Plant, Cell and Environment (2008) 31, 982–994
doi: 10.1111/j.1365-3040.2008.01810.x
SsTypA1, a chloroplast-specific TypA/BipA-type GTPase
from the halophytic plant Suaeda salsa, plays a role in
oxidative stress tolerance
FANG WANG, NAI-QIN ZHONG, PENG GAO, GUI-LING WANG, HAI-YUN WANG & GUI-XIAN XIA
National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences; National Center for
Plant Gene Research, Beijing 100101, China
ABSTRACT
Suaeda salsa is a leaf-succulent euhalophytic plant capable
of surviving under seawater salinity. Here, we report the
isolation and functional analysis of a novel Suaeda gene
(designated as SsTypA1) encoding a member of the TypA/
BipA GTPase gene family. The steady-state transcript level
of SsTypA1 in S. salsa was up-regulated in response to
various external stressors. Expression of SsTypA1 was
restricted to the epidermal layers of the leaf and stem in S.
salsa, and SsTypA1–green fluorescence protein (GFP)
fusion proteins were targeted to the chloroplasts of tobacco
leaves. Ectopic over-expression of SsTypA1 rendered
the transgenic tobacco plants with significantly increased
tolerance to oxidative stress, and this was accompanied
by a reduction in H2O2 content. Enzymatic and Western
blot analyses revealed that the activity and amount of
the thylakoid-bound NAD(P)H dehydrogenase (NDH)
complex in the chloroplasts of leaf cells were enhanced.
Additionally, an in vitro assay demonstrated that SsTypA1
bound to GTP and possessed GTPase activity that was
stimulated by the presence of chloroplast 70S ribosomes.
Together, these results suggest that SsTypA1 may play a
critical role in the development of oxidative stress tolerance, perhaps as a translational regulator of the stressresponsive proteins involved in reactive oxygen species
(ROS) suppression in chloroplast.
Key-words: NDH complex; translational GTPase.
INTRODUCTION
Plants are subject to various types of environmental constrains including high salinity, drought and extreme temperature. Many of these abiotic stresses can cause a
secondary oxidative stress, resulting from rapid accumulation of reactive oxygen species (ROS) (Apel & Hirt 2004;
Sunkar et al. 2007). To protect the cellular components from
ROS damage, plants have developed intrinsic enzymatic
and non-enzymatic mechanisms against oxidative stress,
Correspondence: G.-X.
[email protected]
982
Xia.
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including enhancement of production of a variety of lowmolecular weight antioxidants and antioxidative enzymes
such as superoxide dismutase (SOD), ascorbate peroxidase
(APX), glutathione S-transferase (GST) and catalase
(CAT) (Mittler 2002; Mittler et al. 2004; Foyer & Noctor
2005).
The chloroplast is the site of photosynthesis in plant cells.
Under stress conditions, chloroplasts generate a large quantity of ROS because of limited photosynthetic CO2 fixation.
Several antioxidative enzymes including SOD and APX are
increased under stress conditions in the chloroplast (Mittler
et al. 2004). In addition, the thylakoid-bound NAD(P)H
dehydrogenase (NDH) complex is also involved in the
regulation of redox state in response to environmental
changes (Rumeau, Peltier & Cournac 2007). It was found
that the chloroplast-encoded NDH polypeptides and
NADH dehydrogenase activity of the NDH complex augmented in response to various abiotic stresses (Martin,
Casano & Sabater 1996; Casano et al. 2000; Casano, Martin
& Sabater 2001; Lascano et al. 2003), and ndh-less mutants
exhibited higher sensitivity to photooxidative or oxidative
stresses (Endo et al. 1999; Horvath et al. 2000). A recent
study showed that H2O2 was accumulated in the leaves of
the tobacco ndhC-ndhK-ndhJ mutant and that NDHdependent pathway was involved in the suppression of
ROS generation under temperature stress (Wang et al.
2006).
Tyrosine phosphorylation protein A (TypA/BipA) is a
novel member of the ribosome-binding GTPase superfamily (Margus, Remm & Tenson 2007). BipA was originally
found in Salmonella typhimurium as a protein induced by
the cationic host defence protein BPI (Qi et al. 1995) and
has since been found widely distributed in bacteria and
plants. BipA regulates various cell surface- and virulencerelated components/processes (Farris et al. 1998; Grant
et al. 2003) in enteropathogenic Escherichia coli (EPEC)
and is required for growth of E. coli K12 at low temperature
(Pfennig & Flower 2001). In Sinorhizobium meliloti, TypA
is necessary for survival under some stress conditions and is
also needed for symbiosis of the bacterium with certain
Medicago truncatula lines (Kiss et al. 2004). In plants, TypA
genes were found to be involved in pollen tube growth
in Arabidopsis and in the development of the male
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
The role of a TypA/BipA-type GTPase from S. salsa in oxidative stress tolerance 983
reproductive organ in cucumber (Lalanne et al. 2004; Barak
& Trebitsh 2007). It was shown that BipA proteins from the
EPEC strain had GTPase activity (Farris et al. 1998), and
purified BipA proteins bound to ribosomes (Grant et al.
2003). More recently, BipA was found to function as a translation factor that was required specifically for the expression of the transcriptional modulator Fis in E. coli. Based on
the structural features displayed by fis mRNA, it was proposed that BipA may act to destabilize the strong interactions between the Shine–Dalgarno (S–D) segment of the 5′
untranslated region (UTR) of fis mRNA and the 3′ end of
16S ribosome RNA (Owens et al. 2004).
In this study, we have isolated a cDNA encoding a TypA/
BipA homolog (designated SsTypA1) from Suaeda salsa, a
halophytic plant growing in a high-salinity region in the
northern part of China. We found that the expression of
SsTypA1 was up-regulated by various abiotic stresses;
ectopic over-expression of the gene in tobacco plants conferred enhanced oxidative stress tolerance to the transgenic
plants in which H2O2 levels were decreased; SsTypA1 was a
functional GTPase and its activity was enhanced by the
presence of chloroplast 70S ribosomes, and SsTypA1 overexpression resulted in increase in the activity and amount of
the chloroplastic NDH complex. Our results suggest that
SsTypA1 may play a role in coordinating cellular responses
to environmental stresses by regulating the translation of
mRNA transcripts of genes involved in the ROS control in
chloroplast.
MATERIALS AND METHODS
Plant growth and stress treatments
Seedlings of S. salsa (Shandong ecotype) and tobacco (Nicotiana tabacum L.) were grown in the growth chamber
under 16 h photoperiod (100 mmol photons m-2 s-1) at
24 ⫾ 1 °C and watered weekly with Hogland, and Murashige and Skoog (MS) nutrient solutions, respectively. For
expression pattern analysis, 5-week-old seedlings of S. salsa
were subjected to various treatments: 400 mm NaCl, 400 mm
LiCl, 500 mm mannitol and 20 mm H2O2 for indicated times.
genes. Domain organization of protein was analysed using
the BlastP search, and prediction of the peptide’s subcellular localization was conducted using TargetP (Emanuelsson
et al. 2000) and ChloroP programs (Emanuelsson, Nielsen
& Von Heijne 1999). Multiple alignments of protein
sequences were performed using ClustalX software
(Thompson et al. 1997), with gap opening and extension
penalties being set as 10.0 and 0.05, respectively. The phylogenetic tree of TypA proteins was constructed by the
neighbour-joining method using MEGA version 4.0
(Tamura et al. 2007) with replication in 1000 bootstraps. The
accession numbers of TypA cDNA and protein sequences
are described in Supplementary Text S1.
Northern blot and RT-PCR analyses
Total RNA was fractionated by eletrophoresis on 1.2%
formaldehyde agarose gel, and blotted onto Hybond-N+
membranes (Amersham, Piscataway, NJ, USA) subsequently. The 32P labelled probes used in the experiments
were either the 194 bp 3′ UTR fragments of SsTypA1
cDNA amplified with primer pair 1 (forward 5′-GAG ACC
CTG CAA TAC ACC TTA CCA-3′, reverse 5′-GAT GCT
GTC CAA ACC TGT CCT T-3′) or the 588 bp fragments of
SsTypA1 cDNA amplified with primer pair 2 (forward
5′-GCG GAT CCA ATG ACA TTA GAA ACA TAG CTA
TTG-3′, reverse 5′-GTC GAC ATT GTG GCC CAG GGA
TGC A-3′). Hybridization was carried out at 65 °C according to the Hybond-N+ membrane user manual.
For RT-PCR analysis, total RNA (1 mg) was reverse transcribed using SuperScript III RNase H- Reverse Trancriptase (Invitrogen, Carlsbad, CA, USA) with oligo (dT)
primer according to the manufacturer’s instructions. PCR
amplification was performed with the same primers (primer
pair 1) as used in Northern blot analysis. The 365 bp fragment of SsActin cDNA, which was amplified with primers
(forward: 5′-CCA CCC GAG AGG AAA TAC AG-3′ and
reverse: 5′-TCC GCA AAG ATT ACA TAC CAT A-3′),
was used as quantitative control.
Scanning electron microscopy (SEM)
Five-week-old seedlings of S. salsa, treated with 400 mm
NaCl for 48 h were prepared for cDNA library construction. Total RNA was extracted using a guanidine thiocyanate (GT) method, and poly (A+) RNA was purified with a
Qligotex Direct mRNA Midi Kit (Qiagen, Hilden,
Germany). The cDNA library was constructed and
screened as described previously (Chen et al. 2007). The
cDNA sequences were determined by automatic DNA
sequencer (Genecore, Shanghai, China).
Samples were prepared and analysed as previously
described (Cao et al. 2007) with minor modifications.
Briefly, 2-week-old S. salsa seedlings were fixed in 2.5%
glutaraldehyde, 0.2 m sodium phosphate buffer, pH 7.4 at
4 °C overnight. After dehydration in graduated ethanol
series, the samples were treated with 100% isopentyl
acetate at 4 °C overnight. The materials were then dried
using a critical point dryer (model HCP-2, Hitachi, Tokyo,
Japan), sputter coated with gold in an E-1010 ion sputter
(Hitachi) and observed under a scanning electron microscope (SEM S-3000N, Hitachi).
Phylogenetic analyses of TypA genes
In situ hybridization
DnaSP 4.0 software (Rozas et al. 2003) was employed to
calculate the parameters of DNA polymorphism of TypA
Sense and antisense RNA probes used in the in situ hybridization were prepared by PCR using the same primers
cDNA library construction and screening
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
984 F. Wang et al.
(primer pair 1) as those used for Northern blot analysis. The
PCR fragments were inserted into the pGEM-T easy vector
(Promega, Madison, WI, USA), and the resultant plasmids
were sequenced to select constructs with the fragment
inserted in either sense or antisense orientations. The plasmids were then linearized with NcoI and transcribed in
vitro with SP6 RNA polymerase.
The in situ hybridization was carried out as previously
described (Yang et al. 1999). Sections from 2-week-old S.
salsa leaves, stems and roots were fixed with paraformaldehyde and embedded in paraffin. The hybridized probes
were detected using the anti-DIG–alkaline phosphatase
conjugate and BCIP/nitroblue tetrazolium (NBT) substrate. Sections were observed under an Axioskop II microscope (Zeiss, Jena, Germany) 3 h later.
Subcellular localization of SsTypA1
The cDNA fragments containing the SsTypA1 open reading
frame (ORF) were amplified by PCR using specific primers
with BamHI and SacII sites (forward: 5′-CGG GAT CCG
AGC TCA TGG AGA GTG CAA TTA CAA TC -3′;
reverse: 5′-TCC CCG CGG CCA CCT CTA GAC CTG
CCT TTC TTT GTC AT -3′; restriction sites are underlined), and then ligated to the 5′ end of green fluorescence
protein (GFP) coding region. The fused gene was subcloned
into the pPZP111 expression vector (Hajdukiewicz, Svab &
Maliga 1994) and placed under the control of the CaMV
35S promoter. The resultant construct was designated
pSsTypA1–GFP–PZP.
Fully expanded tobacco leaves were infiltrated with
Agrobacterium tumefaciens harbouring pSsTypA1–GFP–
PZP following the method as previously described (Prokhnevsky, Peremyslov & Dolja 2005). The agroinfiltrated
tobacco leaves were visualized with a laser scanning
confocal microscope (Zeiss, Jena) 2 d later. GFP fluorescence was excited at a wavelength of 488 nm, and chlorophyll autofluorescence was excited at a wavelength of
543 nm.
Tobacco transformation
The cDNA fragments containing the SsTypA1 ORF were
amplified by PCR using specific primers (forward: 5′-CGG
GAT CCG AGC TCA TGG AGA GTG CAA TTA CAA
TC-3′; reverse: 5′-GTC GAC TCA CCT GCC TTT CTT
TGT-3′), and then cloned into the pGEM-T easy vector
(Promega). The resultant plasmid, designated as
pSsTypA1-T, was trimmed with SacI and inserted into
similarly cut pSsTypA1–GFP–PZP. By this means, the
SsTypA1::GFP fusion region in the later plasmid was
replaced with the SsTypA1 ORF fragment. The derived
construct was named as pSsTypA1–PZP.
Recombinant plasmid pSsTypA1–PZP was transformed
into A. tumefaciens strain EHA105, and tobacco plants
were transformed by the method of Agrobacteriummediated leaf disc transformation (Horsch et al. 1985).
Expression and purification of
recombinant proteins
A pMAL Protein Fusion and Purification System (New
England BioLabs, Beverly, MA, USA) was used to express
recombinant maltose-binding protein (MBP)–SsTypA1.
Plasmid pSsTypA1-T was digested with BamHI and SalI,
and the ORF fragment of SsTypA1 was ligated into similarly cut pMAL-c2. The plasmid was transformed into the
E. coli BL21 strain. Cells were grown at 37 °C in LB medium containing 100 mg mL-1 of ampicillin to A600 = 0.6 and
induced with 400 mm isopropyl-d-thiogalactopyranoside at
28 °C for 5 h. The recombinant proteins were purified
according to the protocol provided by the supplier (New
England BioLabs).
Ribosome binding assay
Chloroplasts and 70S ribosomes were prepared using a
method previously described (Bartsch, Kimura & Subramanian 1982) with minor changes. All operations were performed at 0–4 °C. Forty grams of tobacco leaves chilled on
ice was hand chopped, homogenized in 50 mL buffer I
[0.7 m sorbitol in buffer A (10 mm Tris–HCl/pH 7.6, 50 mm
KCl, 10 mm Mg acetate, 7 mm 2-mercaptoethanol)] and
crushed with a Waring (Dayu, Shanghai, China) blender.
The slurry was filtered through eight layers of medical
cloches and centrifuged at 1200 g for 15 min. The pellet of
intact chloroplasts was washed once with buffer II (0.4 m
sorbitol in buffer A) and stored at -80 °C until use. For
further purification of the 70S ribosomes, the chloroplast
pellet was suspended in 10 mL buffer III [2% Triton X-100
(Amresco, Solon, OH, USA) in buffer A] for 30 min and
centrifuged at 30 000 g for 30 min. Crude chloroplast
extracts (supernatant) were layered over buffer IV (1 m
sucrose in buffer A) and centrifuged at 86 000 g for 17 h.
The ribosomal pellet was dissolved in 4 mL buffer V [50 mm
Tris–acetate/pH 8.0, 15 mm Mg acetate, 60 mm K acetate,
30 mm NH4 acetate, 1 mm dithiothreitol (DTT), 0.2 mm ethylenediaminetetraacetic acid (EDTA)] and centrifuged
twice at 30 000 g for 30 min to remove the insoluble materials. The ribosomal supernatant was concentrated and
further purified by filtering through a Nanosep 300K
Device (Pall, Ann Arbor, MI, USA).
The ribosome binding assay was performed using a modified method as previously described (Agafonov et al. 1999).
Sample aliquots (200 mL) containing 0.2 mm MBP–TypA1
proteins with 0.12 mm 70S ribosomes were incubated in
binding buffer [20 mm N-2-hydroxyethylpiperazine-N′-2ethanesulphonic acid (HEPES), 6 mm MgCl2, 150 mm
NH4Cl, 2 mm spermidine, 0.05 mm spermine, 1 mm GTP, pH
7.6] for 10 min at 37 °C, and then filtered through Nanosep
300K Devices by centrifugation at 1000 g for 30 min. The
filtrates were subjected to 10% sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS–PAGE) with subsequent Coomassie blue G-250 staining. The same amounts
of MBP–SsTypA1 proteins and MBP–SsTypA1 proteins
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
The role of a TypA/BipA-type GTPase from S. salsa in oxidative stress tolerance 985
filtrated through the membrane with 300 kDa cut-off
were used as controls.
Measurements were performed on the third leaves of plants
at 0, 0.5, 1, 2 h after high-light treatment and 20 h after
recovery.
Fluorescence-based GTP binding assay
The GTP binding assay was performed using a fluorescent
GTP analog, BODIPY FL-labelled guanosine 5′-O-(3thiotriphosphate), as reported previously (Kimple et al.
2004). The rate of guanine nucleotide exchange on MBP–
SsTypA1 proteins was assayed by monitoring an increase in
fluorescence, which represents the binding of fluorescent
nucleotide analog to catalytic site of SsTypA1. BODIPY
FL–GTP[S] was diluted to 1 mm in assay buffer (10 mm
Tris/pH 8.0, 1 mm EDTA and 10 mm MgCl2), and 1 mL of
the dilution was incubated with 0.1 mm MBP–SsTypA1 proteins in either presence or absence of 10 nm chloroplast 70S
ribosomes. Assay buffer was used to equilibrate the final
volume to 1.1 mL. Samples were transferred into a 3 mL
quartz cuvette with a path length of 1 cm, and the GTP
binding reaction was initiated by rapid mixing. Binding of
the fluorescent nucleotide analog was monitored by a
PerkinElmer LS55 Luminescence Spectrometer (Norton,
OH, USA) with excitation at 485 nm and emission at
530 nm. The fluorescence of BODIPY FL–GTP[S] alone
was used as control.
Methyl viologen (MV) treatment and
H2O2 detection
For oxidative stress treatments, 1-week-, 1-month- and
2-month-old wild-type (WT) and transgenic tobacco plants
were sprayed with 100 mm MV in 0.05% Tween 20 under
illumination at 200 mmol photons m-2 s-1.
The detection of H2O2 was performed essentially as previously described (Wang et al. 2006) with subtle modifications. One-week-old tobacco seedlings germinated on MS
medium were transferred to filter papers soaked with
ddH2O or 100 mm MV for 24 h. The seedlings were then
incubated with 1 mg mL-1 3,3-diaminobenzidine (DAB)
(dissolved in 50 mm TB buffer pH 7.6) at room temperature
in darkness for 20 h. Chlorophyll was removed with 100%
ethanol. For in situ detection of H2O2 in S. salsa, 2-week-old
seedlings were incubated with DAB for 24 h, fixed with 4%
formaldehyde in 0.025 m phosphate buffer pH 7.0, dehydrated through conventional ethanol series and embedded
in historesin (Leica, Wetzlar, Germany).
High-light treatment and chlorophyll
fluorescence analysis
Photooxidative treatments were performed by exposure of
2-month-old tobacco plants to an irradiance of 1000 mmol
photons m-2 s-1 at 20 °C for 2 h. The plants were then transferred to normal conditions (100 mmol photons m-2 s-1,
24 °C) for 20 h. The photosystem II (PSII) activity was estimated by chlorophyll fluorescence parameter (Fv/Fm), using
a portable pulse-modulated fluorometer (PAM-2000, Waltz,
Effeltrich, Germany) after 15 min of dark adaptation.
Zymogram and immunoassay
The third leaf of 2-month-old WT and transgenic tobacco
plants was cut into four segments, incubated in ddH2O or
10 mm H2O2, respectively and then illuminated (100 mmol
photons m-2 s-1) for 20 h. Intact chloroplasts were resuspended in lysis buffer (2% Triton X-100 in buffer A) and
gently stirred for 30 min at 4 °C. The crude chloroplast
extracts were obtained by centrifugation at 30 000 g for
30 min. NAD(P)H dehydrogenase (NDH) zymogram was
performed as previously described (Yao, Ye & Mi 2003).
Crude chloroplast extracts corresponding to 50 mg proteins
were subjected to 8% non-denatured PAGE at 4 °C. The
native PAGE gel was subsequently incubated in 20 mm
Tris–HCl/pH 7.5, 1 mm NADPH and 0.05% NBT for 30 min
at room temperature in the darkness. Bands reflecting
NAD(P)H–NBT oxidoreductase activity were visible as a
purple-blue color.
For the detection of the NDH complex by immunoblotting, the proteins on the native gel were electro-blotted
onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked
at 37 °C for 1 h with 5% powdered skim milk in buffer I
(0.9% NaCl, 100 mm Tris–HCl/pH 7.5), and then incubated
with anti-NDH-K polyclonal antibody (1:1000) for 1 h at
37 °C. The blot was washed twice in buffer I and incubated with anti-rabbit alkaline phosphatase conjugate
(Promega) as secondary antibody at 37 °C for 1 h. The
membrane was then washed twice with buffer I and once
with buffer II (0.9% NaCl, 5 mm MgCl2, 100 mm Tris–
HCl/pH 9.5). The immunoreactive bands were visualized
by incubation of the membrane in BCIP/NBT substrate at
room temperature.
RESULTS
Cloning of SsTypA1 gene and structural
characterization of the protein
To isolate salt tolerance-related genes from S. salsa, a functional identification approach was employed according to
the experimental procedure described in Chen et al. (2007).
Briefly, the cDNAs of S. salsa were inserted into the yeast
expression vector pREP1 under the control of the
thiamine-repressible nmt1 promoter and transformed into
fission yeast cells. Yeast clones showing enhanced salt tolerance were screened after expression induction of the
plant cDNAs by removing the thiamine from the medium.
A number of S. salsa genes able to confer enhanced salt
tolerance to the transgenic yeast cells were isolated. Of
these, a cDNA fragment bestowing tolerance to high salinity was selected for further study. Full-length cDNA that
contained an ORF coding for a protein of 683 amino acids
was obtained by 5′ rapid amplification of cDNA ends
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
986 F. Wang et al.
(a)
Figure 1. DNA polymorphism and
molecular evolution of TypA genes. (a) A
slide window analysis of DNA
polymorphism of TypA genes. Nucleotide
diversity (p), which is defined as the
average number of nucleotide differences
per site between two randomly chosen
DNA sequences, was calculated using
both synonymous and non-synonymous
sites of TypA. Window size was set as
50 bp with step values of 9 bp.
Corresponding domains of SsTypA1
protein are depicted below the curve of p
values. (b) Neighbour-joining tree based
on multiple alignments of the protein
sequences of TypAs. The number above
the internal branches shows the local
bootstrap probability obtained for 1000
repetitions. The scale bar indicates the
estimated number of amino acid
substitutions per site. The TypA/BipA
gene of Escherichia coli was set as an
outgroup.
(b)
(RACE). Sequence analysis indicated that the plant cDNA
encoded for a protein with highest homology to the TypA/
BipA GTP-binding proteins. Thus, the gene was designated
as SsTypA1 (accession no. EU289224). Structural analysis
revealed a TypA/BipA domain of about 200 amino acids
and a chloroplast transit peptide sequence at the Nterminal part of the SsTypA1 protein (Fig. 1a).
A phylogenetic analysis was conducted to study the evolutionary relationships of TypA/BipA proteins from various
organisms in which the proteins have been identified. The
resulting rooted tree presented in Fig. 1b shows that the
proteins share a common ancestor with bacterial TypA and
can be clustered into several groups. Plant TypA proteins
formed a monophyletic group within which SsTypA1,
together with Arabidopsis TypA, constituted a subgroup,
and proteins of the monocot group branched out from the
same origin. The parameters of DNA polymorphism of
TypA genes showed that the TypA/BipA domains encoded
by these genes were highly conserved (Fig. 1a). The low
nucleotide diversity (p) and the short branch length
(Fig. 1b) indicated that TypA proteins from different organisms had close evolutionary relationships and might have
highly conserved cellular roles.
Expression of SsTypA1 is responsive to
various abiotic stresses
As SsTypA1 gene was isolated while screening for salt
tolerance-related genes, we investigated the effect of salinity stress on the expression of SsTypA1 gene in S. salsa by
Northern blot analysis using the 3′ UTR fragments of
SsTypA1 cDNA as a gene-specific hybridization probe.
Figure 2a shows that salt stress resulted in an increase in the
relative amount of SsTypA1 mRNA. Enhanced accumulation of SsTypA1 transcripts occurred within 6–12 h and
decreased 72 h after the treatment. In addition to Northern
blot analysis of SsTypA1 expression after NaCl treatment,
the amounts of SsTypA1 mRNA in response to several
other stressors including H2O2, LiCl and mannitol were
assessed by RT-PCR. As can be seen in Fig. 2b, all these
stress treatments led to accumulation of SsTypA1 transcripts. Among the stressors, however, H2O2 gave rise to
faster and more pronounced activation of SsTypA1 expression. Induction of SsTypA1 expression occurred within 4 h
of H2O2 treatment, but did not become evident until 6 h
after exposure to other stressors. To further extend this
finding, a time-course experiment evaluating the effect of
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
The role of a TypA/BipA-type GTPase from S. salsa in oxidative stress tolerance 987
(a)
(b)
(c)
(d)
Figure 2. Expression patterns of SsTypA1. (a) Northern blot
analysis of SsTypA1 expression in response to 400 mm NaCl for
0, 6, 12, 24, 48 and 72 h, respectively. Total RNAs (10 mg) were
extracted from 5-week-old Suaeda salsa, and hybridized with
radioactive 3′ untranslated region (UTR) of SsTypA1 as
gene-specific probes. Ethidium bromide-stained rRNA was used
for equivalent loading control. (b) RT-PCR analysis of the
SsTypA1 expression under different stress treatments. Total RNA
was isolated from 5-week-old S. salsa treated with 20 mm H2O2
(for 0, 4 and 16 h), 400 mm LiCl and 500 mm mannitol (for 0, 6
and 24 h), respectively. (c) RT-PCR analysis of SsTypA1
expression in response to 20 mm H2O2 for 0, 4, 8, 16 and 24 h,
respectively. (d) RT-PCR analysis of organ-specific expression of
SsTypA1. Total RNA was isolated from roots (R), stems (S) and
leaves (L) of 5-week-old S. salsa plants.
H2O2 on SsTypA1 expression was carried out. Figure 2c
shows that increase of SsTypA1 mRNA took place within
4 h and decreased to the basal level 24 h after treatment.
Quantification analysis indicated that SsTypA1 mRNA
amount at the time points of 4 h and 8 h after H2O2 treatment increase about twofold (data not shown). These
results indicated that expression of the SsTypA1 gene was
responsive to multiple external stimuli, but may be associated more directly or tightly with oxidative stress.
Epidermis-specific expression of SsTypA1
To investigate the expression pattern of SsTypA1 in different organs of S. salsa, RT-PCR was performed using total
RNA extracted from root, stem and leaf of the plant. As
shown in Fig. 2d, SsTypA1 was expressed ubiquitously in
these organs, but the expression level in root was rather low
as compared to stem and leaf. Because of the extremely
small flowers of S. salsa, expression of the gene in the flower
was not examined.
Suaeda salsa is a euhalophytic plant possessing succulent
leaves that are cylindrically shaped. Under SEM, the leaf
epidermis of the plant appeared rugged, and many epidermal protrusions were visualized on the stem surface
(Fig. 3a). To further probe the expression features of the
gene at the tissue level, in situ hybridization was employed
to detect SsTypA1 transcripts in different tissues of S. salsa
organs. Transverse sections of stems and median longitudinal sections of leaves of 2-week-old plants (Fig. 3b) showed
that the accumulation of SsTypA1 transcript was restricted
to the epidermal layer of the leaf and stem. No hybridization signal was detected in any other tissues/cells in these
organs, neither in the epidermis and other tissues of root.
Two independent in situ hybridization experiments were
performed to confirm this observation, and the result was
reproducible, indicating that expression of the SsTypA1
gene was specific to the leaf and stem epidermis in S. salsa.
Because subsequent studies indicated that SsTypA1 may
play a role in ROS tolerance (see below), we examined the
H2O2 levels in different tissues of the stem and leaf in S.
salsa by DAB staining. Transverse sections of stems and
median longitudinal sections of leaves shown in Fig. 3c
demonstrated that the H2O2 content was significantly
higher in the epidermal layer than in other tissues. Incubation of seedlings of S. salsa with an eosin-Y solution resulted
in equal staining of the leaf and stem cells (data not shown),
indicating that differential color intensities observed with
DAB staining were not a consequence of differential cell
permeability of the dye.
Chloroplast-specific localization of SsTypA1
The subcellular localization of SsTypA1 proteins was
determined by a transient expression assay. Agrobacterium
tumefaciens harbouring the plasmid containing the
SsTypA1::GFP was introduced into tobacco leaves by the
method of infiltration. Visualization with a confocal microscope showed that SsTypA1–GFP fusion proteins were targeted mainly to the chloroplasts of the transformed leaf
cells including guard cells, other epidermal cells and mesophyll cells. Such localization of the fusion proteins was
particularly evident in the guard cells containing more
abundant chloroplasts. No fluorescence was detected in the
other organelles, such as the mitochondria and Golgi body
(Fig. 4a). This subcellular distribution of SsTypA1 proteins
is consistent with the sequence-based prediction, which
forecasted chloroplast subcellular localization of SsTypA1
with a probability/certainty score of 0.58.
It is frequently described that, unlike guard cells, other
epidermal cells in higher plants usually do not contain any
or have few small chloroplasts. Thus, the epidermal- and
chloroplast-specific expression of SsTypA1 seemed paradoxical. To clarify the situation, the epidermis of S. salsa was
examined for the presence of chloroplasts by confocal
microscopy. Fluorescence micrographs of epidermal peels
clearly demonstrated the existence of normal-sized chloroplasts within both the leaf and stem epidermal cells
(Fig. 4b). By changing the focal plane, it was confirmed that
the observed chloroplasts existed in the epidermal cells and
were not residues of broken mesophyll cells cohering to the
epidermis. The presence of chloroplasts in epidermal cells
could be a feature evolved in some plants that possess
limited leaf surface area in order to compensate for the
requirement of photosynthesis.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
988 F. Wang et al.
(a)
(b)
(c)
Figure 3. Morphology of the Suaeda salsa seedling and in situ detection of SsTypA1 expression and H2O2 accumulation. (a) Photograph
(1) and scanning electron micrographs of leaf (2) and stem (3) epidermis of 2-week-old S. salsa seedlings. Bars = 400 mm. (b) In situ
hybridization of SsTypA1 in median longitudinal sections of leaves (1 and 2) and transverse sections of stems (3 and 4). Positive
hybridization (hybridization with antisense probe) is displayed as a purple-blue color (note the epidermal layers in 2 and 4). Sections that
hybridized with sense probes were used as negative controls (1 and 3). Bars = 50 mm. (c) In situ detection of H2O2 in the leaves (1 and 2)
and stems (3 and 4) of S. salsa seedlings. H2O2 accumulation was detected as brown areas (2 and 4). Unstained leaves and stems were
used as controls (1 and 3; note the stained epidermis in 2 and 4). Bars = 50 mm.
SsTypA1 is a ribosome-binding GTPase
As SsTypA1 encodes for a putative translational GTPase,
the ribosome binding and GTPase activity of purified
SsTypA1 proteins were tested using the filter and
fluorescent assays (Agafonov et al. 1999; Kimple et al.
2004). Full-length SsTypA1 proteins were expressed as
MBP-tagged polypeptides in E. coli and purified by affinity chromatography. To examine the substantial interaction
between SsTypA1 and the ribosome, MBP–SsTypA1 proteins were incubated with chloroplast ribosomes and then
subjected to a size filter assay. As shown in Fig. 5a, after
incubation with ribosomes, SsTypA1 proteins were no
longer able to pass through the filter device (lane 3), indicating that binding of the proteins to the ribosomes
occurred in the reaction.
GTPase activity and the effect of ribosome addition on
the enzymatic activity of SsTypA1 were examined. As can
be seen in Fig. 5b, SsTypA1 had a basal level of GTPase
activity, but this activity was stimulated to a significantly
higher level when 70S ribosomes were added to the reaction, suggesting that the GTPase activity of SsTypA1 was
ribosome dependent. These results demonstrate that
SsTypA1 was a functional ribosomal-binding GTPase,
implying a role for the protein in translational regulation of
its target proteins.
Ectopic over-expression of SsTypA1 rendered
oxidative stress tolerance to tobacco plants
The SsTypA1 expression induced by various stresses suggests a role for this gene in stress tolerance. Because of lack
of a readily available method for genetic transformation of
S. salsa, an ectopic over-expression strategy was employed
to probe the in vivo function of SsTypA1. A plasmid construct containing the SsTypA1 cDNA under the control of
the 35S promoter was introduced into tobacco plants by
Agrobacterium-mediated transformation. Approximately
20 independent transgenic lines were generated, and two
lines showing increased transgene expression (Fig. 6a) were
selected for subsequent stress tolerance inspection. According to the results shown in Fig. 2, the transgenic tobacco
plants were firstly examined for tolerance to oxidative
stress. As MV is known to cause ROS stress in chloroplasts,
this chemical was used in the experiment. WT and transgenic plants grown for 1 week, or 1 or 2 months were
treated with MV, and the damage of the chemical to the
plants were evaluated. As Fig. 6b shows, the leaves of the
two transgenic lines (lines 15 and 18) expressing a relatively
high level of SsTypA1 acquired a remarkably increased
tolerance to the oxidative stress caused by MV. Six hours
after spraying of the chemical, the leaves of control plants
had withered and curled badly, while such severe injury was
not observed in the leaves of lines 15 and 18. This difference
in MV tolerance was particularly evident for the 2-monthold plants that have larger leaves. Transgenic line 37, in
which SsTypA1 was expressed at a rather low level, showed
a phenotype similar to that of the WT control (data not
shown). These results indicate that the ROS tolerance
gained by transgenic plants was positively correlated with
the expression level of SsTypA1 transgene.
In addition to oxidative stress caused by MV treatment,
the photooxidative stress tolerance of the plants under
high-light stress was also tested by chlorophyll fluorescence
analysis (Fig. 6d). The results indicated that the activity of
PSII was higher in the transgenic plants as compared to
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
The role of a TypA/BipA-type GTPase from S. salsa in oxidative stress tolerance 989
(a)
(b)
under the experimental condition (upper panel). When
treated with 100 mm MV, all plants accumulated higher
amounts of H2O2 (lower panel). However, accumulation of
H2O2 appeared much less in transgenic lines 15 and 18 as
compared to WT plants. This analysis clearly showed that
(a)
(b)
Figure 4. Subcellular localization of SsTypA1. (a)
SsTypA1–green fluorescence protein (GFP) fusion proteins in
stomatal cells of tobacco leaf epidermis. The fusion constructs
were introduced into tobacco leaves by agroinfiltration.
Bar = 10 mm. (b) Confocal laser scanning of chloroplasts in
epidermal cells of Suaeda salsa. 1, bulge cells on stem surface; 2,
epidermal peel of stem; 3, epidermal peal of leaf. Arrows indicate
the chlorophyll autofluorescence images in chloroplasts.
Bars = 20 mm.
the WT control under photooxidative stress, while the
difference was less pronounced during the course of photorecovery. Again, this result demonstrated that SsTypA1
over-expression could render oxidative stress tolerance to
the transgenic plants.
The tolerance of the WT and transgenic plants to salinity
and drought stresses was also analysed. Unlike their resistance to oxidative stress, transgenic plants (lines 15 and 18)
exhibited only a slightly improved tolerance to salt and
drought stresses (data not shown).
SsTypA1 over-expression is correlated with
reduced H2O2 content
The improved oxidative stress tolerance of the transgenic
plants raised the possibility that SsTypA1 expression may
alter the ROS content in the cell. To test this possibility, the
H2O2 levels in SsTypA1 transgenic tobacco plants were
analysed. DAB staining that specifically detects H2O2 was
performed. As shown in Fig. 6c, without stress, both WT and
SsTypA1 transgenic plants displayed a low level of H2O2
Figure 5. Detection of ribosome binding and GTPase activities
of SsTypA1. (a) Binding of SsTypA1 to ribosome. Sodium
dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) of purified maltose-binding protein
(MBP)–SsTypA1 proteins and filtrates passed through
membranes with 300 kDa cut-offs. 1, MBP–SsTypA1 proteins;
2–3, MBP–SsTypA1 (2) and MBP–SsTypA1 + 70S ribosomes (3)
reactions filtered by Nanosep 300K device. The additional bands
at the bottom of the gel are degradation products of MBP from
the fusion proteins. (b) Stimulation of the GTPase activity of
SsTypA1 by 70S ribosome. Time-course of BODIPY FL–GTP[S]
(1 mm) binding to MBP–SsTypA1 (100 nm) in the absence or
presence of chloroplast 70S ribosomes. 䊉, MBP–SsTypA1 basal
activity, reaction with BGTP[S] alone was used as a control; 䊊,
MBP–SsTypA1 GTPase activity in the presence of 70S
robosomes, reaction with ribosomes and BGTP[S] was used as a
control.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
990 F. Wang et al.
(a)
(b)
(c)
(d)
Figure 6. Effect of methyl viologen (MV) and high-light
treatments on wild-type (WT) and SsTypA1 transgenic tobacco
plants. (a) Northern blot analysis of SsTypA1 transcript levels.
Total RNAs (20 mg) were extracted from leaves of WT and
transgenic plants, and hybridized with radioactive SsTypA1
cDNA probes. Ethidium bromide-stained rRNA was used as an
equivalent loading control. (b) Typical leaf injuries caused by
exposure to MV. Two-month-old WT and transgenic plants were
sprayed with 100 mm MV and then illuminated (200 mmol m-2 s-1)
for 6 h. (c) Effects of MV stress on the accumulation of H2O2 in
the leaves of WT and transgenic plants. One-week-old seedlings
were transferred to filter papers soaked with ddH2O or 100 mm
MV for 24 h under illumination at 100 mmol m-2 s-1. H2O2
accumulation in leaves was detected as brown areas. (d) WT and
transgenic tobacco (line 15) plants were illuminated at high light
(1000 mmol m-2 s-1) for 2 h then recovered under a light intensity
of 100 mmol m-2 s-1 for 20 h. The photosystem II (PSII) activity
(Fv/Fm) in the third leaf of tobacco plant was measured after dark
adaptation for 15 min. The data are the mean value ⫾ standard
deviation (SD) of three individual experiments.
the chloroplast-encoded ROS-scavenging proteins. As a
first step towards testing this hypothesis, we searched the
literature as well as the available complete sequence of
the tobacco chloroplast genome for the genes that were
reported to participate in ROS detoxification or possibly
involved in redox balance. It turned out that the number of
candidate proteins that we can consider as SsTypA1’s
targets was limited, as only few members in the known
antioxidant systems are encoded by chloroplast genome
(Shimada & Sugiura 1991; Mittler et al. 2004). Albeit this,
the ndh genes encoding for the subunits of NDH complex
attracted our attention because they represented one of the
largest families of plastid genes and were reported to participate in suppressing the accumulation of ROS in chloroplasts (Casano et al. 2000, 2001;Wang et al. 2006).Therefore,
we conducted an experiment to compare the enzymatic
activity of the NDH complex in the chloroplasts of WT and
transgenic tobacco plants. The results appeared to be consistent with our assumption. As shown in Fig. 7, a band
reflecting NAD(P)H–NBT oxidoreductase activity visualized by native PAGE was obviously stronger in the chloroplasts of transgenic plants (lines 15 and 18) as compared to
the WT control under non-stressed condition. After the
oxidative stress treatment, enzyme activity was augmented
in the WT control and was further increased in the transgenic plants (Fig. 7). These results indicate that NDH activity was induced in response to oxidative stress and, in
addition, that SsTypA1 overproduction led to enhanced
NDH activity. To verify that the enzyme activity was
derived from the NDH complex, Western blotting with
native PAGE gel and antibodies against the NDH-K
subunit was carried out. The results in Fig. 7 show that the
activity band represented the NDH complex. More importantly, the nearly identical patterns of enzymatic and
immune-reaction bands indicate that the enhanced enzyme
activity was a result of increased protein amounts of the
NDH complex.
It should be emphasized that, although our experimental
data provide a line of evidence implying the regulation of
over-expression of SsTypA1 resulted in suppression of
H2O2 production caused by MV stress and, additionally, that
the production of this superoxide was negatively related to
the expression levels of the SsTypA1 transgene.
Increase in the amounts and activities of the
NDH complex in SsTypA1 transgenics
The reduced superoxide level suggested an effect of
SsTypA1 on the mechanism controlling the redox status in
the chloroplast. Based on the ribosome-dependent GTPase
activity and chloroplast location of SsTypA1, we assumed
that SsTypA1 may function in translational regulation of
Figure 7. NAD(P)H dehydrogenase activity and
immunoblotting assays. Proteins (50 mg) isolated from wild-type
(WT) and transgenic plant leaf segments incubated in ddH2O (-)
or 10 mm H2O2 (+) for 20 h were run on a native polyacrylamide
gel electrophoresis (PAGE) gel. Zymogram and immunoassay
were conducted after electrophoresis. Upper panel, activity
staining of NAD(P)H–nitroblue tetrazolium (NBT)
oxidoreductase. Lower panel, immunoblotting analysis of the
NDH complex with antibody against NDH-K.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
The role of a TypA/BipA-type GTPase from S. salsa in oxidative stress tolerance 991
NDH complex by SsTypA1, additional target proteins of
SsTypA1 may also exist in the chloroplast.
DISCUSSION
Stress-induced expression of SsTypA1
It was reported that TypA/BipA proteins have important
roles in bacterial tolerance to environmental stresses
including temperature, SDS and low pH (Kiss et al. 2004).
In this study, we observed that the expression of SsTypA1
was stimulated by various abiotic stresses in S. salsa, and
that over-expression of SsTypA1 significantly improved the
oxidative stress tolerance of the transgenic tobacco plants.
Hence, TypA/BipA proteins represent a new class of stress
tolerance-related proteins functioning in organisms ranging
from prokaryotes to higher eukaryotes.
Compared to the salt and drought stressors, ROS treatment resulted in a more pronounced induction of SsTypA1
expression. Moreover, transgenic tobacco plants overexpressing SsTypA1 exhibited a remarkably enhanced tolerance to oxidative stress, but only a slightly increased
tolerance to other stresses, and the H2O2 level was significantly reduced in SsTypA1 transgenic tobacco plants. As
many external stressors generate ROS and cause secondary
oxidative stress in plant cells, we believe that the bona fide
response of SsTypA1 expression was to oxidative stress. The
increased expression of SsTypA1 in response to salt and
drought may result from the excessive ROS generated by
these stressors.
SsTypA1 was isolated in our attempt to identify genes
mediating adaptation to salt stress in S. salsa. Using fission
yeast as a functional screening system, we found that overexpression of SsTypA1 led to an increase in salt tolerance to
transgenic yeast cells (Supplementary Fig. S1a). Because
yeast cell does not contain chloroplast, SsTypA1 may exsert
its function in mitochondria as it is known that some plant
chloroplastic proteins were targeted into mitochondria
when expressed in yeast (Cheng et al. 2006). Subsequently,
we also observed that over-expression of SsTypA1 conferred enhanced salt tolerance to transgenic tobacco BY-2
cells (Supplementary Fig. S1b). Unlike in yeast and BY-2
cells, over-expression of SsTypA1 mainly resulted in
improved tolerance to oxidative stress, but not to salt stress,
in transgenic tobacco plants. These results suggest that the
suppression of ROS resulting from SsTypA1 production
was not sufficient to coordinate the complex regulating
mechanisms involved in the salt stress response in multicelled tobacco plants, while it could affect the mechanisms
responsible for salt tolerance in the simple single-celled
yeast and BY-2 cells.
Epidermal-specific expression of SsTypA1
The epidermis plays crucial roles in the development of
various organs and in water retention in both animals and
plants. Epidermal-specific expression has been found with
the genes encoding proteins for cuticle wax synthesis and
lipid transfer (Clark & Bohnert 1999; Kurata et al. 2003),
and proteins involved in defending the fungus infection
(Finni et al. 2002). As the outermost layer of the plant body
directly exposed to the environment, the epidermis may
accumulate excessive amount of ROS as compared to other
tissues. Indeed, we observed a higher level of ROS in the
epidemic tissue in S. salsa (Fig. 3c), and some previous
studies also reported the presence of a larger quantity of
ROS in the epidermal tissues (McInnis et al. 2006; Li, Xing
& Zhang 2007). It is therefore possible that the epidermalspecific expression of SsTypA1 is an adaptive response
against oxidative injury caused by the highly stressful environment of this extremophile plant. To support this notion,
we observed that betacyanin, which was thought to play
roles in reduction of ROS level in red beet (SepulvedaJimenez et al. 2004), was accumulated most abundantly in
the epidermis in S. salsa (data not shown).
TypA represents a new member of the
ROS-scavenging system in the chloroplast
The chloroplast is one of the major sources of ROS production in plant cells, especially under stress conditions (Foyer,
Descourvieres & Kunert 1994; Mittler et al. 2004; Foyer &
Noctor 2005). Because of the high susceptibility of the chloroplast to oxidative damage, scavenging of ROS in chloroplasts in response to environmental stresses is critical
(Kaiser 1979). Previous studies found that photosynthetic
capacity and PSII photochemistry were not affected by
salinity treatments in S. salsa, and therefore, it was proposed
that this plant possesses an effective antioxidant response
system for avoiding stress-induced oxidative damage within
the chloroplasts (Lu et al. 2003; Zhang et al. 2005).We found
that SsTypA1 proteins were distributed specifically in the
chloroplasts. Such a subcellular localization of SsTypA1,
together with the effect of its over-expression on the oxidative stress tolerance of the transgenic plants, reflected a
physiological relevance of the protein in the chloroplastic
antioxidant systems presenting in S. salsa.
Some ROS-scavenging enzymes including SOD and APX
were found to play important roles in protecting the chloroplasts against oxidative injure. A number of studies have
shown that expression of these chloroplast-targeted antioxidative enzymes was regulated by transcription factors,
microRNA and other proteins (Mittler et al. 2004; Sunkar,
Kapoor & Zhu 2006). In addition to these nuclear geneencoded antioxidative enzymes, the NDH complex containing 11 chloroplast DNA-encoded subunits and three nuclear
gene-encoded peptides was also proposed to participate in
suppressing the stress-related ROS during the chlororespiratory process and serve as an H2O2 detoxification pathway
within the chloroplast (Casano et al. 2000, 2001). In this
study, we observed that the amount of NDH complex was
regulated by SsTypA1 proteins.Together, these data indicate
that the ROS status in chloroplast is controlled by complex
mechanisms involving genes from both the nuclear and
chloroplast genomes, and TypA1 may represent a new
component of this growing regulatory family.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
992 F. Wang et al.
The possible role of SsTypA1 in the regulation
of ROS-scavenging proteins in the chloroplast
TypA/BipA proteins are widely distributed in bacteria and
plants. So far, very limited research has been carried out on
these proteins in higher plants. Thus, our knowledge of their
cellular roles is poor. Based on our experimental results, we
propose that SsTypA1 may act as a translational regulator
of the stress-responsive proteins involved in ROS control in
the chloroplast, such as peptides in the NDH complex. The
following facts formed the basis of our speculation: (1)
SsTypA1 encodes for a putative translational GTPase, and
our biochemical analysis demonstrated that SsTypA1 was a
functional ribosome-stimulated GTPase; (2) SsTypA1 proteins are located in the chloroplast, and it is known that
translational regulation is a primary step in chloroplast
gene expression (Marin-Navarro, Manuell & Wu 2007;
Raynaud et al. 2007); (3) SsTypA1 is a stress-responsive
gene, and it is understood that translation of chloroplast
mRNAs is regulated in response to a variety of biotic and
abiotic factors (Marin-Navarro et al. 2007; Raynaud et al.
2007); (4) In SsTypA1 transgenic tobacco plants, the
amounts of the NDH complex augmented; and (5) E. coli
BipA1 was proposed to execute its function by destabilizing
the exceptionally high degree of complementary structure
formed between the SD element of fis mRNA and the 3′
end of 16S rRNA, thus allowing ‘fast-track’ translation of
the mRNA (Owens et al. 2004). Based on these points, it is
not unreasonable to assume that SsTypA1 may play a
similar role as its counterpart does in E. coli. More extensive studies are certainly required to evaluate such a function of SsTypA1 in our future studies.
In summary, because of the dual role of ROS, such as
H2O2 in signalling and superoxidation, the steady-state level
of ROS in plant cells needs to be tightly regulated (Mittler
et al. 2004). The data presented here suggest that TypA may
represent a new player in the ROS gene network and that
TypA-mediated post-transcriptional regulation in the chloroplast may represent an important mechanism controlling
the development of oxidative stress tolerance in halophytic
plants such as S. salsa.
ACKNOWLEDGMENTS
We are grateful to Dr Hui Zhang of Shandong Normal
University for providing us generously with the S. salsa
seeds, and Dr Hualing Mi of the Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, for her
kind gift of NDH antibodies. We appreciate Dr Tian-Ying
Yu and Hong-Wei Li of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for their
kind help on the in situ hybridization and chlorophyll fluorescence analysis; Dr Wei Qian of our institute for his constructive help on the phylogenetic analysis; and Dr Yong Hu
of Capital Normal University for the confocal analysis. This
work was supported by the ‘863’ High Technology Development Program (Grant No. 2007AA021405) and the
Projects of Important Science and Technology in Xinjiang
(Grant No. 20073118-3).
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Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994
994 F. Wang et al.
Received 15 December 2007; received in revised form 13 March
2008; accepted for publication 14 March 2008
SUPPLEMENTARY MATERIAL
The following supplementary material is available for this
article:
Figure S1. Over-expression of SsTYPA1 conferred salt tolerance to transgenic yeast and BY-2 cells. (a) cDNA fragment containing the SsTypA1 open reading frame (ORF)
was cloned into pREP5N and the resulted construct was
designated as pREP5N–SsTypA1. Cell densities of fission
yeast transformed with pREP5N and pREP–SsTypA1 were
adjusted to OD600 = 1.0, and spotted on solid MM medium
(control) or the same medium supplemented with 600 mm
NaCl. (b) Viability test of BY-2 cells that were transferred
to liquid LS medium (control) or the same medium supplemented with 500 mm NaCl for 2 h. Fluorescein diacetate
(FDA) was utilized to estimate the cell viability.
Text S1. GenBank accession numbers for the cDNA
sequence used in DNA polymorphism analysis.
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© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 982–994