Plant Cell Physiol. 43(9): 1043–1048 (2002)
JSPP © 2002
The Role of Plant CCTa in Salt- and Osmotic-Stress Tolerance
Akiyo Yamada 1, 3, Mikiko Sekiguchi 1, Tetsuro Mimura 2 and Yoshihiro Ozeki 1
1
Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo,
184-8588 Japan
2
Department of Biological Sciences, Faculty of Science, Nara Women’s University, Kitauoyanishi-machi, Nara-shi, Nara, 630-8506 Japan
;
To find key genes essential for salt tolerance in the
mangrove plant, Bruguiera sexangula, functional screening was performed using Escherichia coli as the host organism. A transformant expressing a cytosolic chaperonincontaining TCP-1a (CCTa) homologue displayed enhanced
salt tolerance. Analysis in E. coli of the functional region
revealed that a sequence of only 218 amino acids, containing the apical domain, is necessary for osmotolerance. Furthermore, this domain shows chaperone activity in vitro.
Therefore, CCTa facilitates the folding of proteins without
ATP or the cage-like structure, and may play an important
role in stress tolerance, at least in B. sexangula.
Keywords: Bruguiera sexangula — CCTa — Mangrove —
Salt-tolerance.
Abbreviations: CCT, chaperonin containing TCP-1; TCP-1,
t-complex peptide-1; CS, citrate synthase; MBP, maltose binding protein.
The nucleotide sequence reported in this paper has been submitted to EMBL/GenBank/DDBJ database under accession number
AB073552.
Salt stress is one of the most serious factors limiting plant
growth and productivity in the world (Boyer 1982). To enhance
salt tolerance in higher plants, genes have been cloned for
numerous key proteins; i.e. late-embryogenesis abundant proteins (Xu et al. 1996), P5CS (Kishor et al. 1995), DREB1A
(Kasuga et al. 1999), and AtNHX1 (Apse et al. 1999). In contrast, there are few reports dealing with genes for salt-stress tolerance in mangrove plants. To find the key genes essential for
salt tolerance in mangrove plants, we constructed a cDNA
library from suspension-cultured cells of the mangrove plant,
Bruguiera sexangula (Mimura et al. 1997a, Mimura et al.
1997b, Kura-Hotta et al. 2001) and undertook functional
screening for key genes essential for salt-tolerance mechanisms, using Escherichia coli as the host organism. This
screening isolated transformants with enhanced salt tolerance
that expressed a component of a cytosolic chaperonin-containing TCP-1a (CCTa) homologue.
3
Chaperonins are a class of molecular chaperones that
mediate the folding of non-native polypeptides with concomitant ATP hydrolysis (Frydman 2001), and are classified as
either type I or type II (Kubota et al. 1995). Type I includes
GroEL from eubacteria (Georgopoulos et al. 1973), HSP60
from mitochondria (Cheng et al. 1989), and the rubisco-subunitbinding proteins from chloroplasts (Hemmingsen et al. 1988).
Type II includes TF55 (Trent et al. 1991) and the thermosomes
(Phipps et al. 1991) from archaebacteria, and the CCT complex (also called TriC, the TCP-1 ring complex) from the
cytosol of eukaryotes (Kubota et al. 1995). Like other chaperonins, the CCT complex is a high-molecular-weight protein,
the subunits of which are arranged in two stacked multimeric
rings with a central cavity. Whereas type I chaperonins, such as
GroEL, are promiscuous, assisting in the folding of many other
proteins, only a few proteins have been described as the natural substrates for the CCT complex, principally actin and tubulin (Ursic et al. 1994). Why salt tolerance in E. coli is enhanced
by the expression of CCTa cannot be explained on the basis of
previous data on eukaryotic CCTa. Furthermore, no information is available concerning the role of the CCT complex in the
salt tolerance of higher plants. In this study, we focused on the
a subunit of the CCT complex, CCTa, the function of which
was investigated in vivo and in vitro.
Eukaryotic CCTa is a highly conserved protein and consists of three domains (Kubota et al. 1999): an equatorial
domain, which contains the ATP-binding site and provides
most of the intersubunit contacts; an apical domain which
binds to peptides; and an intermediate domain, which is located
between the apical and equatorial domains (Fig. 1A). To identify the functional region in B. sexangula CCTa (BsCCTa),
three forward primers and three reverse primers were designed
to amplify several regions of BsCCTa cDNA by PCR (30
cycles of 92°C for 30 s, 45°C for 30 s, and 72°C for 30 s). Forward primers contained a stop codon (TAG), an XbaI endonuclease site (TCTAGA), and a start codon (ATG). Reverse primers contained two stop codons (TAG, TAA, or TGA) and an
XhoI endonuclease site (CTCGAG). The amplified cDNA fragments were digested with XbaI/XhoI, and cloned into the XbaI
and XhoI sites of pBluescript SK (+). Primer sequences are as
follows:
Corresponding author: E-mail, [email protected]; Fax, +81-42-388-7239.
1043
1044
Molecular analysis of plant CCTa
Fig. 1 Structure of BsCCTa cDNA and its deletion clones (A), and regional functional analysis of BsCCTa (B). The transformants were grown
up to exponential phase in liquid 2YT medium containing 86 mM NaCl (normal NaCl concentration), 0.05 mM IPTG, 50 mg ml–1 ampicillin, and
50 mg ml–1 kanamycin. Cell densities of these E. coli transformants were adjusted to 0.1 (A600), and serial dilutions (1 : 10) were made. Each dilution (15 ml) was spotted onto 2YT agar plates supplemented with 50 mg ml–1 kanamycin, 50 mg ml–1 ampicillin, 0.05 mM IPTG, and 350 mM
NaCl or 600 mM sorbitol. Empty pBluescript SK vector was used as the control. Plates were photographed after 12 h incubation at 37°C.
CCT-1F 5¢-GACTCTAGATGGCAATCGCGGCTCAAACTCC-3¢
CCT-192F 5¢-GGGTCTAGATGAAATATCCTATCAAGAGTAT-3¢
CCT-348F 5¢-ATTTCTAGATGCTTGGACAAGCTGAAGAAG-3¢
CCT-546R 5¢-AAGCTCGAGCTACTATTCCTCTTCATTCTGAGTCTC-3¢
CCT-409R 5¢-ACCCTCGAGTTATCAATTAGATTCAAGGGTTCTCTTG-3¢
CCT-260R 5¢-TCTCTCGAGCTATCAGGGATCAGTGACTAAGACTTG-3¢.
The XbaI and XhoI endonuclease sites are underlined and
the start or stop codons are given in bold letters.
The cloned BsCCTa cDNA fragments were then introduced into E. coli and the salt- and osmotic-stress tolerance of
each transformant was tested (Fig. 1). All transformants grew
in the presence of 86 mM NaCl (control). Transformants carrying deletion clones (1–260, 348–546) or an empty plasmid did
not grow in the presence of 350 mM NaCl. On the other hand,
transformants carrying full-length BsCCTa cDNA or one of
two deletion clones (192–546, 192–409) grew in the presence
of 350 mM NaCl or 600 mM sorbitol. These data reveal that a
218 amino acid sequence (192–409), containing the complete
apical domain, is required for the salt-tolerance mechanism
Molecular analysis of plant CCTa
1045
Fig. 2 Construction of plasmids containing MBP-BsCCTa (1–546) and MBP-BsCCTa (192–409) fusion genes driven by the tac promoter (A)
and SDS-PAGE of MBP-BsCCTa (1–546) and MBP-BsCCTa (192–409) fusion proteins and MBP, produced in E. coli and purified by amylose
resin columns (B). Lane M, size marker; lane 1, MBP-BsCCTa (1–546) fusion protein; lane 2, MBP-BsCCTa (192–409) fusion protein; lane 3,
MBP.
of BsCCTa.
To analyze whether BsCCTa proteins have chaperone
activity, BsCCTa (1–546) and BsCCTa (192–409) proteins
were purified with a maltose-binding protein (MBP) tag using
the pMAL Protein Fusion and Purification System (New England BioLabs Inc., MA, U.S.A.). In constructing pMALc2XBsCCT plasmids (encoding 1–546 and 192–409), four primers
were designed to amplify BsCCTa cDNA fragments by PCR
(30 cycles of 92°C for 30 s, 50°C for 30 s, and 72°C for 30 s)
using whole BsCCTa cDNA as template. Primer sequences are
as follows:
pMALCCT1F 5¢-ATGGCAATCGCGGCTCAAACTCCG-3¢
pMALCCT546R 5¢-AAGTCTAGACTACTATTCCTCTTCATTCTGAGT-3¢
pMALCCT192F 5¢-ATCAAATATCCTATCAAGAGTATA-3¢
pMALCCT409R 5¢-AACTCTAGATTATCAATTAGATTCAAGGGTTCTCTT-3¢.
The amplified cDNA fragments were digested with XbaI,
and cloned into the XbaI/XmnI sites of pMALc2X (Fig. 2A).
These plasmids were then introduced into E. coli, where the
protein was expressed. MBP-CCTa proteins were purified
from the E. coli transformants by using amylose resin columns
and following the purification procedure described in the
pMAL Protein Fusion and Purification System instruction
manual. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the purification of the MBPBsCCTa (1–546) and MBP-BsCCTa (192–409) fusion proteins (Fig. 2B). To analyze the functions of these fusion proteins in E. coli, pMALc2X-BsCCT (1–546 or 192–409) was
introduced into E. coli, and the bacterial stress tolerance was
tested (Fig. 3A, B). The expression of these fusion proteins
enhanced salt- and osmotic-stress tolerance in E. coli. Furthermore, the transformant expressing pMAL-BsCCTa (192–409)
enhanced heat-shock tolerance (Fig. 3B). A similar effect was
1046
Molecular analysis of plant CCTa
Fig. 3 Effects of MBP-BsCCTa fusion protein expression on salt- and osmotic-stress tolerance and heat-shock tolerance in E. coli. The methods
for testing NaCl- and osmotic-stress tolerance in these transformants are described in the legend to Fig. 1. For heat-shock tolerance experiments,
cell concentrations of these E. coli transformants were adjusted to 0.1 (A600), then cells were incubated for the indicated times. After incubation,
15 ml of the cell suspensions were spotted immediately onto 2YT agar plates containing 0.05 mM IPTG, 50 mg ml–1 ampicillin, and 50 mg ml–1
kanamycin.
also observed in MBP-free BsCCTa transformants (data not
shown). The difference in the stress tolerance effect between
BsCCTa (192–409) and BsCCTa (1–546) may have been
caused by the protein contents in these transformants or the
structures of these protein fragments. The apical domain of
BsCCTa (192–409) is exposed when compared with the apical
domain of BsCCTa (1–546), which may result in differences in
the interaction of substrate proteins with these apical domains
in E. coli. On the basis of these results, we postulate that BsCCTa has chaperone activity without requiring ATP or formation of the cage-like structure.
To confirm this hypothesis, the chaperone activities of
MBP-BsCCTa (1–546, 192–409) fusion proteins were tested
using citrate synthase (CS) in vitro. CS (from porcine heart)
was purchased from Sigma and its activity was measured as
described previously (Caldas et al. 1998). Fig. 4 shows the
effects of heat-shock treatment (43°C) on CS activity in the
presence of 4 mM MBP-BsCCTa (1–546), 4 mM MBP BsCCT
(142–409), 4 mM MBP, or in the absence of additional proteins
(control). As has been reported previously (Jakob et al. 1993),
CS loses its native conformation during incubation at 43°C. In
accordance with previously presented data (Jakob et al. 1993),
CS activity decreased rapidly under control conditions. After
20 min of heat incubation, CS activity was hardly detectable.
MBP showed weak chaperone activity, which may have arisen
from non-specific interaction between CS and MBP. Similar
effects have been reported for bovine serum albumin (BSA)
(Zahn et al. 1996). In contrast, over 70% of CS activity
remained in the presence of MBP-BsCCTa (1–546) and MBPBsCCTa (142–409) after 40 min of heat incubation. Therefore, MBP-BsCCTa (1–546) and MBP-BsCCTa (142–409)
show distinct chaperone activity in vitro. To confirm these
chaperone activities in detail, the effects of heat-shock treatments (20 min, 43°C) on CS activity were investigated in the
presence of various concentrations of MBP-BsCCTa (1–546),
MBP-BsCCTa (142–409), MBP, BSA, or in the absence of
additional protein (Fig. 5). In the presence of 0–1 mM MBPBsCCTa (1–546, 142–409), CS activity increased in a concentration-dependent manner. In the presence of more than 2 mM
MBP-BsCCTa (1–546 or 142–409), CS activity reached a
fixed value (70–90% activity of untreated CS). MBP and BSA
also showed chaperone activity, but very weakly. Therefore, we
conclude that BsCCTa (1–546) and BsCCTa (142–409) have
chaperone activity in vitro.
The function of the bacterial chaperonin GroEL, which
belongs to the type I chaperonins, has been extensively investi-
Molecular analysis of plant CCTa
1047
Fig. 4 Effect of heat-shock treatment (43°C) on CS activity in the
presence of 4 mM MBP BsCCT (1–546), 4 mM MBP-BsCCT (192–
409), 4 mM MBP, or in the absence of additional protein (control). Percentage of CS activity refers to the CS activity remaining after heatshock treatment (100% CS activity refers to that activity present without heat-shock treatment). Standard errors were within 5%.
Fig. 5 Effect of heat-shock treatment (43°C, 20 min) on CS activity
at various concentrations of MBP-BsCCT (1–546, 192–409), MBP,
BSA, or in the absence of additional protein. Percentage of CS activity refers to the CS activity remaining after heat-shock treatment
(100% CS activity refers to that activity present without heat-shock
treatment). Standard errors were within 5%.
gated. The structures of type I and type II chaperonins have
similar organization of their three domains (apical, intermediate, and equatorial) within the monomer (Llorca et al. 1998). In
accord with our results, Chatellier et al. (1998) showed that the
fragment encompassing the apical domain of GroEL, called the
minichaperone, facilitates the refolding of several proteins in
vivo without requiring GroES, ATP, or the cage-like structure
of multimeric GroEL (Chatellier et al. 1998). Therefore, we
postulate that most chaperonins, including type I and type II,
have chaperone activity in the absence of ATP and without the
formation of the cage-like structure. The eukaryotic CCT complex consists of eight kinds of subunits, and it has been suggested that each subunit has a fixed partner (Liou and Willison
1997). However, the maize a- and e-subunits of CCT proteins
have been detected by Western blot analysis and the expression patterns of these proteins differed according to the prevailing light conditions (Himmelspach et al. 1997). Therefore, the
cytosol may contain incomplete forms of the CCT complex or
monomeric CCT subunits, which play an important role in the
stress-tolerance mechanisms of higher plants. Enhanced saltand osmotic-stress tolerance were observed in BsCCTa transformants of E. coli. Most plant CCT proteins have similar functions because they are highly conserved among the higher
plants. However, such in vivo or in vitro functions have not
been reported previously for CCT subunits. Further analysis of
the CCT subunits will contribute to our understanding of the
salt-tolerance mechanisms of higher plants at the molecular
level.
Acknowledgments
This work was supported in part by the Salt Science Research
Foundation and Grants-in-Aid for Scientific Research (B) to YO (no.
12440224) and for Encouragement of Young Scientists to AY (no.
11740438) from the Ministry of Education, Science, Sports and Culture, Japan.
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(Received February 6, 2002; Accepted May 17, 2002)