NAD,
ascorbate, and glutathione are similar to values obtained
in studies where these compounds have been measured
individually in Arabidopsis [25,36,37].
Analysis of changes in redox couples during development of Arabidopsis revealed that total contents were more
variable than redox states. Standard errors were greater for
the reduction states of NAD and NADP than for those of
ascorbate and glutathione. This can be explained partly by
an additional source of experimental variation because the
reduction state is calculated from two independent extracts
for pyridine nucleotides, whereas in the case of ascorbate
and glutathione it is calculated from assays performed on
aliquots of the same extracts. Changes in redox states of all
four redox couples, either in whole plant tissue or in speciWc
subcellular compartments, can occur in response to
environmental stress and/or abrupt changes in irradiance
[3842]. The current data show that, during Arabidopsis
rosette development in controlled conditions, only limited
change in redox state is observed in the form of slight variation in NAD redox status and a general trend toward
increased oxidation of the glutathione pool (Fig. 6). Glutathione is unique among the four redox couples examined in
that its redox potential is dependent on total concentration
(because the concentration term of the Nernst equation
relates to [GSH]2 / GSSG). Calculations from the data of
Fig. 6 reveal that although the GSH/GSSG ratio changes
during Arabidopsis leaf development, the estimated glutathione redox potential is remarkably stable at around 180
mV (Fig. 7). Thus, it appears that adjustments in total glutathione concentration act to oVset the changes in GSH/
GSSG and that the global redox potentials of all four redox
couples are very stable throughout Arabidopsis shoot
development.

68

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

In contrast to the stability of tissue redox potentials,


the total contents of ascorbate, glutathione, and NAD all
showed clear age- and photoperiod-related trends.
Recent studies, notably of Arabidopsis mutants, have
highlighted the importance of total ascorbate and
glutathione concentrations in processes such as plant
pathogen and plantsymbiont interactions, meristem
development, and light signaling [412,36]. Emerging evidence, particularly from work on yeast and mammalian
cells, points to roles for NAD in modulating development and gene expression [4345]. Other studies in plants
have shown that photoperiod and/or growth irradiance
causes adjustments in some components of leaf redox
state [4648]. The current study shows that both NAD
and ascorbate increased signiWcantly in response to the
transfer of plants to long days, although the transfer had
little immediate eVect on glutathione contents (Fig. 6).
Whereas NAD and ascorbate both decreased at the onset
of Xowering, glutathione behaved in an opposing manner
and contents were highest in the oldest leaves. A comparison of glutathione contents with total thiols shows that
the glutathione contents of the oldest leaves (Fig. 6) were
signiWcantly higher than the total soluble thiol contents
of the youngest leaves (Table 4). Thus, the increase in leaf
glutathione with plant age is not simply the result of its
decreased use, for example, in the synthesis of phytochelatins, which if present should be detected by the DTNB
assay for total acid-soluble thiols [49]. Interestingly, a
role in the control of Xowering recently has been ascribed
to changes in both glutathione and ascorbate concentrations [8,50]. Ascorbate concentration has also been linked
to growth rate, cell diVerentiation, and pathogen resistance in Arabidopsis [6,10,51], whereas enhanced stress
resistance is observed in tobacco mutants with high NAD
contents [19,20]. Decreases in leaf pyridine nucleotide
contents following Xowering in spinach have been
reported [52]. It remains to be seen whether the general
increases in NAD and ascorbate that occur throughout
the vegetative phase of the Arabidopsis life cycle have any
role in controlling growth or determining age-related
diVerences in susceptibility to stress.
Acknowledgments
We thank Dorothe Thominet for excellent technical
assistance and Myroslawa Miginiac-Maslow for helpful
advice on measuring pyridine nucleotides. This work was
funded partly by the French ANR-Gnoplante program
GNP0508, Redoxome.
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