Plant Cell Physiol. 45(1): 1–8 (2004)
JSPP © 2004
Rapid Paper
Level of Glutathione is Regulated by ATP-Dependent Ligation of Glutamate
and Cysteine through Photosynthesis in Arabidopsis thaliana: Mechanism of
Strong Interaction of Light Intensity with Flowering
Ken’ichi Ogawa 1, 2, 3, Aya Hatano-Iwasaki 1, 2, 4, Mototsugu Yanagida 1, 2 and Masaki Iwabuchi 1
1
2
Research Institute for Biological Sciences, Okayama (RIBS Okayama), 7549-1 Yoshikawa, Okayama, 716-1241 Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)
;
stresses caused by excess photons, drought and chilling (Noctor
and Foyer 1998). It also plays a part in tolerance against heavy
metals (Cobbett 2000). GSH is associated with cell division
(Vernoux et al. 2000), root hair growth (Sánchez-Fernández et
al. 1997), tracheary element differentiation (Henmi et al. 2001),
flowering (Ogawa et al. 2001, Ogawa et al. 2002b, Yanagida et
al. 2002) and anthocyanin accumulation (Xiang et al. 2001).
However, the mechanism underlying these events has not been
identified.
The metabolic basis of GSH and cysteine has been characterized in plants (Saito 2000) and animals (Lu 1999). In animals, GSH is synthesized from glutamate, cysteine and glycine
via two reactions taking place in the cytosol, and the first step
is catalyzed by g-glutamylcysteine synthetase (g-ECS) which is
a heterodimer. g-ECS is active when the inter-subunit disulfide
bridge is formed (Huang et al. 1993), which accounts for the
feedback inhibition of g-ECS activity by GSH and, consequently, of GSH biosynthesis. The level of the GSH precursor
cysteine is also under rigid control and cysteine originates from
the protein degradation products cysteine and methionine
(Kwon and Stipanuk 2001). Thus, cysteine availability is considered as one of the limiting factors of GSH biosynthesis in
animals (McBean and Flynn 2001). In plants, there are several
isozymes in the biosynthesis of cysteine and GSH, and cysteine
is synthesized from sulfate absorbed in the root (Saito 2000),
but, on the contrary, a single gene of g-ECS is found in the
genome in Arabidopsis and rice, which is demonstrated by the
g-ECS-null rml1 mutant of Arabidopsis having a trace level of
GSH (Vernoux et al. 2000). All the g-ECS cDNAs that have
been reported contain the transit sequences for plastids,
although the enzymatic activity of g-ECS outside of the plastid
has been reported as well as that inside of the plastid (Noctor
and Foyer 1998). Noctor et al. (2002) showed that most g-ECS
proteins are likely localized in the plastid in Arabidopsis. Plant
g-ECS is a monomer and has unique amino acid sequences
compared to the animal g-ECS (May and Leaver 1994), but it is
still subjected to the feedback inhibition by GSH (May and
Leaver 1994, Noctor et al. 2002). In spite of such extensive
studies, how GSH metabolism is regulated to control growth
Glutathione (GSH) is associated with flowering in Arabidopsis thaliana, but how GSH biosynthesis is regulated to
control the transition to flowering remains to be elucidated. Since the key reaction of GSH synthesis is catalyzed
by g-glutamylcysteine synthetase (g-ECS) and all the g-ECS
cDNAs examined contained extra sequences for plastid targeting, we investigated the relationships among GSH levels,
photosynthesis and flowering. The GSH level in Arabidopsis increased with the light intensity. The ch1 mutants
defective in a light-harvesting antenna in photosystem II
showed reduced GSH levels with accumulation of the GSH
precursor cysteine, and introduction of the g-ECS gene
GSH1 under the control of the cauliflower mosaic virus 35S
promoter (35S-GSH1) into the ch1 mutant altered the GSH
level in response to the g-ECS mRNA level. These indicate
that photosynthesis limits the g-ECS reaction to regulate
GSH biosynthesis. Like the glutathione-biosynthesis-defective
cad2-1 mutant, the ch1 mutants flowered late under weaklight conditions, and this late-flowering phenotype was
rescued by supplementation of GSH. Introduction of the
35S-GSH1 construct into the ch1 mutant altered flowering
in response to the g-ECS mRNA and GSH levels. These
findings indicate that flowering in A. thaliana is regulated
by the g-ECS reaction of GSH synthesis that is coupled with
photosynthesis.
Keywords: Flowering — g-Glutamylcysteine synthetase —
Glutathione — Redox — Photosynthesis.
Abbreviations: g-ECS, g-glutamylcysteine synthetase; GSH,
reduced glutathione; GSSG, oxidized glutathione; TCA trichloroacetic acid.
Introduction
In plants, glutathione (GSH) is involved in cellular redox
homeostasis, in particular, in adaptive responses to oxidative
3
4
Corresponding author: E-mail, [email protected]; Fax, +81-866-56-9454.
Current address: The Institute of Low Temperature Science, Hokkaido University, Sapporo, 060-0819 Japan.
1
2
Photosynthesis and GSH synthesis in development
and development remains to be elucidated in plants and in animals.
We have postulated that the rate of GSH biosynthesis is a
causal factor for flowering in Arabidopsis thaliana because
high GSH levels in a late-flowering mutant, fca, possibly
causes the feed-back inhibition of GSH biosynthesis and
because this idea was inconsistent with the late-flowering phenotype of the glutathione-biosynthesis-defective cad2-1 mutant
(Ogawa et al. 2001). GSH is associated with flowering, but the
background wild type of each mutant is different and the lateflowering phenotype of the cad2-1 mutant was not great
enough to explain that of the fca mutant. Taking into account
that the key enzyme of GSH biosynthesis is likely localized in
plastids, we here investigated the regulation of GSH biosynthesis and levels by light and its relation with flowering behavior
in Arabidopsis, using a chlorophyll b-less mutant, ch1-1, defective in photosynthetic light harvesting.
[This work was preliminarily presented at the International Congress on Photosynthesis held in Brisbane, Australia
2001 (Ogawa et al. 2002a)].
Results
Effect of light intensity on GSH levels and flowering in A. thaliana
The level of GSH in the wild-type Columbia (Col) plants
increased as the light intensity increased, and then reached a
plateau at 100–200 mE m–2 s–1 (Fig. 1B). Likewise, flowering
was hastened with the increase in light intensity, in particular,
in a range from 25 to 100 mE m–2 s–1 (Fig. 1C). The lateflowering phenotype of the cad2-1 mutant defective in the first
enzyme g-ECS (encoded by the gene GSH1) of GSH biosynthesis (Cobbett 2000, Ogawa et al. 2001) was strong at a low
light intensity (Fig. 1C), and this difference in the flowering
phenotype between the mutant and wild-type plants almost disappeared at a light intensity of 500 mE m–2 s–1. The g-ECS
silence line of transgenics harboring the 35S-GSH1 transgene
(Fig. 1A) showed phenotypes similar to those of the cad2-1
mutant (Fig. 1B, C).
Chlorophyll b-less ch1 mutants are deficient in GSH
To verify the relationship between photosynthesis and
GSH biosynthesis further, we investigated the levels of GSH
and its precursor amino thiols using a chlorophyll b-less
mutant, ch1-1, defective in photosynthetic light harvesting
(Tables 1, 2). The ch1-1 mutant lacks the light-harvesting
antenna complex in photosystem II (PSII) and so receives photosynthetic light by the limited number of chlorophyll a in the
PSII core complex. The GSH level in the ch1-1 mutant was
lower than that in the wild types Col and Ler, whereas the Cys
level in the mutant was prominently higher (Table 1). Neither
the activity nor GSH1 mRNA level accounted for the Cys accumulation in the ch1-1 mutant (Table 2, Fig. 2). The ATP level
in the ch1-1 mutant was 3-fold lower and that in the cad2-1
Fig. 1 GSH levels and flowering of the wild-type and glutathionedeficient plants. (A) Results of RT-PCR analysis of GSH 1 in wild type
(Col-0) and a silence line transgenic carrying the 35S-GSH1 transgene
(35S-GSH1 6–8). Plants were grown for 4 weeks on soil at a light
intensity of 100 mE m–2 s–1 were harvested for the RT-PCR analysis. A
typical result of three experiments is shown. (B) Total glutathione levels in wild-type (Col-0) and GSH-synthesis-defective plants (cad2-1
and 35S-GSH1 6–8) grown at various intensities of light. Plants as
indicated were grown on soil at the indicated light intensities. Each
value is the mean ± SE (n = 40–150) of five experiments. (C) Flowering behavior of wild type (Col-0) and GSH-synthesis-defective plants
(35S-GSH1 6–8) grown at various intensities of light. Plants were
grown on soil at the indicated light intensities until the first flower
opened, and then the number of rosette leaves counted as a flowering
index. Each value is the mean ± SE (n = 25–50) of four experiments.
Photosynthesis and GSH synthesis in development
3
Table 1 Contents of aminothiols in ch1-1 mutant and wildtype plants
Genotype
GSH
g-GluCys
Cys
Col
ch1-1 (Col)
Ler
ch1-1 (Ler)
847.5±133.5
385.7± 28.8
423.8± 48.9
358.5± 16.5
5.8±0.3
5.0±1.3
5.6±4.3
2.5±2.5
9.2± 2.3
199.7±31.2
1.7± 1.0
158.5±34.5
Plants were grown on soil for 1 month at a light intensity of 100 mE
m–2 s–1 and assayed as described in Materials and Methods. The background wild type of each mutant is indicated. Units of values are nmol
(g FW)–1. Each value is the mean ± SE (n = 12–21) of three experiments.
Fig. 2 RT-PCR analysis of GSH1 encoding g-ECS in wild-type (Col-0)
and chlorophyll b-less mutant (ch1-1) plants. Five plants grown on soil
for 4 weeks at a light intensity of 100 mE m–2 s–1 were analyzed. A representative result is shown.
Table 2 Activity of g-ECS in ch1-1 mutant and wild-type
plants
Genotype
Col
ch1-1 (Col)
Ler
ch1-1 (Ler)
Relative activity of g-ECS
1.00±0.11
1.60±0.15
1.70±0.12
1.53±0.20
Plant materials are the same as described in Table 1. Values are relative
to the activity of the Col wild-type plant. Each value is the mean ± SE
(n = 12–21) of three experiments. Assay of g-ECS was performed as
described in Materials and Methods.
Table 3 Total ATP contents in ch1-1 and cad2-1 mutant and
wild-type plants
Genotype
Col
ch1-1
cad2-1
ATP, nmol (g FW)–1
0.34±0.05
0.09±0.06
0.67±0.08
Plants were grown on soil for one month at a light intensity of 100 mE
m–2 s–1. Assay of ATP contents were performed as described in Materials and Methods. The background wild type of each mutant is indicated. Each value is the mean ± SE (n = 12–16) of four experiments.
mutant about 2-fold higher than that in wild-type Col (Table 3).
In chloroplasts, ATP generation is dependent on the formation
of DpH across the thylakoid membrane, which is accompanied
by a photosynthetic electron flow from PSII to PSI. Therefore,
these results suggest that the photosynthetic electron flow regulates the rate of GSH biosynthesis through the production of ATP.
Flowering behavior of ch1 mutants
The ch1-1 mutation in both wild types Col and Ler
increased the number of rosette leaves at flowering (Fig. 3A).
The ch1-1 mutation also retarded plant growth, which led to
absolute delay in flowering time. These findings were consistent with the flowering behavior of the cad2-1 mutant (Fig. 1C).
Compared to the wild type, flowering in the ch1-1 mutant was
Fig. 3 Flowering behavior of chlorophyll b-less mutant (ch1-1) and
wild-type (Col-0) plants. (A) Flowering behavior of ch1-1 mutants
with different wild-type background. Plants were grown on soil at a
light intensity of 50 mE m–2 s–1 until the number of rosette leaves at
flowering was counted. Each value is the mean ± SE (n = 20) of three
experiments. (B) Light-dependent flowering behavior of ch1-1 mutant
and wild-type (Col-0) plants. Plants were grown on soil at the indicated intensities of light until the number of rosette leaves at flowering
was counted. Each value is the mean ± SE (n = 50–100) of five experiments.
retarded at low light intensities and was promoted at higher
light intensities (Fig. 3B), suggesting that there is a critical
amount of light harvested in PSII required for floral transition.
4
Photosynthesis and GSH synthesis in development
Fig. 4 Effect of GSH on flowering of the wild-type (Col-0) and chlorophyll b-less mutant (ch1-1) plants. Plants were grown at a light
intensity of 70 mE m–2 s–1 on the MS agar medium supplemented with
GSH at the indicated concentration until the floral stem elongated
beyond 1 cm. Each value is the mean ± SE (n = 40–63) of three experiments.
GSH restores the flowering phenotype of the ch1-1 mutant
GSH was applied to the ch1-1 mutant grown in the MS
agar medium at a light intensity of 70 mE m–2 s–1, at which
flowering and GSH level in the wild type (Col-0) were similar
to those grown at higher light intensities (Fig. 1B, C). Supplementation with GSH resulted in the promotion of flowering
(Fig. 4) and growth (Fig. 5C, D) of the ch1-1 mutant, but had
little effect on those of the wild type (Col-0) (Fig. 4, 5A, B).
The ch1-1 mutant flowered earlier than the wild-type plant
when the light intensity exceeded the threshold level (Fig. 3B)
or when GSH was sufficiently supplied (Fig. 4), suggesting that
there is a flowering pathway suppressed by light, which is
probably through photosynthesis.
Flowering in the ch1-1 mutant is altered in response to the gECS mRNA and GSH levels
We also generated transgenic plants of the ch1-1 mutant
that harbors the 35S-GSH1 transgene, to establish whether the
g-ECS reaction is a key for floral determination. We obtained
both overexpressor and cosuppression lines of the GSH1
mRNA (Fig. 6A). These lines of transgenics showed various
levels of GSH (Fig. 6B) and altered flowering time (Fig. 6C) in
response to the GSH1 mRNA levels (Fig. 6A). The overexpressor lines flowered earlier than the ch1-1 mutant and wild
type at all light intensities tested (Fig. 3B, 6C), and the maximal flowering-promotional effect was obtained at lower light
intensity compared to other transgenic lines or the ch1-1
mutant. On the other hand, the cosuppression lines flowered
later than the ch1-1 mutant at lower light intensities. Altogether, these findings strongly indicate that the g-ECS reaction
is limited by the photosynthetic production of ATP and this
limitation is a key for floral determination.
Fig. 5 Effect of GSH on the growth of wild-type (Col-0) and chlorophyll b-less mutant (ch1-1) plants. Col-0 (A, B) and ch1-1 (C, D)
plants were grown on the medium containing 0 or 500 mM GSH for 2
weeks. Bars = 1 cm.
Discussion
GSH biosynthesis is coupled with photosynthesis
The level of GSH in wild-type plants increased as the light
intensity increased in a certain range (from 25 to 100 mE m–2 s–1),
and then reached a plateau at 100–200 mE m–2 s–1 (Fig. 1B). In
the ch1-1 mutants that are defective in photosynthetic light harvesting, the level of GSH was lowered with accumulation of
the precursor cysteine (Table 1). Altogether, it is suggested that
photosynthesis limits the g-ECS reaction and, as a result, GSH
biosynthesis in a certain range of light intensity. This is consistent with the results that the ch1-1 mutant showed elevated
cysteine and lowered ATP levels (Table 1, 3). Cysteine biosynthesis is also required for ATP (Saito 2000) and is regarded as
the limiting factor for GSH biosynthesis in animals (McBean
and Flynn 2001), but the present results indicate that, in plants,
ATP availability limits the g-ECS reaction rather than cysteine
biosynthesis. Higher ATP level in the cad2-1 mutant might
reflect that GSH biosynthesis is a major sink of ATP in plants.
Flowering is regulated by GSH biosynthesis that is coupled
with photosynthesis
The level of GSH was strongly correlated with flowering
time (Fig. 1B, C, 3A, Table 1). GSH deficiency brought by the
ch1-1 mutation was associated with growth and flowering
retardation (Fig. 4, 5C, D), and this retardation was rescued by
GSH supplementation. These findings indicate that growth and
flowering are regulated by GSH. This idea is not inconsistent
Photosynthesis and GSH synthesis in development
5
with the result that flowering in the ch1-1 mutant was altered in
response to the g-ECS mRNA and GSH levels (Fig. 6).
Growth and flowering in plants are strongly influenced by
environmental conditions such as nutrient availability, temperature and light intensity. These influences are often issues in
agriculture and horticulture, but there has been little information on them (Bernier et al. 1993, Ohto et al. 2001). The
present study showed that GSH controlled flowering time
under different nutrient conditions (soil and agar medium)
where the number of rosette leaves at flowering were strongly
affected, and that GSH levels were regulated by the ATPdependent g-ECS reaction that was coupled with photosynthesis. Based on these findings, we propose a mechanism (Fig. 7)
explaining the general phenomenon that growth and flowering
are strongly affected by light intensity. The second reaction of
GSH biosynthesis that is catalyzed by glutathione synthetase
would occur both inside and outside of the chloroplast, judging
from the existence of EST clones containing both the corresponding gene products. From this viewpoint, it is reasonably
convenient to control the GSH supply for floral transition by
regulating the g-ECS reaction.
Flowering is determined by the balance between the suppressive and promotional effects of light
At higher intensities of light over 100 mE m–2 s–1, the ch1-1
mutant flowered earlier than the wild-type plants (Fig. 3B)
and the ch1-1 mutant supplemented with GSH flowered earlier
than the wild type did (Fig. 4). These results suggest that light
had a suppressive effect on flowering through photosynthesis as
well as promotional effect that involves GSH (Fig. 7). Supplementation of GSH to the wild type did not promote flowering
any more at a light intensity of 70 mE m–2 s–1 (Fig. 3B), at
which the GSH level in the wild type almost reached a plateau
(Fig. 1B) and flowering was similar to those grown at higher
light intensities (Fig. 1C). This suggests that the floweringpromotional pathway involving GSH exists upstream of the
flowering-suppressive pathway. This observation is consistent
with the fact that flowering is suppressed under light compared
to that in the dark (Araki and Komeda 1993).
Fig. 6 Introduction of the 35S-GSH1 transgene into the ch1-1mutant.
The plants 35S-GSH1 1–13, 2–8 and 3–14 having various mRNA levels of GSH1 were selected from transgenic plants (background: the
ch1-1 mutant) carrying the 35S-GSH1 transgene were generated as
described in Materials and Methods. (A) RT-PCR analysis of GSH1 in
transgenic plants carrying the 35S-GSH1 transgene. Plants grown on
soil for 4 weeks at a light intensity of 100 mE m–2 s–1 were analyzed. A
typical result of three experiments is shown. (B) GSH levels of the
transgenic plants. GSH levels in plants grown on soil for 4 weeks at a
light intensity of 100 mE m–2 s–1 were determined by the DTNBrecycling method. Each value is the mean ± SE (n = 10) of two experiments. (C) Flowering behavior of transgenic plants grown on soil at
various intensities of light. The number of rosette leaves was counted
when the first flower opened. Each value is the mean ± SE (n = 31–
100) of three experiments.
Regulation of GSH level
The level of GSH is determined by the balance between
synthesis and consumption (including degradation). It reached
a plateau at 100–200 mE m–2 s–1 as the light intensity increased
(Fig. 1B). This restricted elevation of GSH level somewhat
reflects the feedback inhibition of g-ECS by GSH (May and
Leaver 1994, Noctor et al. 2002), but it should also be noted
that, at a light intensity over 100 mE m–2 s–1, the level of GSH
in the cad2-1 mutant decreased, which indicates that GSH is
consumed by some reactions. For a shade-tolerant plant, Arabidopsis, light intensities over 100 mE m–2 s–1 must be strong
enough to provoke oxidative stress that consumes GSH. It must
also include anthocyanin accumulation because it is associated
with the level of GSH in Arabidopsis (Xiang et al. 2001).
6
Photosynthesis and GSH synthesis in development
Fig. 7 A scheme for the regulation of GSH biosynthesis by light
with reference to flowering, summarizing this study. Thin and broken arrows indicate metabolic flows and translocation, respectively. Thick arrows and T bar indicate promotional and
suppressive pathways, respectively. Proton (H+) translocates into
the thylakoid lumen coupled with electron flow from PSII to PSI,
and the proton gradient across the thylakoid membrane drives ATP
synthesis. This ATP supply restricts GSH biosynthesis at the gECS step. GSH synthesized is used for the floral determination,
and photosynthesis suppresses such a promotional effect of GSH
on flowering. The GSH-required flowering and its suppression by
light are governed by an unknown mechanism. This scheme
explains why light intensity strongly interacts with flowering.
Anthocyanin accumulation restricts light availability, and therefore GSH may also be an indirect limiting factor of flowering
and GSH biosynthesis itself.
Concluding remarks
The present study showed that GSH participates in the
regulation of growth and flowering in plants and its biosynthesis is restricted by the ATP-dependent g-ECS reaction
that is coupled with photosynthesis. It is possible that a certain
flowering-associated protein is activated or inactivated by
glutathionylation like some known enzymes (Cotgreave and
Gerdes 1998, Ito et al. 2003), but the exact mechanism remains
to be elucidated. GSH, together with H2O2, was strongly associated with stress and also postulated as a growth regulator
during the germination period (Ogawa and Iwabuchi 2001).
The mechanism of growth and flowering by GSH would therefore lead to understanding of the general mechanism for a
strong interaction of oxidative stress with plant development.
Materials and Methods
Plant materials and growth conditions
All A. thaliana ecotypes and mutants used in this study without
the cad2-1 mutants were obtained from the Nottingham Arabidopsis
Stock Centre (http://nasc.nott.ac.uk/). The GSH-deficient mutant cad21 was generously provided by Dr. C. S. Cobbett (The University of
Melbourne, Parkville, Australia).
For soil culture, 3–5 plants were grown in a plastic square pot
(6.5´6.5´5 cm) filled with two volumes of vermiculite (Asahi-Kogyo,
Okayama, Japan) in the bottom, one volume of Kureha soil (KurehaEngei-Baido, Kureha Chemical, Tokyo, Japan) in the middle layer and
one volume of vermiculite on the top, in a closed greenhouse at
24±2°C under long-day conditions (16-h light/8-h dark cycle; light
intensity).
For culture on agar medium, plants were grown on 1% (w/v) agar
plates of one-half-strength Murashige-Skoog (MS) medium as described
previously (Ogawa et al. 2001) without sucrose supplementation.
Assay of g-ECS activity and contents of aminothiols
Thiols were extracted and analyzed according to previous methods after modification (Matamoros et al. 1999, Atamna et al. 2000).
The aboveground part of five plants frozen with liquid nitrogen was
ground to a powder using a chilled mortar and pestle. The powder
(20 mg) with 200 ml of 0.1 M HCl in a 1.5-ml Eppendorf tube was
homogenized. The homogenate was centrifuged at 20,000´g for
10 min at 4°C. The supernatant was centrifuged with a microconcentrator Microcon YM-3 (Amicon, Inc., Beverly, MA, U.S.A.) at
14,000´g at 4°C. The filtrates were used for assay of thiols. Thiols
were detected with a model 5200A Coulochem electrochemical detector (ESA, Inc., Chelmsford, MA, U.S.A.) following HPLC with a
reverse phase C18 column (Shiseido, Tokyo, Japan). The mobile phase
consisted of 50 mM sodium phosphate monobasic monohydrate,
0.25 mM octanesulfonic acid, and 3.7% methanol, adjusted to pH 2.7
with phosphoric acid.
Alternatively, when we measured only GSH and GSSG, we used
the DTNB [5,5¢-dithiobis (2-nitrobenzoic acid)]-recycling method
(Ellman 1959). Plants frozen with liquid nitrogen were ground to a
powder using a chilled mortar and pestle. The powder (20 mg) was
homogenized with 100% trichloroacetic acid (TCA) and centrifuged at
20,000´g for 20 min at 4°C. The supernatant was divided into half for
assay of total glutathione (GSH + GSSG) and for GSSG alone. For
assay of total glutathione, TCA was removed from the supernatant by
extracting three times with diethyl ether. For assay of GSSG, 40 mM
N-ethylmaleimide, dissolved in 10 mM sodium phosphate buffer (pH
7.5) containing 5 mM EDTA, was added to the supernatant, and
mixed. Then, TCA and N-ethylmaleimide were removed by extracting
three times with diethylether. The reaction mixture for the DTNBrecycling was composed of 10 mM sodium phosphate buffer, 0.25 mM
NADPH, 0.75 units glutathione reductase and the extracted glutathione. The absorbance change at 412 nm was measured immediately
after the addition of DTNB. Glutathione content was calculated from a
standard curve obtained from authentic GSH and GSSG.
Photosynthesis and GSH synthesis in development
Assay of g-ECS activity was performed for 60 min in a reaction
medium (50 mM Tris-HCl buffer, pH 7.6, 0.25 mM glutamate, 10 mM
ATP, 1 mM dithioerythritol and 2 mM cysteine. Reaction was started
by adding cysteine, and g-ECS activity was defined as cysteinedependently generated PO42– determined by the molybdenum blue
method.
Assay of ATP
The aboveground part of five plants frozen with liquid nitrogen
was ground to a powder using a chilled mortar and pestle. The powder
(20 mg) with 200 ml of 0.1 M HCl in a 1.5-ml Eppendorf tube was
homogenized. The homogenate was centrifuged at 20,000´g for
10 min at 4°C. The supernatant was centrifuged with a microconcentrator Microcon YM-3 (Amicon, Inc., Beverly, MA, U.S.A.) at
14,000´g at 4°C. The filtrates were used for assay of ATP. Total cellular
ATP content was determined by using an ATP determination kit (A6608;
Molecular Probes, Inc., Eugene, OR, U.S.A.) and a MicroLumat Plus
LB (Berthold Japan, Tokyo, Japan) microinjector luminometer.
Flowering index
As a flowering index, the number of rosette leaves on the main
floral shoot was counted when the first flower opened.
Construction of transgenic Arabidopsis with altered levels of g-ECS
The full-length cDNA of GSH1 (the GenBank accession number:
Z29490) encoding g-ECS (EC 6.3.2.2) was generated from two cDNA
fragments whose sequences partially overlapped. The two fragments
were generated by isolating the total RNA from 4-week-old Columbia
wild-type (Col-0) plants, followed by RT-PCR using Prostar firststrand RT-PCR kit (Stratagene, La Jolla, CA, U.S.A.). The primers
used for RT-PCR were as follows: for the fragment containing the 5¢
region of the cDNA, 5¢-ATGCCAAAGGGGAGATACGAC-3¢ and 5¢GGAGACTCGAGCTCTTCAGATAG-3¢; for the other fragment containing the 3¢ region, 5¢-GCTTTCTTCTAGATTTCGACGG-3¢ and 5¢CCTGATCATATCAGCTTCTGAGC-3¢. Underlines indicate the
mutated nucleotides for introduction of XbaI and SacI restriction sites
into the respective resulting fragments. The resulting fragments were
cloned into the pGEM-T vector (Promega, Madison, WI, U.S.A.). The
sequences of the cloned DNA fragments were verified through
sequencing. The XbaI–KpnI and KpnI–SacI fragments that were
derived from the respective DNA fragments were fused at the KpnI
restriction site to generate a 1.7-kbp fragment containing the fulllength GSH1 cDNA. The b-glucuronidase gene of the binary vector
pBI 121 were replaced by the XbaI–SacI fragment to place the GSH1
cDNA under the control of the cauliflower mosaic virus 35S promoter
(35S-GSH1). The construct carrying 35S-GSH1 was mobilized into
Agrobacterium tumefaciens strain LBA4404, and the T-DNA was
introduced into wild-type (Col-0) and ch1-1 mutant plants using the
floral dip transformation method (Clough and Bent 1998). Lines
homozygous for the introduced T-DNA were selected using kanamycin
resistance. To confirm overexpression of the GSH1 gene in the transgenic lines carrying the 35S-GSH1, RT-PCR was performed.
RT-PCR analysis of GSH1
Four-week-old plants were harvested and the total RNA was
extracted from leaf tissues using the RNeasy Plant Mini kit (Qiagen
Inc., Valencia, CA, U.S.A.) and first-strand cDNA synthesis was performed as described above. The primers used in the RT-PCR analysis
were as follows: for GSH1, 5¢-ATCATATAATAAACCCACCCAGAA3¢ and 5¢-CTTGATATGATGCTCCGAAC-3¢; for ACT1 (U39449) as a
control gene, 5¢-GATGATGCACCTAGAGCTGT-3¢ and 5¢-CTCCATGTCATCCCAATTGT-3¢. PCR conditions were as follows: one cycle
of 94°C (2 min); 32 cycles for GSH1 at 94°C (30 s), 50°C (30 s) and
7
72°C (1 min), and 30 cycles for ACT1 at 94°C (30 s) and 55°C (30 s)
and 72°C (1 min); then one cycle of 72°C (5 min). Amplified DNA
was analyzed through an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Hachioji, Japan).
Acknowledgments
We are very grateful to Prof. Dr. Toshiharu Shikanai (NAIST,
Japan) for reading the manuscript and giving helpful comments. We
also thank Prof. Dr. Christopher S. Cobbett (The University of Melbourne, Australia) for providing seeds of the wild-type and cad2-1
mutant plants, and the Nottingham Arabidopsis Stock Centre for providing seeds of the wild-type plants Columbia (Col-0) and Landsberg
erecta (Ler) and mutant ch1-1. Thanks are due to Tamiko Ishihara for
plant care. The present work was supported partially by the “Research
for the Future” Program of The Japan Society for the Promotion of
Science (JSPS-RFTF 00L01605) and (JSPS-RFTF00L01604) and by
the Novartis Foundation (Japan) for the Promotion of Science.
References
Atamna, H., Paler-Martinez, A. and Ames, B.N. (2000) N-t-Butyl hydroxylamine, a hydrolysis product of a-phenyl-N-t-butyl nitrone, is more potent in
delaying senescence in human lung fibroblasts. J. Biol. Chem. 275: 6741–
6748.
Araki, T. and Komeda, Y. (1993) Flowering in darkness in Arabidopsis thaliana. Plant J. 4: 801–811.
Bernier, G., Havelange, A., Houssa, C., Petitjean, A. and Lejeune, P. (1993)
Physiological signals that induce flowering. Plant Cell 5: 1147–1155.
Clough, S.J. and Bent, A.F. (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–
743.
Cobbett, C.S. (2000) Phytochelatin biosynthesis and function in heavy-metal
detoxification. Curr. Opin. Plant Biol. 3: 211–216.
Cotgreave, I.A. and Gerdes, R.G. (1998) Recent trends in glutathione biochemistry: Glutathione–protein interactions: a molecular link between oxidative
stress and cell proliferation? Biochem. Biophys. Res. Commun. 242: 1–9.
Ellman, G.L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70–
77.
Henmi, K., Tsuboi, S., Demura, T., Fukuda, H., Iwabuchi, M. and Ogawa, K.
(2001) A possible role of glutathione and glutathione disulfide in tracheary
element differentiation in the cultured mesophyll cells of Zinnia elegans.
Plant Cell Physiol. 42: 673–676.
Huang, C.S., Chang, L.S., Anderson, M.E. and Meister, A. (1993) Catalytic and
regulatory properties of the heavy subunit of rat kidney g-glutamylcysteine
synthetase. J. Biol. Chem. 268: 19675–19680.
Ito, H., Iwabuchi, M. and Ogawa, K. (2003) The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione. Plant Cell Physiol.
44: 655–660.
Kwon, Y.H. and Stipanuk, M.H. (2001) Cysteine regulates expression of
cysteine dioxygenase and g-glutamylcysteine synthetase in cultured rat hepatocytes. Amer. J. Physiol. Endocrinol Metab. 280: E804–E815.
Lu, S.C. (1999) Regulation of hepatic glutathione synthesis: current concepts
and controversies. FASEB J. 13: 1169–1183.
Matamoros, M.A., Moran, J.F., Iturbe-Ormaetxe, I., Rubio, M.C. and Becana,
M. (1999) Glutathione and homoglutathione synthesis in legume root nodules. Plant Physiol. 121: 879–888.
May, M.J. and Leaver, C.J. (1994) Arabidopsis thaliana g-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli
homologs. Proc. Natl Acad. Sci. USA 91: 10059–10063.
McBean, G.J. and Flynn, J. (2001) Molecular mechanisms of cystine transport.
Biochem. Soc. Trans 29: 717–722.
Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: Keeping active
oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 249–
279.
8
Photosynthesis and GSH synthesis in development
Noctor, G., Gomez, L., Vanacker, H. and Foyer, C.H. (2002) Interactions
between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J. Exp. Bot. 53: 1283–1304.
Ogawa, K. and Iwabuchi, M. (2001) A mechanism for promoting the germination of Zinnia elegans seeds by hydrogen peroxide. Plant Cell Physiol. 42:
286–291.
Ogawa, K., Hatano-Iwasaki, A., Tokuyama, M. and Iwabuchi, M. (2002a) A
possible role of glutathione as an electron source for flowering in Arabidopsis thaliana. In PS2001 Proceedings, S20–30. CSIRO Publishing, Melbourne.
Ogawa, K., Hatano-Iwasaki, A., Yanagida, M. and Henmi, K. (2002b) The
redox-dependent regulation in the bolting and flowering of plants. Flowering
Newslett. 34: 45–51.
Ogawa, K., Tasaka, Y., Mino, M., Tanaka, Y. and Iwabuchi, M. (2001) Association of glutathione with flowering in Arabidopsis thaliana. Plant Cell Physiol. 42: 524–530.
Ohto, M., Onai, K., Furukawa, Y., Aoki, E., Araki, T. and Nakamura, K. (2001)
Effects of sugar on vegetative development and floral transition in Arabidopsis. Plant Physiol. 127: 252–261.
Saito, K. (2000) Regulation of sulfate transport and synthesis of sulfur-containing
amino acids. Curr. Opin. Plant Biol. 3: 188–195.
Sánchez-Fernández, R., Fricker, M., Corben, L.B., White, N.S., Sheard, N.,
Leaver, C.J., Van Montagu, M., Inzé, D. and May, M.J. (1997) Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Proc. Natl Acad. Sci. USA 94: 2745–2750.
Vernoux, T., Wilson, R.C., Seeley, K.A., Reichheld, J.P., Muroy, S., Brown, S.,
Maughan, S.C., Cobbett, C.S., Van Montagu, M., Inzé, D., May, M.J. and
Sung, Z.R. (2000) The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2
gene defines a glutathione-dependent pathway involved in initiation and
maintenance of cell division during postembryonic root development. Plant
Cell 12: 97–110.
Xiang, C., Werner, B.L., Christensen, E.M. and Oliver, D.J. (2001) The biological functions of glutathione revisited in Arabidopsis transgenic plants with
altered glutathione levels. Plant Physiol. 126: 564–574.
Yanagida, M., Mino, M., Iwabuchi, M., and Ogawa, K. (2002) Vernalizationinduced bolting involves regulation of glutathione biosynthesis in Eustoma
grandiflorum. Plant Cell Physiol. 43: s43.
(Received October 1, 2003; Accepted November 3, 2003)