Reduced expression of a gene encoding a Golgi localized monosaccharide transporter (OsGMST1) confers hypersensitivity to salt in rice (Oryza sativa).
ABSTRACT Sugar transport is critical for normal plant development and stress responses. However, functional evidence for the roles of monosaccharide transporters in rice (Oryza sativa) has not previously been presented. In this study, reversed genetics was used to identify OsGMST1 as a member of the monosaccharide transporter family in rice. The predicted 481 amino acid protein has the typical features of a sugar transporter in the plastid glucose transporter subfamily consistent with reduced monosaccharide accumulation in plants with reduced OsGMST1 expression. OsGMST1-green fluorescent protein is localized to the Golgi apparatus. OsGMST1 expression is induced by salt treatment and reduced expression confers hypersensitivity to salt stress in rice. OsGMST1 may play a direct or an indirect role in tolerance to salt stress in rice.
Article: Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport.[show abstract] [hide abstract]
ABSTRACT: The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT-green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter-beta-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type-specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH(4)(+), fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.The Plant Cell 01/2007; 18(12):3476-90. · 8.99 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativa monosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.Bioscience Biotechnology and Biochemistry 04/2003; 67(3):556-62. · 1.28 Impact Factor
Article: Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.).[show abstract] [hide abstract]
ABSTRACT: Monosaccharide transporters mediate the membrane transport of a variable range of monosaccharides, which plays a crucial role in sugar distribution throughout the plant. To investigate the significance of monosaccharide transporters for rice (Oryza sativa L.) seed development, cDNA of a new putative monosaccharide transporter gene OsMST4 was isolated. The deduced OsMST4 protein shows typical features of monosaccharide transporters, and shares high homology with other plant homologues. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that OsMST4 is a functional monosaccharide transporter capable of transporting glucose, fructose, mannose and galactose. Transcriptional analysis revealed that OsMST4 is expressed in all tested organs/tissues. In developing caryopses, its expression is high at the early and middle grain filling stages, and declines gradually to low levels after that. Further analysis revealed that it is expressed in both the maternal tissue and the filial tissue, with its highest expression in embryo. Cellular location in young caryopses through RNA in situ hybridization showed that OsMST4 mRNA mainly accumulates in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm. The expression pattern of OsMST4 was further confirmed by histochemical analysis of the OsMST4-promoter-beta-glucuronidase (GUS) transgenic rice plants. These data indicate that OsMST4 is actively involved in monosaccharides supply for seed development during the course of grain filling. In addition, the cell type-specific expression patterns of OsMST4 in other sink and source tissues were also investigated, and its corresponding physiological roles were discussed.Plant Molecular Biology 12/2007; 65(4):439-51. · 4.15 Impact Factor
Journal of Experimental Botany, Vol. 62, No. 13, pp. 4595–4604, 2011
doi:10.1093/jxb/err178Advance Access publication 25 May, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Reduced expression of a gene encoding a Golgi localized
monosaccharide transporter (OsGMST1) confers
hypersensitivity to salt in rice (Oryza sativa)
Hong Cao1,*, Siyi Guo1,2,*, Yunyuan Xu1, Kun Jiang3, Alan M. Jones3,4and Kang Chong1,5,†
1Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing
2Graduate University of Chinese Academy of Sciences, Beijing 100049, China
3Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
4Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
5National Research Center for Plant Gene, Beijing 100093, China
* These authors contributed equally to this paper.
yTo whom correspondence should be addressed. E-mail: Chongk@ibcas.ac.cn
Received 7 March 2011; Revised 1 May 2011; Accepted 3 May 2011
Sugar transport is critical for normal plant development and stress responses. However, functional evidence for the
roles of monosaccharide transporters in rice (Oryza sativa) has not previously been presented. In this study,
reversed genetics was used to identify OsGMST1 as a member of the monosaccharide transporter family in rice. The
predicted 481 amino acid protein has the typical features of a sugar transporter in the plastid glucose transporter
subfamily consistent with reduced monosaccharide accumulation in plants with reduced OsGMST1 expression.
OsGMST1-green fluorescent protein is localized to the Golgi apparatus. OsGMST1 expression is induced by salt
treatment and reduced expression confers hypersensitivity to salt stress in rice. OsGMST1 may play a direct or an
indirect role in tolerance to salt stress in rice.
Key words: Golgi, monosaccharide transporter, NaCl stress, Oryza sativa L., rice, SGB1.
In higher plants, sugars function as an energy source, signal
molecules, building blocks of polysaccharide structures, and
osmotic regulators. Sugars are synthesized in the photo-
trophic tissues (source) of the plant and then exported to
heterotrophic tissues (sink), and this process requires several
transport steps across membranes (Lalonde et al., 1999;
Williams et al., 2000). Sugar transporters play key roles in
sugar allocation at both the subcellular level and in
long-distance transport via the phloem (Buttner, 2007).
Long-distance transport is required for sinks to import
photosynthetic assimilates from sources in the form of sucrose
(Sauer, 2007). In sink tissues, released sucrose travels to sink
cells via plasmodesmata and/or sucrose transporters (SUCs)
or via monosaccharide transporters (MSTs) after hydrolysis
to glucose and fructose by cell wall-bound invertases
(Williams et al., 2000; Weschke et al., 2003). As such, the
process of long-distance transport involves both cell-to-cell
transport as well as to different organelles in a single cell. The
sugar transport mode is dependent on the plant developmen-
tal stage and the sugar location (Williams et al., 2000). SUCs
and MSTs are the main sugar transporters in plants.
MSTs mediate the transport of a wide range of mono-
saccharides including glucose, fructose, maltose, raffinose,
and sugar alcohols (Turgeon and Medville, 2004; Klepek
et al., 2005). In Arabidopsis, 53 monosaccharide transporter
(-like) genes group into seven subfamilies: (i) STP (plasma
membrane monosaccharide transporter), (ii) ERD-like,
(iii) VGT (vacuolar glucose transporter), (iv) PLT (polyol
ª 2011 The Author(s).
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transporter), (v) INT (inositol transporter), (vi) TMT
(tonoplast monosaccharide transporter), and (vii) pGlcT
(plastid glucose transporter) (Johnson et al., 2006; Buttner,
2007). Most MSTs have 12 membrane-spanning domains
separated by a cytoplasmic loop between transmembrane
helices 6 and 7. In addition to transport across the plasma
membrane, monosaccharides are also transported across
organelle membranes, such as the inner plastid envelope
(Weber et al., 2000; Niittyla et al., 2004) and tonoplast
(Wormit et al., 2006). MSTs on the Golgi apparatus (Wang
et al., 2006) and mitochondria (Szarka et al., 2004) have
also been reported. The rice genome encodes a total of 65
MST genes that group into the seven known subfamilies as
defined by the Arabidopsis clades (Johnson and Thomas,
2007). The localization of a few of these transporters are
known but, to date, nothing is known about their physio-
logical functions (Toyofuku et al., 2000; Ngampanya et al.,
2003; Mamun et al., 2006; Oliver et al., 2007; Wang et al.,
2007, 2008; Cho et al., 2010).
In plants, sugars are signalling molecules that regulate
plant growth and development and responses to biotic and
abiotic stresses (Moore and Sheen, 1999; Rolland et al.,
2002, 2006; Gibson, 2005). There is evidence that abiotic
stresses affect the expression of plant monosaccharide
transporters. The transcript level of TMT1 increased in
response to salt, drought, and cold treatment in Arabidopsis,
accumulation during cold stress (Wormit et al., 2006). The
expression of ERD6, which encoded a putative sugar
transporter of Arabidopsis, was up-regulated by dehydration
and cold treatment (Kiyosue et al., 1998). AtESL1 was also
an abiotic stress-inducible monosaccharide transporter gene
in Arabidopsis (Yamada et al., 2010). In rice, expression of
OsMST7 and OsMST8 were affected by cold treatment
during male gametophyte development (Mamun et al.,
2006; Oliver et al., 2007). Therefore, some MSTs may play
a pivotal role during stress responses.
The Golgi-localized hexose transporter SGB1 (Wang
et al., 2006) belongs to the pGlcT subfamily in Arabidopsis.
The gain-of-function mutant sgb1-1Dis a suppressor of
agb1, a null mutant of the b subunit of G protein. sgb1 and
agb1 exhibited similar phenotypes with shorter hypocotyls
and open hooks. SGB1 may transport glucose for cell wall
biosynthesis in Arabidopsis during cell division (Wang et al.,
2006). To date, SGB1 is the only known monosaccharide
transporter localized in the Golgi apparatus in plants.
OsGMST1, encoded by locus Os02g17500 with unknown
function, showed the highest sequence identity (61%) with
SGB1 by a BLASTP search. The function of OsGMST1,
a Golgi-localized monosaccharide transporter in rice, is
Materials and methods
Rice plants (Oryza sativa L. ssp. Japonica cv. Zhonghua 10)
were grown under field conditions or in a greenhouse with a 16/8 h,
30/28 ?C, light/dark photoperiod.
Molecular cloning, vector construction, and the generation of
The cDNA fragment of OsGMST1 was cloned by reverse
transcription PCR (RT-PCR) using specific primers (forward
primer 5#-TCGCCCTTACTGGTCACA-3#), and then it was
ligated into the binary vector pUN1301 digested with BamHI and
KpnI in an antisense orientation to enable expression to be driven
by the maize (Zea mays) ubiquitin promoter. This constructed
vector (Ubi::antiOsGMST1) was genetically transformed into rice
mediated by Agrobacterium tumefaciens EHA105 as previously
described by Ge et al. (2004). The T2generations of transgenic
plants were used for further functional characterization.
Heterologous expression of OsGMST1 in yeast
The complete open reading frame of OsGMST1 was ligated with
vector pEX-Tag (Meyer et al., 2000) digested by EcoRI in both the
sense and antisense orientations, and then the recombinant vectors
or empty vector were transformed into the yeast hexose trans-
porter-deficient mutant EBY. VW4000 (Wieczorke et al., 1999), to
create strains S1, S2, AS1, and Vector. Transformed cells were
pregrown in YEPM medium (1% yeast extract, 2% peptone, 2%
maltose) to OD600of 1.0. Serial dilutions of cell suspensions were
streaked on uracil-deficient solid medium containing 0.67% yeast
nitrogen base with ammonium sulphate, supplemented with
leucine, tryptophan, histidine, and various sugars, such as maltose,
glucose, fructose, mannose, galactose, xylose, and ribose, and were
incubated at 30 ?C for 2 or 3 d.
Subcellular localization of the OsGMST1-GFP fusion protein
A transient expression vector 35S::OsGMST1-GFP was con-
structed, in which OsGMST1 was fused with the 5#-end of GFP
driven by the 35S promoter. Protoplast transformation was as
previously described (Sheen, 2001; Bart et al., 2006). Briefly, leaf
sheaths from 10-d-old etiolated rice seedlings were cut and digested
by cellulase RS and macerozyme R-10 (Yakult, Japan), and the
released protoplast cells were transformed by polyethylene glycol
(PEG) mediation and then were incubated in darkness overnight.
To observe the auto fluorescence of chloroplasts, transformed
protoplast cells were incubated under dim light. Mitochondria
were stained with 20 nM fluorescent dye MitoTracker Red
CMXRos (Molecular Probes) for 2 min, and then visualized at an
excitation wavelength of 543 nm and an emission wavelength of
599 nm. Chlorophyll autofluorescence was used as the chloroplast
marker. Then, cells were observed under confocal microscope
(LSM 510 Meta, Carl Zeiss).
Quantitation of mRNA steady-state levels
Total RNA was prepared from seedlings using Trizol reagent
(Invitrogen, USA) and first strand cDNA was synthesized with
M-MLV reverse transcriptase (Promega, USA) according to the
manufacturer’s instructions, and PCR was used to clone genes by
specific primers. Real-time PCR was performed by using SYBR
Green Master Mix PCR reagent (Toyobo, Japan) and the
MX3000P detector (Stratagene, USA). In the analysis of relative
transcript level, gene-specific primers of OsGMST1 were used with
the rice a-TUBULIN gene (Accession EF575922) as the internal
Tissue expression pattern analysis
Total RNA was extracted from different tissues, and the
OsGMST1 transcript level was analysed by real-time PCR.
OsGMST1::GUS transgenic plants were generated, and GUS
staining of various tissues was performed by the histochemical
method as previously described by Jefferson et al. (1987). Different
organs of OsGMST1::GUS transgenic seedlings were incubated in
4596 | Cao et al.
5-bromo-4-chloro-3-indolyl-b-glucuronic acid buffer at 37 ?C for
12 h. After staining, the tissues were destained in 70% ethanol
several times until chlorophyll was removed and images were
visualized on an Olympus SZX9 microscope.
Genomic DNA was extracted from 14-d-old seedlings of wild-type
and transgenic rice. 20 lg of DNA was digested with EcoRI or
HindIII, electrophoresed on a 0.7% agarose gel, and transferred to
a nylon membrane (Hybond N+; Amersham Pharmacia Biotech,
Buckinghamshire, UK) under alkaline conditions. [a-32P]dCTP-
labelled GUS amplified from pUN1301 was used as a probe for
hybridization. Membrane was stored in –70 ?C for 2–4 d, and then
exposed to X-ray film (Eastern Kodak, Rochester NY).
Expression pattern analysis with stress treatment
To detect the transcript level of OsGMST1, seedlings of wild-type
rice plants were used. Two-week-old seedlings grown in Kimura
B nutrient solution (Kato-Noguchi and Ino, 2005; Chen et al.,
2006) were treated with 200 mM NaCl or 18% PEG4000, followed
by sampling at 0, 3, 5, 7, 12, 24, and 36 h. For the cold stress
treatment, soil-grown seedlings were transferred to a chamber at
4 ?C with a 12/12 h light/dark photoperiod, and sampled at the
same time as the two stresses above.
Stress tolerance assays
Seeds of wild-type or transgenic rice plants were soak in water at
28 ?C for 2 d, and germinated seeds were sown in the 96-well plates
from which the bottoms were removed. The plate was floated in
Kimura B nutrient solution, and seedlings were incubated in
a growth chamber with a 30/28 ?C, 16/8 h light/dark photoperiod.
For the NaCl treatment, 14-d-old or 21-d-old seedlings were
treated in culture solution containing 150 mM or 200 mM NaCl
for 9–11 d, and the treated seedlings were recovered for 7 or 8 d
followed by survival rate analysis. For PEG treatment, 3-week-old
seedlings were transferred to culture solutions containing 20%
(w/v) PEG4000. For cold treatment, 3-week-old seedlings were
incubated at 4 ?C in a growth chamber for 4 d, and then removed
to normal growth conditions for 7 d. For the seed germination
assay, T2homozygous transgenic seeds were germinated on 1/2 MS
medium supplemented with 0, 150, and 200 mM NaCl for 4–6 d,
and were scored for germination based on whether the shoot
length exceeded half that of the seed length.
Measurement of glucose/fructose/sucrose levels in leaves
Two-week-old seedlings of wild-type and transgenic rice were treated
with 0 mM and 150 mM NaCl for 24 h, and then leaves were cut and
weighed for fresh weight determination. Sugar extraction from the
leaves was performed as described with modifications (Strand et al.,
1999). Briefly, leaves were ground to powder in liquid nitrogen, and
then extracted with 80% ethanol at 80 ?C for 40 min, and this
extraction was repeated once. Extracts were evaporated to dryness
then dissolved in sterile water. Spectroscopic quantification and
calculation of the sugar contents were performed according to the
protocol of D-glucose/D-fructose/sucrose kits (Biosentec, France).
OsGMST1 encodes a putative monosaccharide
The OsGMST1 full-length cDNA is 2196 bp, with an
opening reading frame of 1446 bp, encoding a protein of
481 amino acids (Fig. 1A) with a predicted molecular mass
of 52 kDa and an isoelectric point of 8.45. It is a putative
monosaccharide transporter, and has the sugar transport
proteins signature 1 (Fig. 1A, underlined sequence) by the
ExPASy (Expert Protein Analysis System) proteomics
server (http://ca.expasy.org/prosite/). The plant membrane
protein database Aramemnon (http://aramemnon.botanik.
uni-koeln.de/) predicted 10–12 transmembrane domains,
form¼toppred) predicted 12 hydrophobic transmembrane
domains with a hydrophobicity value above 1.0 (Fig. 1A,
double-arrow lines) arranged in two sets separated by
a central hydrophilic loop (Fig. 1C). The transmembrane
topology structure is consistent with the monosaccharide
transporters in plants (Bush, 1999), yeast (Bisson et al.,
1993), and mammals (Bell et al., 1993).
Phylogenetic analysis (Fig. 1B) shows that the putative
Arabidopsis paralogues to OsGMST1 are encoded by
At1g67300, SGB1, At1g05030, and AtpGlcT and the pro-
teins have 67%, 65%, 44%, and 43% identity to OsGMST1,
respectively. These four MST proteins are in the pGlcT
subfamily (Buttner, 2007). SGB1 (suppressor of G protein
beta1) was identified as a Golgi-localized hexose transporter
in this subfamily (Wang et al., 2006). Wang and coworkers
showed that SGB1 genetically complemented the phenotype
of the agb1 mutant lacking the b subunit of G protein and
that the transcript-null mutant, sgb1-2, mimicked the agb1
2 d etiolated phenotypes with shorter hypocotyls and open
hooks (Wang et al., 2006). It has been proposed that, in rice
(OspGlcT) belong to the pGlcT subfamily (Johnson and
Thomas, 2007) with sequence identity approximately 36%
(see Supplementary Fig. S1 at JXB online). Tobacco pGlcT
and maize pGlcT belong to the pGlcT subfamily and are
also closely related to OsGMST1 (36.33% and 36.16%
identity, respectively). Yeast RGT2 and human SCL2A are
similar to OsGMST1.
Expression of OsGMST1 in sgb1-2 mutants rescued the
short hypocotyl phenotype to the wild type (P¼0.634)
(Fig. 1D, E), indicating that OsGMST1 is paralogous to
SGB1. As was the case for SGB1 (Wang et al., 2006),
OsGMST1 did not rescue the growth phenotype of a yeast
strain EBY.VW4000 (Wieczorke et al., 1999) which lacked
22 monosaccharide transporter genes, HXT1-17, GAL2,
STL1, AGT1, MPH2, and MPH3 (see Supplementary
Fig. S2 at JXB online).
OsGMST1 is localized on the Golgi apparatus
To identify the subcellular localization of OsGMST1, a
transient expression vector harbouring OsGMST1-GFP
fusion protein driven by the 35S promoter (35S::OsGMST1-
GFP) was constructed. Protoplast cells were prepared from
leaf sheaths of 7–10 d dark grown rice seedlings, and then
transiently transformed with recombinant or empty vector as
a control. Fluorescence images were observed under the
confocal microscope. OsGMST1-GFP fluorescence was dis-
tributed in cells in a punctate pattern (Fig. 2A, B), but GFP
alone was distributed throughout the cytoplasm (Fig. 2C, D).
OsGMST1 may play a role in salt stress tolerance in rice | 4597
To test the hypothesis that OsGMST1 is localized to the
Golgi as is SGB1 (Wang et al., 2006), OsGMST1-GFP was
co-expressed with the Golgi marker sialyltransferase (ST)
tagged red fluorescence protein (RFP). Most of the red and
green fluorescence merged (Fig. 2E, F, G, H), indicating that
OsGMST1 was in the Golgi body. By contrast, OsGMST1-
GFP fluorescence did not co-localize with the mitochondrion
fluorescence dye MitoTracker Red (Fig. 2I, J, K, L) or with
chlorophyll autofluorescence (Fig. 2M, N, O, P).
OsGMST1 is ubiquitiously expressed
Transgenic rice plants expressing b-glucoronidase gene
driven by the OsGMST1 promoter showed that OsGMST1
was expressed in all developmental stages from germination
to flowering (Fig. 3A). Furthermore, quantitative PCR
confirmed high levels of OsGMST1 mRNA in all the organs
tested, such as the shoot, root, culm, panicle, leaf, and leaf
sheath before and after heading (Fig. 3B), suggesting that
OsGMST1 is ubiquitously expressed.
OsGMST1 expression is salt responsive
The OsGMST1 promoter sequence (1500 bp upstream of
the start codon ATG) contains stress-responsive related
cis-elements, such as ABRE, a MYB recognition site,
a MYC recognition site, and a GCC box (see Supplemen-
tary Fig. S3 at JXB online) based on predictions using the
PLACE database (http://www.dna.affrc.go.jp/PLACE). As
shown in Fig. 3C, the transcript level of OsGMST1 in
seedlings was induced by NaCl treatment but unaffected by
low temperature (4 ?C) and PEG4000. Salt treatment
conferred a sustained increase in the steady-state level of
the OsGMST1 mRNA (Fig. 3C).
Reduced expression of OsGMST1 confers
hypersensitivity to NaCl
Reduced expression of OsGMST1 by antisense technology
was performed (see Supplementary Fig. S4A at JXB online;
see the Materials and methods). Three independent trans-
genic rice lines, AS-L4, L12, L18 (see Supplementary
Fig. S4B at JXB online) were confirmed by quantitative
PCR to have reduced expression (see Supplementary
Fig. S4C at JXB online). In AS-L12 and AS-L18, the
expression of OspGlcT (Os01g04190) and Os09g23110,
which have the highest nucleic acid sequence identity
(46.8% and 39.8%) with OsGMST1 were not affected
(see Supplementary Fig. S4D at JXB online). In addition,
Fig. 1. Sequence characterization of OsGMST1 and OsGMST1 complements the short hypocotyl phenotype of sgb1-2. (A) Deduced
amino acid sequence of OsGMST1; numbers on the right refer to the positions of amino acid residues. Sugar transporter signature 1
predicted for OsGMST1 is indicated by the red underline. Double-headed arrows show the transmembrane regions with a high
hydrophobicity value. (B) Phylogenic analysis of OsGMST1 homologues from rice and other organisms by MEGA 4.0. The predicted
protein sequence is compared with OsMST1-8, Os09g23110, OspGlcT, and OsTMT1-4 from Oryza sativa, AtSGB1, At1g67300,
At1g05030, and AtpGlcT from Arabidopsis thaliana, ScRGT2 from Saccharomyces cerevisiae, HsSLC2A3 from Homo sapiens, NtpGlcT
from Nicotiana tabacum, and ZmpGlcT from Zea mays. (C) OsGMST1 is a membrane protein predicted by the TopPred program,
showing 12 putative transmembrane helix domains. The red line and the green line indicate upper and lower cutoff of hydrophobicity,
respectively. (D) Etiolated seedlings of Col-0 (WT), sgb1-2, and a transgenic line over-expressing OsGMST1 in the sgb1-2 mutant
background. Bar¼2 mm. (E) The hypocotyl length of etiolated seedlings. A total of 36 hypocotyls were measured for each line (wild-type,
mutant, and transgenic). Error bars represent SE. ** indicates P <0.01 by Student’s t test. (This figure is available in colour at JXB online.)
4598 | Cao et al.
the expression of other genes might not be affected in the
OsGMST1 with other genes is lower than 30%, and there is
no gene that has high similarity with OsGMST1 in a short
sequence stretch by BLAST search in rice. The development
and morphology of the T2 progeny was no different from
the wild type (WT) under normal growing conditions (see
Supplementary Fig. S4E at JXB online).
There were no differences in the seed germination rate
between WT and the transgenic lines without NaCl stress
(Fig. 4A). Treatment with 150 mM and 200 mM NaCl,
however, reduced the germination rate of AS-L12 and AS-
L18 (Fig. 4A, B). At 200 mM NaCl, the germination rate of
the wild type was 90%, while the germination rates of the
AS-L12 and AS-L18 lines were reduced to 40% and 50%,
respectively (P <0.01) (Fig. 4B), and this difference was
maintained over several days (P <0.01) (Fig. 4C).
Two-week-old rice seedlings of WT, AS-L12, and AS-L18
were treated with 200 mM NaCl as a supplement in Kimura
B nutrient solution (see the Materials and methods) for 9 d,
and then recovered in Kimura B nutrient solution alone for
8 d. Reduced expression of OsGMST1 also conferred
(Fig. 4D). The survival rate of AS-L12 (40% and 20% in
150 mM NaCl and 200 mM NaCl, respectively) and AS-
L18 (50% and 30%) were lower than the WT (70% and 50%)
Fig. 2. Subcellular localization of OsGMST1-GFP transiently expressed in rice protoplasts. (A, B) A rice protoplast cell expressing
OsGMST1-GFP (A) and its DIC image (B), showing a punctuate expression pattern. Bars¼10 lm. (C, D) A rice protoplast cell expressing
GFP (C) as control and its DIC image (D), showing its distribution in nucleus, membrane, and cytoplasm. Bars¼10 lm. (E–H) A rice
protoplast cell expressing OsGMST1-GFP (E), Golgi localized ST-RFP (F) ,a merged image (G), and its DIC image (H). Bars¼10 lm. (I–L)
A rice protoplast expressing OsGMST1-GFP (I), stained by mitochondria dye MitoTracker Red (J), a merged image (K), and its DIC image
(L). Bars¼10 lm. (M–P) A rice protoplast cell expressing OsGMST1-GFP (M), the chlorophyll autofluorescence (N), a merged image (O),
and its DIC image (P). Bars¼10 lm. This figure is available in colour at JXB online.)
OsGMST1 may play a role in salt stress tolerance in rice | 4599
with treatment of 150 mM and 200 mM NaCl (P <0.01)
(Fig. 4E). By contrast, the tolerance of OsGMST1 antisense
plants to PEG and cold stress was the same as that of WT
plants (Fig. 4F, G). Just as for SGB1, increased expression
of OsGMST1 had no effect (see Supplementary Fig. S5 at
JXB online). The observation that OsGMST1 overexpres-
sion in rice seedlings did not confer increased salt tolerance
suggests that OsGMST1 is not rate limiting.
Knockdown expression of OsGMST1 reduced glucose
and fructose content in leaves
Because OsGMST1 is a putative monosaccharide trans-
porter and its transcript is up-regulated by salt stress, the
leaf sugar contents were determined in normal and salt
stress conditions. As shown in Fig. 5A, under normal
growth conditions, the glucose content was 4.0 mg g?1fresh
weight in the WT, and 3.0 and 3.3 mg g?1fresh weight in
the antisense transgenic plants, representing 25% and 20%
reduced glucose content in knockdown plants (P <0.01).
Fructose content was reduced by 25% and 12% of the WT
(P <0.01) (Fig. 5B), whereas sucrose levels in transgenic rice
plants were 1.5–2-fold of that in the WT, 10.0–14.0 versus
7.0 mg g?1fresh weight, respectively (P <0.01) (Fig. 5C).
The data indicated that sugar levels were interrupted in
OsGMST1 knockdown rice, although they showed no
visibly different phenotype compared with WT plants.
To analyse the differences in sugar accumulation in plants
under NaCl stress further, the sugar levels were quantified
after the wild-type and OsGMST1 knockdown rice were
treated with 150 mM NaCl for 24 h. In leaves of WT plants,
salt stress caused glucose and fructose contents to increase
50% and 60% compared with that of the untreated control
leaves, respectively (P < 0.01) (Fig. 5A, B), and sucrose rose
to 3-fold that of the untreated leaves (P <0.01) (Fig. 5C). In
the antisense transgenic lines, glucose and fructose levels
were not statistically increased by salt stress (Fig. 5A, B). In
addition, sucrose contents in antisense plants accumulated
only 0.5–1.0-fold higher than the untreated leaves in
contrast to the 2-fold accumulation of sucrose in WT plants
(P <0.01) (Fig. 5C). These results indicate that accumula-
tion of sugars is, in part, by OsGMST1.
OsGMST1 is a Golgi-localized monosaccharide
transporter in rice
Monosaccharide transporters play a pivotal role in the
translocation and distribution of monosaccharides through-
out the plant. In rice, 12 MSTs, OsMST1–8 and OsTMT1–4
have been reported. Nevertheless, functional evidence for
their roles in rice is not presented. In this study, we
identified OsGMST1 as a Golgi-localized monosaccharide
transporter in rice. Although the transport activity of
OsGMST1 has not been characterized, we speculate that it
is a sugar transporter based on its sequence characteristics
(Fig. 1) and the effect on the sugar content when this gene
expression is reduced in rice (Fig. 5). The orthologue of
OsGMST1 in Arabidopsis is SGB1 (Wang et al., 2006),
which is a Golgi-localized hexose transporter. Furthermore,
expression of OsGMST1 in the sgb1-2 mutant can rescue
the hypocotyl phenotype (Fig. 1). Therefore, we conclude
Fig. 3. Expression pattern analysis of OsGMST1. (A) GUS histochemical staining in OsGMST1-promoter::GUS transgenic rice plants,
showing a germinating seedling (a), young root (b), young leaf (c), mature root (d), mature leaf (e), leaf sheath (f), node (g), internode
(h), panicle (i), pistil (j), and pollen gains (k). (a–f, h, i) Bars¼5 mm; (g, j) 1 mm; (k) 0.1 mm. (B) Quantitative real-time PCR analysis of relative
OsGMST1 transcript levels in shoots, roots, leaves, leaf sheaths, culms, and panicles, and data are the percentage of a-TUBULIN
expression. The results are means 6SD of triplicate assays. (C) Real time PCR analysis of OsGMST1 transcript level under salt stress, PEG,
and cold treatment with different time-courses. Error bars indicates SD of three replicates. This figure is available in colour at JXB online.)
4600 | Cao et al.
that the biochemical function of OsGMST1 is the same or
similar to SGB1 function. Some monosaccharide trans-
porter genes are only expressed in source tissues (Buttner
et al., 2000), and in sink tissues (Schneidereit et al., 2003,
2005; Scholz-Starke et al., 2003; Mamun et al., 2006; Oliver
et al., 2007), or in both (Wormit et al., 2006; Wang et al.,
2007). OsGMST1 is expressed ubiquitously (Fig. 3A, B),
suggesting that it may be involved in sugar translocation in
both source and sink tissues of rice.
OsGMST1 may play a role in salt-stress tolerance
Under abiotic stress conditions, soluble sugars derived from
starch breakdown accumulate in plants to increase stress
tolerance (Yano et al., 2005; Lee et al., 2009; Yamada et al.,
2010). The Arabidopsis sex1 mutant, which was unable to
accumulate sugars when exposed to freezing stress, displayed
impaired freezing tolerance (Yano et al., 2005). OsGMST1
knockdown rice seedlings are hypersensitive to NaCl stress
compared with the WT (Fig. 4), as the NaCl-induced
accumulation of glucose and fructose is impaired in
knockdown plants, and the accumulation amount of
sucrose is also different from the WT (Fig. 5). Sugars
generate adenosine 5’-triphosphate (ATP) and other impor-
tant metabolites needed for biosynthesis and growth during
O2deficiency stress of rice (Lee et al., 2009). Sugars can also
function as signals that affect gene expression under stress
conditions (Hanson and Smeekens, 2009; Hey et al., 2010),
so there is cross-talk between sugar signalling and stress.
The interaction of stress and sugar signalling is essential for
plants to tolerate stress. Sucrose non-fermenting-1-related
protein kinases (SnRKs) play a central role in the in-
teraction of the two signalling pathways, which serve as
metabolic sensors to adjust energy homeostasis under stress
Fig. 4. Salt tolerance test of OsGMST1 antisense transgenic rice plants. (A) Seed germination of WT and OsGMST1 knockdown
transgenic rice plants on MS medium containing 0 mM and 200 mM NaCl after 6 d. (B) Germination rate of WT and OsGMST1
knockdown transgenic rice seeds with 0, 150, and 200 mM NaCl treatment for 6 d. Data are means 6SD of three repeats, 20 seeds in
each repeat. * and ** indicate P <0.05 and P <0.01, respectively by Student’s t test. (C) Seed germination rate of WT and OsGMST1
knockdown transgenic rice seeds with 200 mM NaCl treatment in 0–6 d. Data are means 6SD of three repeats, 20 seeds in each
repeat. ** indicate P <0.01 by Student’s t test. (D) Growth of WT and transgenic seedlings before (left) and after (right) NaCl treatment.
2-week-old seedlings were treated with 200 mM NaCl for 9 d, and then recovered for 8 d. Bars¼5 cm. (E) Survival rate of WT and
transgenic seedlings after being treated with 150 mM and 200 mM NaCl. 15 seedlings at 2 weeks old were used in each repeat. Error
bars are SE of three replicates. ** indicates P <0.01 by Student’s t test. (F) PEG stress tolerance test of WT and OsGMST1 knockdown
seedlings. Three-week-old seedlings were treated with 20% PEG for 15 d, and then recovered for 8 d. Bars¼5 cm. (G) Cold
stress tolerance test of WT and knockdown seedlings. 3-week-old seedlings were treated with 4 C for 4 d, and then recovered for 7 d.
Bars¼5 cm. This figure is available in colour at JXB online.)
OsGMST1 may play a role in salt stress tolerance in rice | 4601
(Halford et al., 2003; Halford and Hey, 2009). In rice, the
SnRK1-dependent sugar-sensing cascade regulates sugar
and energy production in order to enable rice growth under
flooding stress (Lee et al., 2009). In OsGMST1 knockdown
rice plants, the accumulation of sugars are affected under
NaCl stress, suggesting that sugar signal transduction may
be interrupted. Consequently, the expression of genes
regulated by sugars may not be activated (or depressed) to
provide energy for rice.
Expression of tonoplast localized TMT1 and TMT2 is
induced by cold treatment, and glucose uptake into isolated
leaf mesophyll vacuoles of WT plants is promoted by cold
stress. In addition, tmt knockout lines accumulated less
glucose and fructose compared with the WT plants (Wormit
et al., 2006), analogous to results found with reduced
expression of OsGMST1.
OsGMST1 resides in the Golgi apparatus (Fig. 2) and
contributes to monosaccharide transport, and NaCl stress
up-regulates the expression of OsGMST1 transcriptionally
(Fig. 3C). One possible explanation is that NaCl stress
OsGMST1 and, consequently, the activated OsGMST1
upon salt stress import sugars into the Golgi or export them
out of the organelle to keep cytosolic sugar at homeostasis.
In conclusion, it is hypothesized that sugar homeostasis is
affected in OsGMST1 knockdown transgenic rice, which
putatively results in altered sugar contents, so the seedlings
show a NaCl hypersensitive phenotype.
In summary, OsGMST1 is a novel Golgi-localized mono-
saccharide transporter that may play a role in salt stress
tolerance in rice.
Supplementary data can be found at JXB online.
Supplementary Fig. S1. Alignment of OsGMST1 with
other members of the rice pGlcT (plastid glucose trans-
Supplementary Fig. S2. Growth of hexose transporter-
deficient yeast cells transformed with sense and antisense
OsGMST1 on plates supplemented with sugars.
Supplementary Fig. S3. Distribution of stress related
cis-elements in the OsGMST1 promoter region.
Supplementary Fig. S4. Molecular identification and
phenotype observation of OsGMST1 knockdown transgenic
Supplementary Fig. S5. Molecular identification and
phenotype observation of OsGMST1 overexpressed trans-
genic rice plants.
The authors thank Professor Norbert Sauer of Germany for
providing the vector pEX-Tag and Professor Eckhard Boles
of Germany for providing the yeast strain EBY.VW4000.
This work was supported by the Foundation for Innovative
Research Groups of the National Natural Science Founda-
tion of China (30821007). Work in the Jones laboratory was
supported by grantsto
(R01GM065989), DOE (DE-FG02-05er15671), and NSF
(MCB-0723515 and MCB-0718202).
Bart R, Chern M, Park CJ, Bartley L, Ronald PC. 2006. A novel
system for gene silencing using siRNAs in rice leaf and stem-derived
protoplasts. Plant Methods 2, 13–21.
Bell GI, Burant CF, Takeda J, Gould GW. 1993. Structure and
function of mammalian facilitative sugar transporters. Journal of
Biological Chemistry 268, 19161–19164.
Bisson LF, Coons DM, Kruckeberg AL, Lewis DA. 1993. Yeast
sugar transporters. Critical Reviews in Biochemistry and Molecular
Biology 28, 259–308.
Bush DR. 1999. Sugar transporters in plant biology. Current Opinion
in Plant Biology 2, 187–191.
Fig. 5. Effect of NaCl treatment on sugar content in rice leaves. Glucose (A), fructose (B), and sucrose level (C) in the leaves of WT and
OsGMST1 knockdown transgenic rice plants under 0 mM and 150 mM NaCl treatment. Two-week-old seedlings were treated with
0 mM and 150 mM NaCl for 24 h, and then the sugar levels of the leaves (mg g?1FW) were measured as described in the Materials and
methods. Twelve plants were used in each experiment. Data are the means of three independent repeats 6SD. The confidence level for
significant differences in sugar content between 0 mM and 150 mM NaCl is indicated by **a(P <0.01); and between WT and knockdown
plants under non-salt stress treatment (0 mM NaCl) confidence is indicated by **b(P <0.01).
4602 | Cao et al.
Buttner M. 2007. The monosaccharide transporter(-like) gene family
in Arabidopsis. FEBS Letters 581, 2318–2324.
Buttner M, Truernit E, Baier K, Scholz-Starke J, Sontheim M,
Lauterbach C, Huss VAR, Sauer N. 2000. AtSTP3, a green
leaf-specific, low affinity monosaccharide-H+ symporter of Arabidopsis
thaliana. Plant and Cell Physiology 23, 175–184.
Chen RF, Shen RF, Gu P, Dong XY, Du CW, Ma JF. 2006.
Response of rice (Oryza sativa) with root surface iron plaque under
aluminium stress. Annals of Botany 98, 389–395.
Cho JI, Burla B, Lee DW, et al. 2010. Expression analysis and
functional characterization of the monosaccharide transporters,
OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa. New
Phytologist 186, 657–668.
Ge L, Chen H, Jiang JF, Zhao Y, Xu ML, Xu YY, Tan KH, Xu ZH,
Chong K. 2004. Overexpression of OsRAA1 causes pleiotropic
phenotypes in transgenic rice plants, including altered leaf, flower, and
root development and root response to gravity. Plant Physiology 135,
Gibson SI. 2005. Control of plant development and gene
expression by sugar signaling. Current Opinion in Plant Biology 8,
Halford NG, Hey S, Jhurreea D, Laurie S, McKibbin RS, Paul M,
Zhang Y. 2003. Metabolic signalling and carbon partitioning: role of
Snf1-related (SnRK1) protein kinase. Journal of Experimental Botany
Halford NG, Hey SJ. 2009. Snf1-related protein kinases (SnRKs) act
within an intricate network that links metabolic and stress signalling in
plants. Biochemical Journal 419, 247–259.
Hanson J, Smeekens S. 2009. Sugar perception and signaling: an
update. Current Opinion in Plant Biology 12, 562–567.
Hey SJ, Byrne E, Halford NG. 2010. The interface between
metabolic and stress signalling. Annals of Botany 105, 197–203.
Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions:
beta-glucuronidase as a sensitive and versatile gene fusion marker in
higher plants. The EMBO Journal 6, 3901–3907.
Johnson DA, Hill JP, Thomas MA. 2006. The monosaccharide
transporter gene family in land plants is ancient and shows differential
subfamily expression and expansion across lineages. BMC
Evolutionary Biology 6, 64–83.
Johnson DA, Thomas MA. 2007. The monosaccharide transporter
gene family in Arabidopsis and rice: a history of duplications, adaptive
evolution, and functional divergence. Molecular Biology and Evolution
Kato-Noguchi H, Ino T. 2005. Possible involvement of momilactone
B in rice allelopathy. Journal of Plant Physiology 162, 718–721.
Kiyosue T, Abe H, Yamaguchi-Shinozaki K, Shinozaki K. 1998.
ERD6, a cDNA clone for an early dehydration-induced gene of
Arabidopsis, encodes a putative sugar transporter. Biochimica et
Biophysica Acta 1370, 187–191.
Klepek YS, Geiger D, Stadler R, Klebl F, Landouar-Arsivaud L,
Lemoine R, Hedrich R, Sauer N. 2005. Arabidopsis POLYOL
TRANSPORTER5, a new member of the monosaccharide transporter-
like superfamily, mediates H+-symport of numerous substrates,
including myo-inositol, glycerol, and ribose. The Plant Cell 17,
Lalonde S, Boles E, Hellmann H, Barker L, Patrick JW,
Frommer WB, Ward JM. 1999. The dual function of sugar carriers.
Transport and sugar sensing. The Plant Cell 11, 707–726.
Lee KW, Chen PW, Lu CA, Chen S, Ho TH, Yu SM. 2009.
Coordinated responses to oxygen and sugar deficiency allow rice
seedlings to tolerate flooding. Science Signaling 2, ra61.
Mamun EA, Alfred S, Cantrill LC, Overall RL, Sutton BG. 2006.
Effects of chilling on male gametophyte development in rice. Cell
Biology International 30, 583–591.
Meyer S, Melzer M, Truernit E, Hummer C, Besenbeck R,
Stadler R, Sauer N. 2000. AtSUC3, a gene encoding a new
Arabidopsis sucrose transporter, is expressed in cells adjacent to the
vascular tissue and in a carpel cell layer. The Plant Journal 24,
Moore B, Sheen J. 1999. Plant sugar sensing and signaling:
a complex reality. Trends in Plant Science 4, 250–250.
Ngampanya B, Sobolewska A, Takeda T, Toyofuku K,
Narangajavana J, Ikeda A, Yamaguchi J. 2003. Characterization of
rice functional monosaccharide transporter, OsMST5. Bioscience,
Biotechnology, and Biochemistry 67, 556–562.
Niittyla T, Messerli G, Trevisan M, Chen J, Smith AM,
Zeeman SC. 2004. A previously unknown maltose transporter
essential for starch degradation in leaves. Science 303, 87–89.
Oliver SN, Dennis ES, Dolferus R. 2007. ABA regulates apoplastic
sugar transport and is a potential signal for cold-induced pollen sterility
in rice. Plant and Cell Physiology 48, 1319–1330.
Rolland F, Baena-Gonzalez E, Sheen J. 2006. Sugar sensing and
signaling in plants: conserved and novel mechanisms. Annual Review
of Plant Biology 57, 675–709.
Rolland F, Moore B, Sheen J. 2002. Sugar sensing and signaling in
plants. The Plant Cell 14, SupplementS185–S205.
Sauer N. 2007. Molecular physiology of higher plant sucrose
transporters. FEBS Letters 581, 2309–2317.
Schneidereit A, Scholz-Starke J, Buttner M. 2003. Functional
characterization and expression analyses of the glucose-specific
AtSTP9 monosaccharide transporter in pollen of Arabidopsis. Plant
Physiology 133, 182–190.
Schneidereit A, Scholz-Starke J, Sauer N, Buttner M. 2005.
AtSTP11, a pollen tube-specific monosaccharide transporter in
Arabidopsis. Planta 221, 48–55.
Scholz-Starke J, Buttner M, Sauer N. 2003. AtSTP6, a new
pollen-specific H+-monosaccharide symporter from Arabidopsis. Plant
Physiology 131, 70–77.
Sheen J. 2001. Signal transduction in maize and Arabidopsis
mesophyll protoplasts. Plant Physiology 127, 1466–1475.
Strand A, Hurry V, Henkes S, Huner N, Gustafsson P,
Gardestrom P, Stitt M. 1999. Acclimation of Arabidopsis leaves
developing at low temperatures. Increasing cytoplasmic volume
accompanies increased activities of enzymes in the Calvin cycle
and in the sucrose-biosynthesis pathway. Plant Physiology 119,
OsGMST1 may play a role in salt stress tolerance in rice | 4603
Szarka A, Horemans N, Banhegyi G, Asard H. 2004. Facilitated
glucose and dehydroascorbate transport in plant mitochondria.
Archives of Biochemistry and Biophysics 428, 73–80.
Toyofuku K, Kasahara M, Yamaguchi J. 2000. Characterization
and expression of monosaccharide transporters (OsMSTs) in rice.
Plant and Cell Physiology 41, 940–947.
Turgeon R, Medville R. 2004. Phloem loading. A reevaluation of the
relationship between plasmodesmatal frequencies and loading
strategies. Plant Physiology 136, 3795–3803.
Wang HX, Weerasinghe RR, Perdue TD, Cakmakci NG,
Taylor JP, Marzluff WF, Jones AM. 2006. A Golgi-localized hexose
transporter is involved in heterotrimeric g protein-mediated early
development in Arabidopsis. Molecular Biology of the Cell 17,
Wang Y, Xiao Y, Zhang Y, Chai C, Wei G, Wei X, Xu H, Wang M,
Ouwerkerk PB, Zhu Z. 2008. Molecular cloning, functional
characterization and expression analysis of a novel monosaccharide
transporter gene OsMST6 from rice (Oryza sativa L.). Planta 228,
Wang Y, Xu H, Wei X, et al. 2007. Molecular cloning and expression
analysis of a monosaccharide transporter gene OsMST4 from rice
(Oryza sativa L.). Plant Molecular Biology 65, 439–451.
Weber A, Servaites JC, Geiger DR, Kofler H, Hille D, Groner F,
Hebbeker U, Flugge UI. 2000. Identification, purification, and
molecular cloning of a putative plastidic glucose translocator. The
Plant Cell 12, 787–802.
Weschke W, Panitz R, Gubatz S, Wang Q, Radchuk R, Weber H,
Wobus U. 2003. The role of invertases and hexose transporters in
controlling sugar ratios in maternal and filial tissues of barley caryopses
during early development. The Plant Journal 33, 395–411.
Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP,
Boles E. 1999. Concurrent knock-out of at least 20 transporter genes
is required to block uptake of hexoses in Saccharomyces cerevisiae.
FEBS Letters 464, 123–128.
Williams LE, Lemoine R, Sauer N. 2000. Sugar transporters in
higher plants: a diversity of roles and complex regulation. Trends in
Plant Science 5, 283–290.
Wormit A, Trentmann O, Feifer I, Lohr C, Tjaden J, Meyer S,
Schmidt U, Martinoia E, Neuhaus HE. 2006. Molecular
identification and physiological characterization of a novel
monosaccharide transporter from Arabidopsis involved in vacuolar
sugar transport. The Plant Cell 18, 3476–3490.
Yamada K, Osakabe Y, Mizoi J, Nakashima K, Fujita Y,
Shinozaki K, Yamaguchi-Shinozaki K. 2010. Functional analysis of
an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion
transporter for monosaccharides. Journal of Biological Chemistry 285,
Yano R, Nakamura M, Yoneyama T, Nishida I. 2005.
Starch-related alpha-glucan/water dikinase is involved in the
cold-induced development of freezing tolerance in Arabidopsis.
Plant Physiology 138, 837–846.
4604 | Cao et al.