Botanical Studies (2007) 48: 35-41.
*
Corresponding author: E-mail: zhangmy2005@yahoo.com.
cn; Tel/Fax: 0086-20-37252891.
INTRODUCTION
Like the synthesis and cycling of glutathione (GSH),
GSH transport systems also have important roles
in sustaining the normal development of plants and
protecting them from biotic and abiotic stresses (Foyer
et al., 2001). GSH is not produced at equivalent rates by
all tissues, or even by all cells within a tissue (May et al.,
1998; Noctor et al., 2002). The trichomes on the stem
and leaf surface of Arabidopsis, for example, show much
higher expression of enzymes involved in the synthesis
of cysteine and GSH; and have GSH contents 2-3 times
higher than the surrounding basal and epidermal cells
(Gutierrez-Alcala et al., 2000).
The GSSG (oxidized glutathione) and GS-conjugated
transport systems on the plasma membrane associated with
systemic transport have been previously reviewed (Foyer
et al., 2001). The differential intercellular partitioning of
the GSH metabolism has been observed in maize (Doulis
et al., 1997), and in broad bean leaf tissues (Jamai et al.,
1996). GSH is also a major means of transporting reduced
sulfur over long distances, within both the xylem and the
phloem (Rennenberg et al., 1979; Herschbach et al., 2000).
Long distance GSH transport has also been implicated
in different roles, including phloem-mediated shoot-to-
root allocation of and demand-driven control of sulfate
uptake and/or loading into the xylem stream by the roots
in herbaceous plants (Lappartient and Touraine, 1997).
Recently, two plant GSH transporters, BjGT1 from
Brassica juncea (Bogs et al., 2003) and OsGT1 from
Oryza sativa (Zhang et al., 2004), were shown to be able
to promote transport of GSH, GSSG, and glutathione
conjugates when they were used to complement yeast
mutants with defects in GSH transport. However, the
physiological functions and expression patterns of the rice
glutathione transporter (OsGT1) (Zhang et al., 2004) were
not studied. Here, Arabidopsis transgenic for the reporter
gene GUS under the control of the OsGT1 promoter
was used to analyze the expression patterns of OsGT1
at various developmental stages and after treatment with
NaCl, heavy metals, abscisic acid (ABA), naphthalene
acetic acid (NAA), salicylic acid (SA), gibberellin (GA),
and low temperature.
MATERIALS AND METHODS
Construction of different deletion OsGT1
promoter-GUS fusion
The promoter region of OsGT1 was identified from
the database of rice genome sequences: accession no.
AP001168. The 2246 bp putative OsGT1 promoter was
amplified from genomic DNA of Oryza sativa cv., nip-
ponbar and confirmed by sequencing. The sequence data
from this article have been deposited at NCBI under ac-
cession number AY338469. To generate promoter deletion
fragments of the putative OsGT1 promoter, sets of primers
were used to amplify different-length fragments of the pro-
moter. The 2230-bp fragment P
13
was from -2246 to -17 bp
Tissue and inducible expression of a rice glutathione
transporter gene promoter in transgenic Arabidopsis
Ya-Qin WANG
1
, Shu-Ying ZHU
2
, Ying WANG
2
, and Ming-Yong ZHANG
2,
*
1
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, P.R. China
2
South China Botanical Garden, the Chinese Academy of Sciences, Xinkelu 723, Guangzhou 510650, P.R. China
(Received January 17, 2006; Accepted July 7, 2006)
ABSTRACT.
The promoter of a rice glutathione transporter (OsGT1) was fused to the reporter gene s-gluc-
uronidase (GUS) and introduced into Arabidopsis to analyze promoter and expression patterns. Deletion of
various length-promoter studies indicated that a 1246 bp fragment upstream of start codon (ATG) of OsGT1
is sufficient to activate the expression of GUS gene. The GUS assays revealed that the OsGT1 promoter is
expressed in an organ and tissue-specific manner. GUS was being expressed in the root stele, the veins of
cotyledons, the anthers and flower petals, but not in leaves or stems under normal growth conditions. GUS ex-
pression activated by the OsGT1 promoter was strongly stimulated by NaCl and low temperature, moderately
by abscisic acid and naphthaleneacetic acid, and not at all by salicylic acid and gibberellic acid. Expression
was inhibited by cadmium but not by copper. The function of OsGT1 in glutathione transport in normal or
stress conditions is discussed.
Keywords: Arabidopsis; Flower; Glutathione transporter; Promoter; Rice; Root.
MOLECULAR BIOLOGY
pg_0002
36
Botanical Studies, Vol. 48, 2007
before the start codon (ATG) of OsGT1 cDNA (AF393848,
A of the start codon was marked as +1), and was ampli-
fied using the forward primer HP
1
(5¡¦-CCCAAGCTTAT-
GGAACATGGGAAACAC-3¡¦) and the reverse primer
BP
2213
(5¡¦-CGCGGATCCAGCCTTCTCAGTTCTCAC-
3¡¦); the 989-bp fragment P
56
(from position -1263 to
-273 bp) was obtained using the forward primer HP
985
(5¡¦-CCCAAGCTTAAAATCTTTAGGAGCATA-3¡¦) and
the reverse primer BP
1956
(5¡¦-CGCGGATCCTCTAAAT-
TACTGCCATCC-3¡¦); the 1973-bp fragment P
16
(from
position -2246 to -273 bp) was obtained using HP
1
and
BP
1956
primers; and the 1246-bp fragment P
53
(from posi-
tion -1263 to -17 bp) was obtained using HP
985
and BP
2213
primers. The introduced Hind III site for HP
1
and HP
985
primers and BamH I site for BP
1956
and BP
2213
primers
were underlined. The four fragments of OsGT1 promoter
were cloned into the vector pBI 101 using the Hind III +
BamH I sites (Clontech) to generate the promoter-GUS
fusions (Figure 1A). The constructs were named P
13
-GUS,
P
16
-GUS, P
53
-GUS, and P
56
-GUS, respectively, and they
were checked by restriction digestion before transfer to A.
tumefaciens (LBA4404) by the electroporation method.
Arabidopsis transformation
Transformation of Arabidopsis thaliana ecotype Co-
lumbia was carried out by the floral dip method (Clough
and Bent, 1998). Transgenic plants were selected on the
basis of hygromycin resistance. Four to ten Arabidopsis
plants were transformed for each construct, and mature
seeds were harvested. Hygromycin-resistant transformants
were germinated on MS media supplemented with hygro-
mycin and transplanted to pots. After a three-generation
selection, the seedlings (T
3
) were used for analyses.
Southern analysis
Total genomic DNA was isolated from young leaves of
T
3
plants by the CTAB method (Murray and Thompson,
1980) for Southern blot. Southern analyses were per-
formed following the standard protocol of Sambrook et al.
(1989). Full-length of OsGT1 promoter (P
13
) was used as a
probe for Southern blot.
GUS activity assays
GUS activity in transgenic plants harvested at differ-
ent developmental stages or after different treatments was
assessed as described by Jefferson (1987). Histochemical
staining was carried out using X-Gluc as a substrate. For
the fluorogenic assay, 4-methylumbelliferyl glucuronide
(4MU) was used as a substrate (Jefferson, 1987). Four in-
dependent transgenic lines were tested for each construct.
Protein content was measured by the method of Bradford
(1976) using bovine serum albumin as a standard.
Inducible treatments of transgenic Arabidopsis
T
3
seedlings of P
53
-GUS were grown on sand in a 15
cm diameter pot, and watered with 1/4 MS liquid every 2
days at 22¢XC under a 16 h/d photoperiod. 14 day-old seed-
lings were used for treatments. One hundred milliliters of
100 £gM abscisic acid (ABA), 100 £gM naphthalene acetic
acid (NAA), 100 £gM salicylic acid (SA), 100 £gM gibber-
ellin (GA), 120 mM NaCl, 100 £gM CuSO
4
, 90 £gM, and
250 £gM CdSO
4
were added to the sand in each pot. 4¢XC
treatments were conducted in the growth chamber in 1500
lux light. Twenty-four hours after the treatments, the roots
were harvested, washed with distilled water, and deep-
frozen before protein extraction. Fifteen plants from five
pots were used for each treatment.
Transcripts of OsGT1 in rice
Oryza sativa cv. IR64 was used to analyze the tran-
script of OsGT1. Leaves, roots and panicles (flowers) of
IR64 at the heading stage under normal growth conditions
in Guangzhou, China, were used to analyze the transcripts
of OsGT1. For treatments, seeds of IR64 were germinated
and grown in 1/2 MS liquid media for one week, before
being treated with 100 £gM SA, 100 £gM ABA, 120 mM
NaCl, 90 £gM CdSO
4
and 4¢XC for 24 h under natural light.
Total RNA was isolated with TaKaRa RNAiso Reagent
(TaKaRa, China). DIG-labeled full-length anti-sense RNA
probes of OsGT1 were generated in vitro transcription
from the cloned OsGT1 cDNA in pGEM-T easy vector
(Promega) by SP6 RNA polymerase following the proto-
cols of the DIG Northern Starter Kit (Roche).
RESULTS AND DISCUSSION
Analysis of the OsGT1 promoter
The 2246 bp putative promoter region of OsGT1 was
obtained by PCR, and it was analyzed for using cis-
elements in the cis-acting regulatory DNA elements da-
tabases: http://www.dna.affrc.go.jp/sigscan/signal1.pl.
(Higo et al., 1999) and http://oberon.rug.ac.be:8080/plant-
CARE/. Various putative cis-sequences were found in the
2.23-kb putative OsGT1 promoter (see the note of NCBI
GeneBank accession no. AY338469). Interestingly, the
consensus sequences of nine pollen specific-expression
cis-elements (POLLEN1LELAT52) and eighteen root
specific-expression cis-elements (ROOTMOTIFTAPOX1)
were found in the promoter (Figure 1A). Meanwhile,
several other inducible cis-elements were also found,
including light response elements, an enhancer-like ele-
ment of anoxic specific inducibility (GC-motif), an ABA-
responsive element (ABRE), an auxin-responsive element
(AuxRR-core), a gibberellin-responsive element (P-
box), an ethylene-responsive element (ERE), and a SA-
responsive element (TCA-element). These cis-elements
might contribute to the anther and root tissue-specific ex -
pressions and the inducible expressions of the transgenic
plants on which we will be focused.
The OsGT1 promoter confers tissue-specific
expression in transgenic Arabidopsis
To investigate OsGT1 promoter, four deleted promoter
fragments were generated by PCR, and they (P
13
, P
16
, P
53
pg_0003
WANG et al. ¡X OsGT1 promoter in
Arabidopsis
37
and P
56
) were 2230, 1973, 1246 and 989 bp in length,
respectively (Figure 1A). These fragments were inserted
into the front of the s-glucuronidase (GUS) gene in the
vector pBI 101. Arabidopsis was transformed with these
constructs. Four to ten transformed Arabidopsis lines were
obtained for each construct. Southern analysis confirmed
that all the constructs were integrated into the Arabidopsis
genome (Figure 1B).
To understand the tissue and developmental expression
of GUS activated by the OsGT1 promoter, the transgenic
Arabidopsis plants were grown on sterile 1/2 MS solid
media at 22¢XC under 16 h light/day regimen. The seed-
lings were tested for GUS activity, using the histochemical
stain method, at different developmental stages. The earli-
est GUS activity was observed in the cotyledons of 2-day-
old seedlings of P
53
-GUS (Figure 2 C1). Five days after
germination, GUS expression was present in the roots and
the cotyledons of all P
13
-GUS (Figure 2 A2-A4) and P
53
-
GUS (Figure 2 C2-C4) transgenic Arabidopsis, but not in
P
16
-GUS (Figure 2 B1-B4) or P
56
-GUS (Figure 2 D1-D4).
Under these growth conditions, regardless of develop-
mental stage, the GUS staining pattern of P
13
-GUS and
P
53
-GUS was similar, suggesting that the 1246 bp region
upstream of the OsGT1 cDNA start codon (from -1263
to -17 bp in P
53
-GUS; Figure 1A) was sufficient to drive
the specific expression of GUS gene in roots and flowers.
Furthermore, 10 of the 18 root-specific motifs and 8 of
the 9 pollen specific motifs were located in this region.
However, when the 273-bp region immediately upstream
of the start codon of OsGT1 was deleted (to create P
16
-
GUS and P
56
-GUS), the promoter was no longer able to
drive expression of the GUS gene in transgenic Arabi-
dopsis. This observation indicates that the 256-bp region
from -273 to -17 bp of the OsGT1 promoter is essential
to activating GUS expression. The eighteen root motifs
(ATATTT, ROOTMOTIFTAPOX1) and nine pollen motifs
(POLLEN1LELAT52) in the OsGT1 promoter (see note
of AY338469) could be involved in the root and anther
specific-expression of GUS. POLLEN1LELAT52 motif
was one of the two co-dependent regulatory elements
responsible for pollen-specific activation of tomato lat52
gene (Bate and Twell, 1998), and ROOTMOTIFTAPOX1
motif was found for the root specificity and strength of the
rol D promoter in roots of tobacco (Elmayan and Tepfer,
1995). These results might further support the reports of
Hertig et al. (1991) and Santamaria et al. (2001), where
the ATATTT motif (ROOTMOTIFTAPOX1) was required
for expression in root tissues. The role of these motifs in
the OsGT1 promoter needs to be further studied.
The transgenic Arabidopsis P
13
-GUS and P
53
-GUS
showed an identical expression pattern in flowers, cotyle-
dons and roots (data not shown). GUS staining occurred
in the veins of cotyledons (Figure 3A). Flowers showed
GUS-staining in the anthers and in the veins of the petals
(Figure 3B), but not in the pistil. Root staining revealed
GUS-activity throughout the main root though not in the
root caps or root hairs (Figure 3A, C and E). Tissue slices
of roots showed strong GUS-staining in the stele, but not
in the cortex, epidermis, or endodermis (Figure 3D, E).
These results indicated that the OsGT1 promoter was ex-
Figure 1. A, Schematic diagram of OsGT1 promoter deletions fused to the GUS gene. Different length regions of the OsGT1 promot-
er were amplified by PCR and fused to the GUS gene of the binary vector pBI 101. The negative numbers indicated the bp upstream
from start codon of OsGT1 (the A of start codon ATG of OsGT1 was marked +1). B, Southern analysis of transgenic Arabidopsis. The
genomic DNA of each construct was isolated from T
3
generation and digested with Hind III; the full-length of OsGT1 promoter was
used as a probe. No Hind III cut site is in the promoter. Un-transgenic Arabidopsis was as control (CK).
pg_0004
38
Botanical Studies, Vol. 48, 2007
pressed in a tissue- and organ-specific manner in the trans-
genic Arabidopsis.
To confirm the above transgenic results, rice IR64
was used to analyze the organ expression of OsGT1 by
Northern blot. Under normal growth conditions, OsGT1 is
expressed in roots and panicles (flowers) of rice, but not in
the leaves (Figure 5, normal). These results suggested that
the OsGT1 promoter has a similar expression pattern in
rice and Arabidopsis, and OsGT1 is mainly expressed in
the roots and flowers of rice.
The constitutive expression of GUS driven by OsGT1
promoter in the stele of roots might indicate that OsGT1
is involved in long distance GSH transport in the root
stele. GSH¡¦s importance to root development was shown.
The Arabidopsis rml1 (root meristemless) mutant, which
lacks an enzyme involved in GSH synthesis, abolishes cell
division in the root but not in the shoot (Vernoux et al.,
2000), indicating that GSH is essential for postembryonic
root development in the root apical meristem after
germination. The expression of OsGT1 in cotyledon
suggests that GSH transport takes place from cotyledons
to the growing organs and that it may be important for
Figu re 3. Histochemical localization of
GUS ac tivity in the transgenic Arabi-
dopsis (P
53
-GUS) transformed with GUS
gene under the control of the 1246 bp
OsGT1 promoter region. A, whole plant;
B, flower; C, whole root; D, cross section
of a root; E, longitudinal section of a root.
Ant, anther; Cor, cortex; Cot, cotyledon;
End, endodermis; Epi, epidermis; Hyp,
hypocotyl; Pet, petal.
Figure 2. GUS staining patterns of trans-
genic Arabidopsis at different develop-
m ental s tages . Plants were transformed
with OsGT1 promoter fragments fused to
GUS gene. The trans genic Arabidopsis
were grown on 1/2 MS solid media at 22¢X
C under a 16 h/day photoperiod. Four in-
dependent transgenic lines were analyzed
for each cons truct; the GUS staining of
the different transgenic lines was similar
within the same constructs.
pg_0005
WANG et al. ¡X OsGT1 promoter in
Arabidopsis
39
the growth of roots and other organs. OsGT1 promoter
drives the GUS expression in the anthers of transgenic
Arabidopsis (Figure 3A), and OsGT1 expression was
in panicles (flowers) of rice (Figure 5 Normal). These
findings indirectly support the controlling role GSH has
in Arabidopsis flowering (Ogawa et al., 2001), which is
associated with the rate of GSH biosynthesis and/or the
levels of GSH in Arabidopsis. Our results indicate the
involvement of not only GSH synthesis but also of GSH
transport in normal development of roots and flowering
organs. OsGT1 is grouped into the oligopeptide transporter
family (OPT) (Zhang et al., 2004). The AtOPT3 gene of
the OPT family was also shown to be expressed in pollen
and is essential for embryo development (Stacey et al.,
2002). Together with our results, this indicates that GSH
and other oligopeptides are transported into anthers and
embryos.
Inducible expression of GUS driven by OsGT1
promoter
Because GSH is involved in resistance of plants to bi-
otic and abiotic stress (Noctor et al., 2002), the transgenic
Arabidopsis (P
53
-GUS) was used to check the response of
the OsGT1 promoter to environmental factors. GUS activ-
ities driven by P
53
-GUS were approximately twofold up-
regulated by NaCl and low temperature and approximately
onefold up-regulated by ABA and NAA. GUS activity was
down-regulated by high concentrations of cadmium, but
unaffected by copper (Figure 4). These results indicate that
GSH transport might be involved in protecting plants from
saline and low temperature stresses. Given the induction
by ABA, the response of OsGT1 to saline stress and low
temperature might be linked to ABA-dependent pathways.
To confirm this result of the transgenic Arabidopsis, one-
week-old rice seedlings were treated with NaCl, low tem-
perature, ABA, SA, and CdSO
4
. Transcripts of OsGT1 in
rice seedlings also could be strong up-regulated by NaCl,
and down-regulated by Cd (Figure 5, treatment).
Although GSH is known to chelate heavy metals (Per-
rin and Watt, 1971), we found GUS activity directed by
OsGT1 promoter was inhibited by Cd (Figure 4). Bogs et
al. (2003) also showed that Cd inhibited the expression of
BjGT1. The amount of BjGT1 protein in Brassica juncea
leaves is decreased by a 96 h exposure of the root sys-
tem to Cd. However, we could not find the transcription
response of OsGT1 gene in rice plants to Cd (Figure 5).
These results might indicate that OsGT1 is not involved
in the detoxification of toxic metals. They confirm that
OsGT1 is distinct from ABC transporters (Zhang et al.,
2004), another family of GS-conjugate transporters (Rea et
al., 1998), because the sequence of OsGT1 is not homolo-
gous with ABC transporters and lacks signature sequences
(Walker motifs) characteristic of ABC transporters (Zhang
et al., 2004). However, when the transgenic Arabidopsis
were sprayed with the above chemicals, GUS staining
could still not be detected in leaves and stems (data not
shown).
Figure 5. Expression of OsGT1 in rice under normal growth condition and treatments. For normal, the roots, leaves and panicles of
the heading Oryza sativa cv. IR64 were grown in field at Guangzhou, China. For treatment, one-week old IR64 seedlings were treated
by 4¢XC, 120 mM NaCl, 90 £gM CdSO
4
, 100 £gM salicylic acid (SA) and 100 £gM ABA for 24 h. 20 £gg of total RNA were loaded for
Nothern analysis with OsGT1 probe.
Figure 4. Effects of different treatments on the GUS activity in
the P
53
-GUS transgenic Arabidopsis line. P
53
-GUS transgenic
Arabidopsis were grown on sand at 22¢XC under a 16 h/day pho-
toperiod. The 14-day-old seedlings were rinsed in the pots with
100 mL of 100 £gM abscisic acid (ABA), 100 £gM naphthalene
acetic acid (NAA), 100 £gM salicylic acid (SA), 100 £gM gibber-
ellin (GA), 120 mM NaCl, 100 £gM CuSO
4
(Cu), 90 £gM and 250
£gM CdSO
4
(Cd),
or treated with low temperature (4¢XC). After
treatment for 24 h, roots were analyzed. All treatments were in-
dependently repeated twice.
pg_0006
40
Botanical Studies, Vol. 48, 2007
In conclusion, our data show that the heterogenous
OsGT1 promoter from a Monocotyledonous plant could
drive tissue-specific expression and responses to environ-
mental signals in the Dicotyledonous Arabidopsis, and
OsGT1 mainly expresses in roots and flowers of rice and
might responses to the saline and cold stress.
Acknowledgement. We thank Serge Delrot of University
of Poitiers, France for supplying vectors and MT Mei and
CX Zhuang of Agriculture University of South China for
Southern blots. This work was supported
by the director
foundation of South China Botanical Garden, Guangdong
Science Foundation (06026070, 2006A20101004)
National Science Fund (30670169) and "863" Project
(2006AA10Z168).
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pg_0007
WANG et al. ¡X OsGT1 promoter in
Arabidopsis
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Zhang, M.Y., A. Bourbouloux, O. Cagnac, C.V. Srikanth, D.
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