全 文 ：Received 26 Apr. 2004 Accepted 18 Jun. 2004
Supported by the National Natural Science Foundation of China (G39980022) and the State Key Basic Research and Development Plan of
China (G1999011700).
* These authors contributed equally to this work.
** Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1212-1219
Molecular Cloning and Identification of a Heat Shock Cognate
Protein 70 Gene, Thhsc70, in Thellungiella halophila
ZHANG Xia1, 3*, GUO Shan-Li1, 2*, YIN Hai-Bo1, XIONG Dong-Jin3, ZHANG Hui1, ZHAO Yan-Xiu1**
(1. Key Laboratory of Plant Stress Research, Shandong Normal University, Jinan 250014, China;
2. College of Life Sciences, Liaocheng University, Liaocheng 252059, China;
3. College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047, China)
Abstract: Heat shock cognate proteins 70 (hsp70s) act as molecular chaperones. Some hsp70s are also
expressed in unstressed plants, known as hsc70. To gain further knowledge about the hsc70, the Thellungiella
halophila hsc70 (Thhsc70) gene that encoded the cytosolic hsc70 in salt cress (T. halophila (C. A. Mey.) O.
E. Schulz) was identified. In unstressed plants the expression of Thhsc70 was shown to be tissue-specific.
The Thhsc70 gene was induced by heat and cold stresses, but almost not by salt and drought stresses.
Overexpression of Thhsc70 could increase thermotolerance and chilling tolerance in transgenic Arabidopsis
plants.
Key words: heat shock cognate proteins 70; heat stress; molecular chaperone; Thellungiella halophila
Exposure of plants to heat shock or to other environ-
mental stresses results in the synthesis of a set of specific
proteins known as heat shock proteins (hsps) (Vierling，
1991; Boston et al., 1996). Hsps are subdivided into several
classes according to their molecular mass. The 70-kD fam-
ily of hsps (hsp70) is a major, well characterized group of
molecular chaperones.
Hsp70s are encoded by a highly conserved multi-gene
family, which function in some major subcellular compart-
ments of the cell. According to their different locations,
hsp70s can be divided into four major subgroups (Guy and
Li, 1998; Sung et al., 2001a), each localized to endoplasmic
reticulum (ER), cytosol, plastid and mitochondria. Hsp70s
are also localized to other subcellular compartments, such
as glyoxysome and protein bodies (Wimmer et al., 1997;
Sung et al., 2001a).
A hallmark of many hsp70 genes is their strong and
universal induction in response to heat shock. Most plant
hsp70s show strong and rapid induction after 30 min to 2 h
at 37-45 ℃ (Li et al., 1999; Sung et al., 2001a). Several
plant hsp70s are also induced by cold shock (Li et al., 1999).
Some hsp70s are also expressed in unstressed cells, known
as heat shock cognate protein 70 (hsc70), indicating that
they play an essential role in the maintenance of normal cell
functions as well (Storozhenko et al., 1996). The most
important function of hsp70s is as a molecular chaperone
by helping newly synthesized proteins fold properly, main-
taining an extended conformation of proteins during
translocation, and preventing unfolded proteins from un-
dergoing nonproductive aggregation (Boston, 1996;
Miernyk, 1997).
Hsp70s participate in different subcellular processes
according to their different locations in subcellular
compartments. The ER lumen harboring an hsp70 homolog,
BiP, is considered to be involved in precursor protein trans-
location across the ER membrane, protein folding, assem-
bly and binding malfolded proteins in the ER lumen (Sung
et al., 2001a). Recently it has been reported that
overexpression of an endogenous BiP in tobacco relieved
ER stress by restoring secretory protein processing and
folding (Leborgne-Castel et al., 1999). Chloroplast hsp70
homologs, which were found in the outer envelope
membrane, the stroma and the thylakoid lumen, were con-
sidered to participate in import process. When chloroplast-
localized hsp70 was overexpressed, reactivation of PSⅡ
af te r photoinhibi t ion was enhanced. Whereas
underexpression resulted in reducing reactivation of PSⅡ
(Schroda et al., 1999).
There are limited functional studies of plant cytosolic
hsp70s. Studies in vitro showed that plant cytosolic hsp70s
ZHANG Xia et al.: Molecular Cloning and Identification of a Heat Shock Cognate Protein 70 Gene, Thhsc70, in Thellungiella
halophila 1213
associate with nascent polypeptides of secretory precur-
sors (Miernyk et al., 1992). Lee and Schnölf (1996) reported
that during heat shock the expression of cytosolic hsp70 in
Arabidopsis was reduced by introducing antisense mRNA
of a tobacco (Nicotiana tabacum) hsp70 under the control
of a soybean (Glycine max) heat shock promoter. Due to
the reduced expression of hsp70, transgenic plants lost
their induced thermotolerance and showed a prolonged heat
shock response. Recently, Sung and Guy (2003) generated
transgenic Arabidopsis over-/under-expressing the major
Arabidopsis cytosolic heat shock cognate protein70
(hsc70), Arabidopsis-Thellungiella halophila cytosolic
hsc70 (Athhsc70-1), to identify its functions. They found
that altering expression of Athsc70-1 brought negative con-
sequences to plant growth and development. Finding no
viable transgenic lines for constitutive underexpression of
Athsc70-1 suggested that reduction of Athsc70-1 expres-
sion was detrimental to plant viability. But constitutive
overexpress ion o f Athsc70-1 enhanced basa l
thermotolerance, affected plant size and altered the root
system.
Salt cress (T. halophila), a close relative of Arabidopsis,
is able to withstand dramatic salinity shock up to 500 mmol/
L NaCl and grow in salt far in excess of the capability of
Arabidopsis (Zhu, 2001). It appears that stress tolerance in
T. halophila is largely the result of basic biochemical and
physiological mechanisms, and therefore T. halophila can
be adopted as a halophytic model for stress-tolerance
research. In the ESTs of T. halophila (Wang et al., 2004),
we found several putative heat shock cognate protein 70
genes. To better understand the roles of hsc70s in T.
halophila tolerance to abiotic stress, here we identified the
Thhsc70 gene that encoded an hsc70 localized to cytosol
in T. halophila , and demonstrated the result of
overexpression of Thhsc70 on thermotolerance and chill-
ing tolerance in transgenic Arabidopsis plants.
1 Materials and Methods
1.1 Plant materials and culture conditions
Seeds of Thellungiella halophila (C. A. Mey.) O. E.
Schulz (collected from Dongying, Shandong Province,
China) were sown in 9 cm plastic pots filled with a 2:1:1
mixture of soil/perlite/vermiculite and watered with Hoagland
solution. The experiment was carried out in a greenhouse
with a photoperiod of 16 h of light and 8 h of dark, a tem-
perature of 25 ℃/20 ℃, a relative humidity of 60%/80%.
1.2 cDNA clone and sequence information
A full-length cDNA clone for the Thhsc70 gene was
obtained from a NaCl-treated cDNA library of T. halophila
by the large-scale partial sequencing of randomly selected
cDNA clones or ESTs. The cDNA clone in pBK-CMV
phagemid vector was sequenced by the dideoxy chain ter-
mination method using standard T3, T7 primers and cloned
specific primers based on already sequenced regions. Prim-
ers for Thhsc70 were 5- AAGATGCGAGAGATTGCC-3 (P1)
and 5- GCTCCTTGGACTATCTTG-3 (P2). The sequences
were submitted to GenBank (GenBank accession number
AY524794).
1.3 Stress treatments
Heat stress experiments were performed with four-week-
old T. halophila at 40 ℃ for 4 h. Cold treatments were
carried out at 4 ℃ for up to 72 h. For salt stress experiments,
plants were watered profusely with 200 mmol/L NaCl for up
to 48 h. For drought stress, water was withheld. Whole
plant samples from control and treated plants were col-
lected at different time points, immediately frozen in liquid
nitrogen and stored at -70 ℃, and subsequently used for
RNA isolation.
1.4 Southern blotting analysis and Northern blotting
analysis
Genomic DNA was isolated as described by Murray and
Thomepson (1980) and total RNA was extracted according
to the method of Chomczynski and Sacci (1987). DNA/RNA
samples were transferred to an N+-Hybond nylon membrane.
Hybridizations were carried out as described by Sambrook
et al. (1989) with 32P-EcoRⅠ-XhoⅠ Thhsc70 cDNA probes.
1.5 Plasmid construction and plant transformation
The Thhsc70 cDNA was cut with BamHⅠ and KpnⅠ
and ligated to pROKⅡ, which contained a 35S promoter,
open with the same enzymes to yield pROK-hsc70. The
resulting plasmid was mobilized to Agrobacterium
tumefaciens strain GV3101 and used for plant
transformation. Arabidopsis (ecotype Columbia) adult
plants (five weeks old) were infected with the A. tumefaciens
by floral dipping method (Clough and Bent, 1998) and grew
in the greenhouse. These seeds collected were screened in
MS medium supplemented with 30 mg/mL kanamycin.
1.6 Assays for thermotolerance and chilling tolerance
The Thhsc70 overexpression lines (OE lines) were ger-
minated on MS medium supplemented with 30 mg/mL
kanamycin. Two-week-old plants were transferred to soil,
and the stress treatments were performed two weeks after
transfer. Thermotolerance assays were conducted as de-
scribed by Sung and Guy (2003), and then take the mea-
surements of electrolyte leakage. Cold treatments were car-
ried out at 4 ℃ for up to 5 d. Malondialdehyde (MDA)
contents were measured using a thiobarbituric acid reac-
tion (Heath and Packer, 1968).
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041214
2 Results
2.1 Isolation and characterization of Thhsc70 gene from
T. halophila cDNA library
We had initiated the cloning of a large number of salt-
induced genes by randomly sequencing a 200 mmol/L NaCl-
treated for 48 h cDNA library of T. halophila aerial part
tissue. From this library, we identified a heat shock cognate
protein 70 cDNA clone. The Thhsc70 gene includes a
1 953-bp reading frame flanked by 5- and 3-noncoding se-
quences of 39 and 203 nucleotides, respectively. The en-
coded polypeptide is 651 amino acids, with the predicted
molecular mass about 70 kD.
Database search reveals that Thhsc70 has the highest
amino acid sequence similarity to hsc70-1 of A. thaliana.
Apart from the three PROSITE signature sequences for the
hsp70 protein family (Sung et al., 2001a), in C-terminal re-
gion Thhsc70 contains a motif for the cytosolic group,
EEVD, which quite possibly acts as an organelle-specific
co-chaperone interaction determinant.
2.2 Southern blotting analysis of the Thhsc70 gene
The copy number and genomic structure of the Thhsc70
gene were assessed by Southern blotting analysis. Total
genomic DNA was digested with some restriction endonu-
cleases and probed with the EcoRⅠ-XhoⅠ 3 fragment of
the Thhsc70 clone. Thhsc70 was multicopy in T. halophila
genome and likely to be a member of a gene family
(Fig.1).
2.3 Northern blotting analysis of the Thhsc70 gene
The expression of Thhsc70 was investigated in leaves,
stems and roots in the absence of stress in order to deter-
mine whether it was expressed in particular tissue(s).
Figure 2A shows that the Thhsc70 expression was high in
leaves, low in stems but almost absent in roots.
Fig.1. Southern blotting analysis of the Thellungiella halophila
heat shock cognate protein 70 (Thhsc70) gene in T. halophila.
Genomic DNA (10 mg per lane) was digested with the indicated
restriction enzymes. The gel blot was hybridized with 32P-EcoRI-
XhoI Thhsc70 cDNA.
Fig.2. Northern blotting analysis of Thellungiella halophila heat shock cognate protein 70 (Thhsc70) in T. halophila. Tissue-specific
(A), heat stress (B), cold stress (C), salt stress (D) and drought stress (E). Total RNA (20 mg) was blotted after separation and hybridized
with 32P-labelled Thhsc70 cDNA probe. The ethidium biomide-stained rRNA band in the agarose gel is shown as a loading control.
ZHANG Xia et al.: Molecular Cloning and Identification of a Heat Shock Cognate Protein 70 Gene, Thhsc70, in Thellungiella
halophila 1215
Heat induction of Thhsc70 gene was confirmed by North-
ern blotting analysis. Figure 2B shows a rapid increasing of
Thhsc70 gene expression upon heat stress. The maximum
level of the mRNA was observed 0.5 h after the onset of the
heat shock at 40 ℃. But after 2 h the steady-state mRNA
level reduced. As shown in Fig.2C, it was indicated that the
Thhsc70 gene was upregulated during 12 h to 72 h cold
stress at 4 ℃.
We also analyzed the expression of Thhsc70 upon salt
stress and drought stress by Northern hybridization. In
Fig.2D, upon 200 mmol/L NaCl stress the mRNA level of
Thhsc70 gene transiently increased at 3 h treatment. After
12 h the mRNA level dropped to the basal level of unstressed
plants. But as shown in Fig.2E, the expression of Thhsc70
upon drought stress showed no significant changes.
2.4 Genetic transformation
Five-week-old Arabidopsis plants were infected with A.
tumefaciens carrying the plasmid pROK-hsc70 and the
empty vector pROKⅡ respectively, by the floral dipping
method. Several transgenic homozygous lines that were all
tolerant to kanamycin (30 mg/mL) were selected, named H20-
8, H27-1, H72-12, etc., and used for molecular and physi-
ological analysis and further experiments. Constitutive
overexpression of Thhsc70 had the lower transformation
efficiency compared with that of the empty vector control.
It was noticed that during screening of primary
transformants numerous kanamycin-resistant seedlings of
Thhsc70 OE plants had smaller stature and more branched
root systems compared with empty vector-transformed
plants (pROK)(data not shown).
2.5 Molecular characterization of the transgenic plants
Northern blotting analysis revealed the presence of
Thhsc70 mRNA in several homozygous transgenic lines.
The levels of expression among different transgenic lines
were different. For example, expression level of line H27-1
was more than that of line H20-8, which might relate with
the copy number and inserted position of the inserted gene.
The pROK line showed no hybridization signal in the North-
ern analysis, as shown in Fig.3.
2.6 Increased thermotolerance and chilling tolerance of
Thhsc70 OE plants
Three strong Thhsc70 OE lines (H20-8, H27-1, H72-12)
were selected for further analysis. Thermotolerance of the
three lines and pROK line was tested by giving a 10-min
heat shock in water at temperatures ranging from 42 ℃ to
48 ℃. Line H27-1 and pROK plants treated with room
temperature (RT), 42 ℃, and 48 ℃ are shown in Fig.4A.
These OE lines showed lower electrolyte leakage and sur-
vived a 10-min heat shock at 42 ℃, but pROK plants showed
greater electrolyte leakage and showed no sign of survival
3 d after heat shock (Fig.4A, B).
To test chilling tolerance, these OE lines and pROK line
were also performed in cold stress experiment at 4 ℃ for up
to 5 d, and then the MDA content was determined. As
shown in Fig.4C, at different treatment periods, OE lines
showed lower MDA content than pROK line, which sug-
gested that Thhsc70 OE lines had increasing chilling toler-
ance compared with the control line.
3 Discussion
Plants inevitably experience a wide array of environ-
mental stresses. Therefore plants developed mechanisms
to respond to different types of stresses, including heat
stress. Heat-shock response is a conserved reaction of cells
against elevated temperatures by transiently reprogram-
ming cellular activities to cease normal protein synthesis
and to synthesize a set of heat-shock proteins (HSPs)
(Schoffl et al., 1998). It is known that HSPs have a molecu-
lar chaperone activity, which could account for a protec-
tive effect at high temperature.
The differential expression of multiple HSP genes has
been reported in several plants. Rizhsky et al. (2002) re-
ported the expressions of hsp70, hsp90 and some small
hsps of tobacco induced by drought stress and heat shock.
Sung et al. (2001b) reported two members of Arabidopsis
hsp70 gene family, mthsc70-2 and cphsc70-2, induced by
heat stress and cold treatment. All the results above con-
tribute to the speculation that HSP gene plays an impor-
tant role in the maintenance of cell function under stress
conditions.
We identified the expression of Thhsc70 that encoded a
cytosolic hsc70. In unstressed T. halophila, the Thhsc70
expression was high in leaves, low in stems but almost
absent in roots (Fig.2A). This suggested that Thhsc70 was
Fig.3. Expression of Thellungiella halophila heat shock cognate
protein 70 (Thhsc70) in empty vector-transformed plants (pROK)
and some OE lines by Northern blot. Twenty micrograms (20 mg)
of total RNA was analyzed by RNA gel blotting. The blot was
hybridized (60 ℃) with the gene-specific DNA probe for Thhsc70.
The ethidium biomide-stained rRNA band in the agarose gel is
shown as a loading control.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041216
ZHANG Xia et al.: Molecular Cloning and Identification of a Heat Shock Cognate Protein 70 Gene, Thhsc70, in Thellungiella
halophila 1217
expressed in tissue specificity and the expression in un-
stressed cells indicated that it played an essential role in
the maintenance of normal cell functions as well. The ex-
pressions of Thhsc70 in response to abiotic stresses were
also analyzed. The Thhsc70 gene was strongly induced by
heat shock at 40 ℃. The maximum level of the mRNA was
observed 0.5 h after the onset of the heat shock. But after 2
h the steady-state mRNA level reduced. Upon cold stress
Thhsc70 was induced to high level for up to 72 h. The
temperature responses demonstrated that cytosolic
Thhsc70 gene was responsible for molecular chaperone
activity under heat stress and cold stress. Under 200
mmol/L NaCl stress the mRNA level of Thhsc70 transiently
increased at 3 h treatment. It is noticeable that after 3 h
treatment, the steady-state mRNA level dropped to the basal
level of unstressed plants. It is perhaps due to the transient
response of Thhsc70 to salt stress. But upon drought
stress, the expression of Thhsc70 showed no significant
changes. These results suggested that the Thhsc70 might
less contribute to plant tolerance to osmotic stress.
To better understand the function of the Thhsc70 gene
we overexpressed Thhsc70 in Arabidopsis. It was noticed
that constitutive overexpression of Thhsc70 had lower trans-
formation efficiency compared with that of the empty vec-
tor control. Similar findings have been obtained from
overexprssion of bacterial hsc70, DnaK, in Escherichia coli
(Blum et al., 1992) and Synechococcus sp. strain PCC7942
(Nimura et al., 2001). During screening of primary
transformants we found that numerous kanamycin-resis-
tant seedlings of Thhsc70 OE plants had smaller stature
and more branched root systems compared with empty
vector-transformed plants. It appeared that constitutive
overexpression of Thhsc70 was deleterious to plant viability.
Sung and Guy (2003) also reported that constitutive
overexpression of Athsc70-1 brought negative conse-
quences to plant growth and development, and constitu-
tive expression of a full-length Athsc70-1 antisense RNA
resulted in no viable transgenic plants. All these suggested
that prolonged interaction of hsp70 with substrates might
be equally harmful to optimal cell functioning as inadequate
or premature termination of the interaction.
It is reported that expression of hsp70 gene positively
correlated with acquisition of thermotolerance (Feder et al.,
1996; Lee and Schöffl, 1996; Nollen et al., 1999), and the
overexpression of hsp70s often resulted in enhanced
thermotolerance (Feder et al., 1996; Nollen et al., 1999). In
our study, when Thhsc70 was constitutively overproduced
in Arabidopsis plants, thermotolerance and chilling toler-
ance were both enhanced. Thermotolerance is a quantita-
tive trait (Ottaviano et al., 1991). However, knockout mu-
tant studies of hsp101 in Arabidopsis (Hong and Vierling,
2000) and maize (Zea mays) (Nieto-Sotelo et al., 2002), dem-
onstrated that alteration of a single gene could result in
significant changes in thermotolerance. In the present
study, we found that overexpression of Thhsc70 promotes
enhanced thermotolerance at the whole plant level (Fig.4A,
B). On the other hand, as shown in Fig.4C, the MDA con-
tent of OE lines, which represented the superoxidation de-
gree of the plasma membrane, were lower than the control
line. It demonstrated that Thhsc70 OE plants also improved
chilling tolerance compared with the control line.
This is the first report on cloning and identification of a
Thhsc70 gene in the halophytic T. halophila as known.
Our study reveals that the Thhsc70 is pivotal in normal
growth and development under non-stressed and stressed
conditions and Thhsc70 is linked with enhanced
thermotolerance and chilling tolerance.
References:
Blum P, Ory J, Bauernfeind J, Krska J. 1992. Physiological con-
sequences of DnaK and DnaJ overproduction in Escherichia
coli. J Bacteriol, 174: 7436-7444.
Boston R S, Viitanen P V, Vierling E. 1996. Molecular chaperones
and protein folding in plants. Plant Mol Biol, 32: 191-222.
Chomczynski P, Sacchi N. 1987. Single-step method of RNA
isolation by acid guanidiumthiocyanate-phenol-chlorofrom
extraction. Anal Biochem, 162: 156-159.
Clough S J, Bent A F. 1998. Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J, 16: 735–743.
Feder M E, Cartano N V, Milos L, Krebs R A, Lindquist S L.
1996. Effect of engineering hsp70 copy number on hsp70
expression and tolerance of ecologically relevant heat shock in
larvae and pupae of Drosophila melanogaster. J Exp Biol,
199: 1837-1844.
Guy C L, Li Q B. 1998. The organization and evolution of the
spinach stress 70 molecular chaperone gene family. Plant Cell,
Fig.4. Thellungiella halophila heat shock cognate protein 70 (Thhsc70) OE line and empty vector-transformed plants (pROK) line
treated at different temperatures (A). Thermotolerance of Thhsc70 OE lines assessed by electrolyte leakage assay. pROK, empty vector-
transformed Arabidopsis (B). MDA content of OE lines and pROK line at cold treatment (C).
←
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041218
10: 539-556.
Heath R L, Packer L. 1968. Photoperoxidation in isolated
chloroplasts. Arch Biochem Biophys, 125: 189-198.
Hong S W, Vierling E. 2000. Mutants of Arabidopsis thaliana
defective in the acquisition of tolerance to high temperature
stress. Proc Natl Acad Sci USA, 97: 4392-4397.
Leborgne-Castel N, Edith P W M, Dooren J V, Crofts A J, Denecke
J. 1999. Overexpression of BiP in tobacco alleviates endo-
plasmic reticulum stress. Plant Cell, 11: 459-469.
Lee J H, Schöffl F. 1996. An Hsp70 antisense gene affects the
expression of HSP70/HSC70, the regulation of HSF and the
acquisition of thermotolerance in transgenic Arabidopsis
thaliana. Mol Gen Genet, 252: 11-19.
Li Q B, Haskell D W, Guy C L. 1999. Coordinate and non-
coordinate expression of the stress 70 family and other mo-
lecular chaperones at high and low temperature in spinach and
tomato. Plant Mol Biol, 39: 21–34.
Miernyk J A. 1997. The 70 kD stress-related proteins as molecu-
lar chaperones. Trends Plant Sci, 2: 180-187.
Miernyk J A, Duck N B, Shatters Jr R G, Folk W R. 1992. The
70-kilodalton heat shock cognate can act as a molecular chap-
erone during the membrane translocation of a plant secretory
protein precursor. Plant Cell, 4: 821–829.
Murray M G, Thompson W F. 1980. Rapid isolation of high
molecular weight plant cDNA. Nucleic Acids Res, 8: 4321–
4325.
Nieto-Sotelo J, Martínez L M, Ponce G, Cassab G I, Alagón A,
Meeley R B, Ribaut J M, Yang R. 2002. Maize hsp101 plays
important roles in both induced and basal thermotolerance
and primary root growth. Plant Cell, 14: 1621-1633.
Nimura K, Takahashi H, Yoshikawa H. 2001. Characterization of
the DnaK multigene family in the cyanobacteuium
synechococcus sp. strain PCC7942. J Bacteriol, 183: 1320-
1328.
Nollen E A, Brunsting J F, Roelofsen H, Weber L A, Kampinga H
H. 1999. In vivo chaperone activity of heat shock protein 70
and thermotolerance. Mol Cell Biol, 19: 2069-2079.
Ottaviano E, Sari-Gorla M, Pé E, Frova C. 1991. Molecular
markers (RFLPs and HSPs) for the genetic dissection of
thermotolerance in maize. Theor Appl Genet, 81: 713-719.
(Managing editor: ZHAO Li-Hui)
Rizhsky L, Liang H J, Mittler R. 2002. The combined effect of
drought stress and heat shock on gene expression in tobacco.
Plant Physiol, 130: 1143-1151.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laboratory Manual. 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
Schoffl F, Prändl R, Reindl A. 1998. Regulation of the heat-shock
response. Plant Physiol, 117: 1135-1141.
Schroda M, Vallon O, Wollman F A, Beck C F. 1999. A chloro-
plast-targeted heat shock protein 70 (hsp70) contributes to
the photoprotection and repair of photosystemII during and
after photoinhibition. Plant Cell, 11: 1165-1178.
Storozhenko S, De Pauw P, Kushnir S, van Montagu M, Inze D.
1996. Identification of an Arabidopsis thaliana cDNA encod-
ing a HSP70-related protein belonging to the HSP110/SSE1
subfamily. FEBS Lett, 390: 113-118.
Sung D Y, Guy C L. 2003. Physiological and molecular assess-
ment of altered expression of Hsc70-1 in Arabidopsis. Evi-
dence for pleiotropic consequences. Plant Physiol, 132: 979-
987.
Sung D Y, Kaplan F, Guy C L. 2001a. Plant Hsp70 molecular
chaperones: protein structure, gene family, expression and
function. Physiol Plantarum, 113: 443-451.
Sung D Y, Vierling E, Guy C L. 2001b. Comprehensive expres-
sion profile analysis of the Arabidopsis hsp70 gene family.
Plant Physiol, 126: 789-800.
Vierling E. 1991. The roles of heat shock proteins in plants. Annu
Rev Plant Physiol Plant Mol Biol, 42: 579-620.
Wang Z L, Li P H, Fredricksen M, Gong Z Z, Kim C S, Zhang C
Q, Bohnert H J, Zhu J K, Bressan R A, Hasegawa P M, Zhao
Y X, Zhang H. 2004. Expressed sequence tags from
Thellungiella halophila, a new model to study plant salt-
tolerance. Plant Sci, 166: 609-616.
Wimmer B, Lottspeich F, van der Klei I, Veenhuis M, Gietl C.
1997. The glyoxysomal and plastid molecular chaperones (70-
kDa heat shock protein) of watermelon cotyledons are en-
coded by a single gene. Proc Natl Acad Sci USA, 94: 13624–
13629.
Zhu J K. 2001. Plant salt tolerance. Trends Plant Sci, 6: 66-71.