Download Conservation of the Cold Shock Domain Protein

Scientific Correspondence
Conservation of the Cold Shock Domain Protein
Family in Plants1
Dale Karlson and Ryozo Imai*
Winter Stress Laboratory, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1,
Toyohira-ku, Sapporo, 062–8555, Japan
In this paper, we report the widespread occurrence
of the nucleic acid-binding cold shock domain (CSD)
in plants and identify the first eukaryotic homologs
that are nearly identical to bacterial cold shock proteins (CSP). Using Arabidopsis as a model system, we
determined that its four unique CSD genes are differentially regulated in response to low temperature.
Prokaryotic response to low temperature has been
extensively studied in Escherichia coli and is accompanied by a spectacular accumulation of nucleic acidbinding CSPs (Graumann and Marahiel, 1998; Yamanaka et al., 1998; Bae et al., 2000). CspA, the most
prominent of the nine-member E. coli CSP family,
accumulates up to 10% of total proteins during cold
stress (Jiang et al., 1997). The three-dimensional
structure of E. coli CspA forms a five-stranded
␤-barrel structure (Newkirk et al., 1994; Schindelin et
al., 1994) that contains two consensus RNA-binding
motifs (RNP1 and RNP2), which facilitate nucleic
acid recognition/binding (Schroder et al., 1995).
CspA has been hypothesized to prevent RNA secondary structure formation (Jiang et al., 1997),
thereby enhancing translation at low temperature.
The CSD, which encompasses bacterial CSPs, is the
most conserved nucleic acid-binding domain and is
capable of binding single-stranded DNA/RNA and
double-stranded DNA (Graumann and Marahiel,
1996). The CSD is proposed to be an ancient structure
that was present before the divergence of prokaryotes and eukaryotes (Graumann and Marahiel,
1998). It is interesting to note that cyanobacteria lack
CSD proteins, however, they contain RNA-binding
domain proteins (RBD; Sato, 1995). RBD proteins are
thought to have evolved a similar three-dimensional
functional surface for nucleic acid binding through
convergent evolution (Graumann and Marahiel, 1996)
and may have replaced CSD proteins (Graumann and
Marahiel, 1998). Bacterial CSP homologs show high
homology to the well-characterized eukaryotic Y-box
proteins (Wolffe, 1993; Bae et al., 2000), which contain
an N-terminal CSD and C-terminal auxiliary domains
1
This work was supported in part by the Ministry of Agriculture, Forestry, and Fisheries (biodesign grant no. 1207) and by the
Science and Technology Agency of Japan (fellowship to D.K.).
* Corresponding author; e-mail [email protected]; fax 81–11– 857–
9382.
www.plantphysiol.org/cgi/doi/10.1104/pp.014472.
12
that facilitate a broad range of in vivo functions such
as RNA masking and transcriptional and translational
regulation (Sommerville, 1999). Surprisingly, within
the plant kingdom, only four proteins are documented
to contain a CSD. Arabidopsis (AtGRP2 and
AtGRP2b), tobacco (Nicotiana tabacum; NtGRP; Kingsley and Palis, 1994), and wheat (Triticum aestivum;
WCSP1; Karlson et al., 2002) contain an N-terminal
CSD in addition to Gly-rich domains that are interspersed by CX2CX4HX4C (CCHC) retroviral-like zinc
fingers. As noted by Guy (1999), Arabidopsis and
tobacco CSD proteins were not studied in any extent
for relation to low temperature or nucleic acid binding. With our recent entry to this class (WCSP1), we
provided the first evidence for cold regulation of a
plant CSD protein and functionally characterized its
nucleic acid-binding activity (Karlson et al., 2002).
In the present study, a comparative (tBLASTn)
GenBank expressed sequence tag (EST) database
search was conducted in an effort to identify novel
plant sequences that contain CSDs. Highly conserved
CSDs were identified within 19 genera that represent
lower plants, monocots, dicots, and woody plants.
Multiple homologs were found within individual
species, which is indicative of small gene families.
ESTs were placed into two groups based upon presence (Type-I) or absence (Type-II) of C-terminal auxiliary domains and multi-aligned with ClustalX software (Fig. 1, A and B, respectively). Because of the
limited number of high-quality sequence data and
incomplete open reading frames (ORFs), only putative amino acid sequences from N-terminal CSDs
were used for multiple sequence alignment and phylogenetic analysis (Type-I; Fig. 1A). It is important to
note that high-quality data from several Type-I ESTs
extended well beyond the CSD, revealing Gly-rich
domains and variable quantities of C-terminal CCHC
zinc fingers (not shown). In Arabidopsis, AtGRP2
(At4g38680) and AtGRP2b (At2g21060) contain two
Gly-rich regions and two CCHC zinc fingers, however, seven CCHC zinc fingers are interspersed within
Gly-rich regions of two undesignated proteins
(At4g36020 and At2g17870). Interestingly, C-terminal
CCHC zinc fingers were not found in the lower plant
EST sequences (Chlamydomonas reinhardtii and Ceratopteris richardii), and their Gly-rich domain composition
appears to be different from higher plant CSD
proteins.
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Scientific Correspondence
Figure 1. Multiple alignment of deduced amino acid sequences of CSD homologs. A, Alignment of Type-I N-terminal CSDs
encoded by EST sequences. Four previously characterized plant CSD proteins (AtGRP2, AtGRP2b, NsGRP2, and WCSP1)
and E. coli CspA are included as references. Note that these ESTs are not complete ORFs and include only N-terminal CSDs.
B, Alignment of Type-II putative amino acid sequences encoded by complete ORFs. Note that these are nearly identical in
size and homology to prokaryotic CSPs. EST sequences are listed with an abbreviated genus name, species name, and
corresponding GenBank accession numbers. The abbreviations and corresponding genera are: A, Arabidopsis; B, Brassica;
C, Ceratopteris; C, Chlamydomonas; G, Gly; G, Gossypium; H, Hordeum; L, Lycopersicum; L, Lotus; M, Medicago; M,
Mesembryanthemum; O, Oryza; P, Pinus; S, Solanum; S, Sorghum; S, Secale; T, Triticum; and Z, Zea. Identical conserved
consensus amino acids are indicated by asterisks, whereas conserved substitutions are indicated by colons and periods.
Consensus regions corresponding to the five ␤-sheets of E. coli CSPs are overlined, and critical core hydrophobic residues
are circled in red. Homology plots are illustrated below multiple alignments.
Of outstanding interest was the discovery that
plants also contain complete ORFs that encode putative CSD proteins that are nearly identical to prokaryotic CSPs in size and sequence (Type-II; Fig. 1B).
Contrary to prokaryotes, all eukaryotic CSD proteins
characterized thus far contain additional C-terminal
auxiliary domains such as Arg-Gly repeats, CCHC
zinc fingers, basic/aromatic islands, and additional
CSDs (Salvetti et al., 1998; Graumann and Marahiel,
Plant Physiol. Vol. 131, 2003
1998; Sommerville, 1999). It is important to note that
the wheat and barley (Hordeum vulgare) ESTs encode
putative proteins solely composed of a CSD. Furthermore, Type-II ESTs were detected only within wheat
and barley and are not within the Arabidopsis
genome.
Previous three-dimensional structural analyses identified residues that are critical for hydrophobic core
formation in E. coli CSP five-stranded ␤-barrel CSD
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Scientific Correspondence
structure (Yamanaka et al., 1998). As designated by
red circles, these residues are almost completely conserved within all identified EST sequences (Fig. 1).
Within Type-I ESTs, Brassica rapa contained a single
exceptional amino acid in the fourth ␤-strand region
where Phe is present instead of Val. A major exception occurred within the third ␤-strand region, where
Leu was present in plant CSDs, whereas, Val is
present in the E. coli consensus (Yamanaka et al.,
1998; Fig. 1A). Because of the hydrophobic nature of
Leu, it is possible that this highly conserved substitution does not compromise three-dimensional structure. Similar to bacteria, Type-II EST sequences contained a conserved Val residue in this same position
(Fig. 1B). Because of the conservation of critical hydrophobic core residues, it is likely that the threedimensional structure is conserved within both Type-I
and II plant CSDs, thereby rendering them competent
for putative nucleic acid-binding functions.
Yamanaka et al. (1998) previously reported that the
loop region between ␤3- and ␤4-strand was the most
diverse among E. coli CSPs and may determine specific in vivo function. Unlike bacteria, eukaryotic ho-
Figure 3. Semiquantitative RT-PCR analysis of four Arabidopsis CSD
genes in response to cold treatment. Total RNA was extracted from
leaves harvested from plants before and subsequent to 4, 12, 24, and
48 h of 4°C treatment and used as template for gene-specific amplification of AtGRP2, AtGRP2b, At4g36020, At2g17870, Cor47, and
AAc1. Inversed images from equally loaded ethidium bromidestained gels revealed that AtGRP2, At4g36020, and At2g17870 increase in response to cold, whereas AtGRP2b is down-regulated in
the same time course. Cor47 and actin 1 (AAc1) were used as
positive controls for low temperature and constitutive responses,
respectively.
Figure 2. Phylogenetic analysis of plant CSD homologs. The multiple alignment was analyzed by ClustalX with a bootstrapped
neighbor-joining method and displayed with TreeViewPPC software.
The phylogenetic tree was rooted with E. coli CspA as the outgroup,
and individual branch lengths indicate evolutionary distance of the
sequences. Two major groupings were detected within plant CSDs:
CSDs that contain additional C-terminal auxiliary domains (Type-I)
and sequences that are composed solely of a CSD (Type-II).
mologs typically contain four additional basic residues within this same region. However, plant CSDs
are moderately conserved between ␤3- and ␤4strands and do not contain additional residues (Fig.
1). This observation is similar to Caenohabditis elegans
LIN-28, a eukaryotic CSD protein that also contains
two C-terminal CCHC zinc fingers (Yamanaka et al.,
1998). Type-I EST sequences showed the highest diversity within the N terminus and within putative
turn regions between ␤-strands 1-2, 2-3, and 4-5, the
significance of which is unknown.
Phylogenetic analysis of the novel plant CSDs revealed general evolutionary trends, where monocots,
dicots, and closely related genera (i.e. Brassica spp./
Arabidopsis) and species (i.e. T. aestivum/Triticum
turgidum) were similarly grouped. Type-II ESTs,
which encode a complete CSD protein, were the most
closely related to bacterial CspA (Fig. 2). Because of
the limitations of EST sequence data, it is critical to
note that the phylogenetic tree was generated as a
comparison of N-terminal CSDs.
Using Arabidopsis as a model plant, we investigated the response of a complete plant CSD gene
family to low temperature stress. Genome data
analysis confirmed that Arabidopsis contains four
unique CSD proteins (AtGRP2-At4g38680, AtGRP2bAt2g21060, At2g17870, and At4g36020). Plants were
grown under continuous illumination in a controlled
growth chamber (25°C) and were sampled before and
4, 12, 24, and 48 h subsequent to their transfer to a
separate pre-equilibrated growth chamber (4°C). Total leaf RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 ␮g was used as a
template for semiquantitative reverse transcriptase
(RT)-PCR as described by (Cheng et al., 2002). Gene-
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Plant Physiol. Vol. 131, 2003
Scientific Correspondence
specific primers were used to amplify individual
genes from synthesized cDNA. Within the tested
time frame of low temperature treatment, the transient increase of At2g17870 was similar to the positive cold-responsive control (Cor47; Gilmour et al.,
1992; Fig. 3). These data contrasted the slower increase of AtGRP2 and At4g36020 and the apparent
down-regulated response of AtGRP2b. Our RT-PCR
data are consistent with E. coli CSPs, where individual CSPs are regulated differentially in response to
low temperature (Yamanaka et al., 1998).
Unlike heat shock, conserved responses to low
temperature stress are largely unknown within prokaryotes and eukaryotes. It is interesting to consider
the structural conservation of CSD proteins within
prokaryotes and eukaryotes and to assess whether
this is because of a convergent role for nucleic acidbinding function or for a similar in vivo functional
role in relation to low temperature stress. Characterization and functional analyses of newly identified
homologs will allow us to assess the importance of
the CSD in plants response to low temperature stress.
Our previous functional analysis of WCSP1 (Karlson
et al., 2002) and the high conservation of critical
amino acids within the CSD ESTs supports the supposition that plant CSDs are capable of binding nucleic acids. The responsiveness of WCSP1 and multi-
Plant Physiol. Vol. 131, 2003
ple Arabidopsis CSD genes to low temperature
support the notion that common mechanisms for
cold adaptation may exist within plants and bacteria.
Received September 10, 2002; returned for revision October 2, 2002; accepted
October 2, 2002.
LITERATURE CITED
Bae W, Xia B, Inouye M, Severinov K (2000) Proc Natl Acad Sci USA 97:
7784–7789
Cheng N, Pittman J, Shigaki T, Hirschi K (2002) Plant Physiol 128:
1245–1254
Gilmour SJ, Artus NN, Thomashow MF (1992) Plant Mol Biol 18: 13–21
Graumann PL, Marahiel MA (1996) BioEssays 18: 309–315
Graumann PL, Marahiel MA (1998) Trends Biochem Sci 23: 286–290
Guy C (1999) J Mol Microbiol Biotechnol 1: 231–242
Jiang W, Hou Y, Inouye M (1997) J Biol Chem 272: 196–202
Karlson D, Nakaminami K, Toyomasu T, Imai R (2002) J Biol Chem 277:
35248–35256
Kingsley PD, Palis J (1994) Plant Cell 6: 1522–1523
Newkirk K, Feng W, Jiang W, Tejero R, Emerson SD, Inouye M, Montelione GT (1994) Proc Natl Acad Sci USA 91: 5114–5118
Salvetti A, Batistoni R, Deri P, Rossi L, Sommerville J (1998) Dev Biol 201:
217–229
Sato N (1995) Nucleic Acids Res 23: 2161–2167
Schindelin H, Jiang W, Inouye M, Heinemann U (1994) Proc Natl Acad Sci
USA 91: 5119–5123
Schroder K, Graumann P, Schnuchet A, Holak TA, Marahiel MA (1995)
Mol Microbiol 16: 699–708
Sommerville J (1999) Bioessays 21: 319–325
Wolffe AP (1993) Bioessays 16: 245–251
Yamanaka K, Fang L, Inouye M (1998) Mol Microbiol 27: 247–255
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Copyright © 2003 American Society of Plant Biologists. All rights reserved.
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