Cold Shock Domain Protein 3 Regulates Freezing Tolerance in Arabidopsis thaliana
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.025791
ISSN1083-351X
AutoresMyung‐Hee Kim, Kentaro Sasaki, Ryozo Imai,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoIn response to cold, Escherichia coli produces cold shock proteins (CSPs) that have essential roles in cold adaptation as RNA chaperones. Here, we demonstrate that Arabidopsis cold shock domain protein 3 (AtCSP3), which shares a cold shock domain with bacterial CSPs, is involved in the acquisition of freezing tolerance in plants. AtCSP3 complemented a cold-sensitive phenotype of the E. coli CSP quadruple mutant and displayed nucleic acid duplex melting activity, suggesting that AtCSP3 also functions as an RNA chaperone. Promoter-GUS transgenic plants revealed tissue-specific expression of AtCSP3 in shoot and root apical regions. When exposed to low temperature, GUS activity was extensively induced in a broader region of the roots. In transgenic plants expressing an AtCSP3-GFP fusion, GFP signals were detected in both the nucleus and cytoplasm. An AtCSP3 knock-out mutant (atcsp3-2) was sensitive to freezing compared with wild-type plants under non-acclimated and cold-acclimated conditions, whereas expression of C-repeat-binding factors and their downstream genes during cold acclimation was not altered in the atcsp3-2 mutant. Overexpression of AtCSP3 in transgenic plants conferred enhanced freezing tolerance over wild-type plants. Together, the data demonstrated an essential role of RNA chaperones for cold adaptation in higher plants. In response to cold, Escherichia coli produces cold shock proteins (CSPs) that have essential roles in cold adaptation as RNA chaperones. Here, we demonstrate that Arabidopsis cold shock domain protein 3 (AtCSP3), which shares a cold shock domain with bacterial CSPs, is involved in the acquisition of freezing tolerance in plants. AtCSP3 complemented a cold-sensitive phenotype of the E. coli CSP quadruple mutant and displayed nucleic acid duplex melting activity, suggesting that AtCSP3 also functions as an RNA chaperone. Promoter-GUS transgenic plants revealed tissue-specific expression of AtCSP3 in shoot and root apical regions. When exposed to low temperature, GUS activity was extensively induced in a broader region of the roots. In transgenic plants expressing an AtCSP3-GFP fusion, GFP signals were detected in both the nucleus and cytoplasm. An AtCSP3 knock-out mutant (atcsp3-2) was sensitive to freezing compared with wild-type plants under non-acclimated and cold-acclimated conditions, whereas expression of C-repeat-binding factors and their downstream genes during cold acclimation was not altered in the atcsp3-2 mutant. Overexpression of AtCSP3 in transgenic plants conferred enhanced freezing tolerance over wild-type plants. Together, the data demonstrated an essential role of RNA chaperones for cold adaptation in higher plants. Many plant species acquire substantial freezing tolerance after exposure to low but non-freezing temperatures, a process known as cold acclimation (1.Sakai A. Larcher W. Frost Survival of Plants: Responses and Adaptations to Freezing Stress. Springer-Verlag, New York1987Crossref Google Scholar). Cold acclimation (CA) 2The abbreviations used are: CAcold acclimationCORcold-regulatedCAcold acclimationCSPcold shock proteinCSDcold shock domainGUSβ-glucuronidaseMHCmajor histocompatibility complexmRNPmessenger ribonucleoprotein complexNAnon-acclimationWTwild typeX-gluc5-bromo-4-chloro-3-indolyl-β-d-glucuronideGSTglutathione S-transferaseGFPgreen fluorescent protein.2The abbreviations used are: CAcold acclimationCORcold-regulatedCAcold acclimationCSPcold shock proteinCSDcold shock domainGUSβ-glucuronidaseMHCmajor histocompatibility complexmRNPmessenger ribonucleoprotein complexNAnon-acclimationWTwild typeX-gluc5-bromo-4-chloro-3-indolyl-β-d-glucuronideGSTglutathione S-transferaseGFPgreen fluorescent protein. induces cellular and physiological changes including alteration in gene expression (2.Guy C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 187-223Crossref Scopus (1139) Google Scholar). Cold-regulated (COR) genes are highly expressed during cold acclimation and contribute substantially to acquiring freezing tolerance. The C-repeat-binding factors (CBF) or dehydration responsive element-binding protein 1 (DREB1) have been identified as transcription activators for COR gene expression (3.Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2342) Google Scholar, 4.Jaglo-Ottosen K.R. Gilmour S.J. Zarka D.G. Schabenberger O. Thomashow M.F. Science. 1998; 280: 104-106Crossref PubMed Scopus (1355) Google Scholar). They play a key role in the signal transduction pathway for cold acclimation and ectopic expression of CBF genes in plants confers tolerance against freezing and other related stresses (3.Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2342) Google Scholar, 4.Jaglo-Ottosen K.R. Gilmour S.J. Zarka D.G. Schabenberger O. Thomashow M.F. Science. 1998; 280: 104-106Crossref PubMed Scopus (1355) Google Scholar). In addition to the major CBF-dependent pathway, CBF-independent pathways are also thought to be necessary for cold acclimation (5.Xin Z. Browse J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7799-7804Crossref PubMed Scopus (332) Google Scholar, 6.Zhu J. Shi H. Lee B.H. Damsz B. Cheng S. Stirm V. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9873-9878Crossref PubMed Scopus (201) Google Scholar, 7.Zhu J. Verslues P.E. Zheng X. Lee B.H. Zhan X. Manabe Y. Sokolchik I. Zhu Y. Dong C.H. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9966-9971Crossref PubMed Scopus (155) Google Scholar). Elucidating the role of these non-CBF pathways is thus required to fully understand cold acclimation mechanisms in plants.Cold acclimation is also observed in diverse organisms including bacteria, where it is known as the cold shock response. The major cold shock protein (CSP) in Escherichia coli, CspA, is dramatically induced immediately following a temperature downshift and accumulates to represent up to 10% of total soluble protein (8.Phadtare S. Tyagi S. Inouye M. Severinov K. J. Biol. Chem. 2002; 277: 46706-46711Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9.Phadtare S. Alsina J. Inouye M. Curr. Opin Microbiol. 1999; 2: 199-215Crossref Scopus (271) Google Scholar). Nine members of the CSP gene family (cspA to cspI) have been identified in E. coli, and four of these have been shown to be induced by cold shock (10.Yamanaka K. Fang L. Inouye M. Mol. Microbiol. 1998; 27: 247-255Crossref PubMed Scopus (273) Google Scholar). The three-dimensional structure of CSP proteins consists of a closed five-stranded anti-parallel β-barrel capped by a long flexible loop and contains two consensus RNA-binding motifs (RNP1 and RNP2) that contribute to bind nucleic acids (8.Phadtare S. Tyagi S. Inouye M. Severinov K. J. Biol. Chem. 2002; 277: 46706-46711Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11.Weber M.H. Fricke I. Doll N. Marahiel M.A. Nucleic Acids Res. 2002; 30: 375-378Crossref PubMed Scopus (16) Google Scholar, 12.Kloks C.P. Spronk C.A. Lasonder E. Hoffmann A. Vuister G.W. Grzesiek S. Hilbers C.W. J. Mol. Biol. 2002; 316: 317-326Crossref PubMed Scopus (105) Google Scholar). CSPs can destabilize the secondary structures in RNA and thus function as RNA chaperones to regulate transcription and translation in bacteria (11.Weber M.H. Fricke I. Doll N. Marahiel M.A. Nucleic Acids Res. 2002; 30: 375-378Crossref PubMed Scopus (16) Google Scholar, 13.Graumann P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Bacterial CSPs are considered to be the most ancient form of the RNA binding protein, which is also found in eukaryote proteins as a RNA-binding domain called the cold shock domain (CSD) (13.Graumann P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar).Among the most widely studied eukaryotic CSD proteins is the Y-Box protein family. All vertebrate Y-box proteins contain a variable N-terminal domain, cold shock domain, and a C-terminal auxiliary domain. Y-box protein was originally identified as a protein binding to the Y-box sequence (CTGATTGG) of the major histocompatibility complex (MHC) class II gene promoter (14.Didier D.K. Schiffenbauer J. Woulfe S.L. Zacheis M. Schwartz B.D. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 7322-7326Crossref PubMed Scopus (356) Google Scholar). Subsequent studies have shown that Y-box proteins are transcription factors that can negatively or positively regulate gene expression in several genes (15.Higashi K. Inagaki Y. Suzuki N. Mitsui S. Mauviel A. Kaneko H. Nakatsuka I. J. Biol. Chem. 2003; 278: 5156-5162Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16.Lasham A. Moloney S. Hale T. Homer C. Zhang Y.F. Murison J.G. Braithwaite A.W. Watson J. J. Biol. Chem. 2003; 278: 35516-35523Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Despite the function as a transcription factor, the majority of the human Y-box protein YB-1 is found in the cytosol as part of the messenger ribonucleoprotein complex (mRNP). YB-1 stimulates or inhibits translation depending on the YB-1/mRNA ratio (17.Nekrasov M.P. Ivshina M.P. Chernov K.G. Kovrigina E.A. Evdokimova V.M. Thomas A.A. Hershey J.W. Ovchinnikov L.P. J. Biol. Chem. 2003; 278: 13936-13943Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Pisarev A.V. Skabkin M.A. Thomas A.A. Merrick W.C. Ovchinnikov L.P. Shatsky I.N. J. Biol. Chem. 2002; 277: 15445-15451Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Another class of eukaryotic CSD protein is the newly emerging LIN28 family. Initially identified as a heterochronic gene in Caenorhabditis elegans (19.Moss E.G. Lee R.C. Ambros V. Cell. 1997; 88: 637-646Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar), LIN28 is now known to function in the enhancement of translation (20.Polesskaya A. Cuvellier S. Naguibneva I. Duquet A. Moss E.G. Harel-Bellan A. Genes Dev. 2007; 21: 1125-1138Crossref PubMed Scopus (237) Google Scholar), biogenesis of miRNA (21.Viswanathan S.R. Daley G.Q. Gregory R.I. Science. 2008; 320: 97-100Crossref PubMed Scopus (1172) Google Scholar), and generation of induced pluripotent stem cells (22.Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar).Highly conserved CSD is also found in a diverse genera of lower and higher plants (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). In wheat, the wheat cold shock protein 1 (WCSP1) accumulates in crown tissue during cold acclimation (24.Karlson D. Nakaminami K. Toyomasu T. Imai R. J. Biol. Chem. 2002; 277: 35248-35256Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and is composed of a CSD and a glycine-rich domain containing three CCHC zinc fingers (Fig. 1A). WCSP1 displays activity to bind ssDNA, dsDNA, and RNA, and unwinds nucleic acid duplexes (24.Karlson D. Nakaminami K. Toyomasu T. Imai R. J. Biol. Chem. 2002; 277: 35248-35256Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 25.Nakaminami K. Sasaki K. Kajita S. Takeda H. Karlson D. Ohgi K. Imai R. FEBS Lett. 2005; 579: 4887-4891Crossref PubMed Scopus (34) Google Scholar, 26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar). Heterologous expression of WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold sensitive phenotype. WCSP1 was also demonstrated to have transcriptional anti-termination activity in E. coli (26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar). These studies indicated that WCSP1 functions as a RNA chaperone to destabilize RNA secondary structures. However, the detailed functions of WCSP1 in planta remain to be elucidated.Arabidopsis thaliana has four CSD proteins that displayed differential regulation in response to low temperature (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). Two of these proteins (AtGRP2/AtCSP2/At4g38680 and AtGRP2b/AtCSP4/At2g21060) contain two CCHC zinc fingers and the other two (AtCSP1/At4g36020 and AtCSP3/At2g17870) contain seven CCHC zinc fingers within the glycine-rich region (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). AtCSP2 has been subject to further characterization (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar, 29.Kim J.S. Park S.J. Kwak K.J. Kim Y.O. Kim J.Y. Song J. Jang B. Jung C.H. Kang H. Nucleic Acids Res. 2007; 35: 506-516Crossref PubMed Scopus (196) Google Scholar) and shown to unwind a nucleic acid duplex and partially complement the E. coli cspA, cspB, cspE, cspG quadruple deletion mutant (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). AtCSP2 is regulated by developmental cues, as well as low temperature (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar), and is possibly involved in flowering time control (28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar). AtCSP2 mRNA (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar) and protein levels (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar) increased during cold acclimation, and localization of AtCSP2::GFP was shown to be in the nucleolus and cytoplasm (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar).To elucidate the regulatory mechanism of Arabidopsis CSD proteins during cold acclimation, we have chosen the Arabidopsis AtCSP3 (At2g17870) protein for further characterization. AtCSP3 was shown to function as an RNA chaperone, sharing this biochemical function with bacterial CSPs and wheat WCSP1. In vivo functional analyses with overexpressors and a knock-out mutant as well as expression analyses indicate that AtCSP3 regulates freezing tolerance in Arabidopsis during cold acclimation independent of the CBF/DREB1 pathway.DiscussionCold shock domain proteins have been identified in a variety of organisms ranging from bacteria to mammals. In higher plants, cold shock domain proteins are involved in the cold response and share conserved functions with bacterial CSPs (26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar, 27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). In the current study, we have shown that the Arabidopsis AtCSP3 functions as a RNA chaperone and is involved in the acquisition of freezing tolerance.Expression analysis indicated that the level of AtCSP3 transcripts increased in response to cold in both shoots and roots (Fig. 2, A and B), with a maximum level of expression at around 12 h of cold treatment. The fact that AtCSP3 expression was not modulated by drought, NaCl, or ABA (data not shown), suggested that AtCSP3 function is mainly associated with cold stress. The spatial expression pattern of AtCSP3 was characterized using a reporter gene fusion construct (Fig. 2B and supplemental Fig. S2). AtCSP3 promoter-GUS expression was limited to shoot and root apical regions of vegetative plants, which are considered to be tissues that primarily sense environmental cues. The shoot apical meristem was reported to sense low temperature signals during vernalization (36.Schwabe W.W. J. Exp. Bot. 1954; 5: 389-400Crossref Scopus (20) Google Scholar, 37.Curtis O.F. Chang H.T. Amer. J. Bot. 1930; 17: 1047-1048Google Scholar), whereas root tips are also known to sense environmental signals such as gravity and water availability (38.Takahashi H. J. Plant Res. 1997; 110: 163-169Crossref PubMed Google Scholar). In response to cold, GUS and GFP expression was extended to a broader region of root (Fig. 2B, and supplemental Fig. S2K). Because root growth was inhibited during cold treatment, this extension was not due to cell division of GUS-expressing cells. It is thus plausible that a signal from root tip is transduced toward the basal part of root to induce expression of AtCSP3.Transgenic expression of AtCSP3-GFP driven by the native AtCSP3 promoter revealed that AtCSP3 was localized to both the nucleus and cytoplasm (Fig. 2C and supplemental Fig. S2K). The nuclear and cytoplasmic localization is consistent with a role for AtCSP3 involving interaction with mRNA. Arabidopsis LOS4 encodes a DEAD-box RNA helicase and is required for efficient export of RNA from the nucleus to the cytoplasm (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar). LOS4 also localizes to the nucleus and cytoplasm, and might be important for nuclear pore remodeling under cold temperatures (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar, 40.Gong Z. Dong C.H. Lee H. Zhu J. Xiong L. Gong D. Stevenson B. Zhu J.K. Plant Cell. 2005; 17: 256-267Crossref PubMed Scopus (272) Google Scholar). RNA helicases and RNA chaperones are involved in various steps of RNA metabolism (41.Sommerville J. Bioessays. 1999; 21: 319-325Crossref PubMed Scopus (144) Google Scholar). In Bacillus subtilis, CspB physically interacts with cold-induced DEAD-box RNA helicases, CshA and CshB (42.Hunger K. Beckering C.L. Wiegeshoff F. Graumann P.L. Marahiel M.A. J. Bacteriol. 2006; 188: 240-248Crossref PubMed Scopus (92) Google Scholar). It will be interesting to determine if plant CSD proteins interact with RNA helicases.The atcsp3-2 mutant plant was more sensitive to freezing than wild type under both NA and CA conditions (Fig. 3, D–F). A change in freezing tolerance may reflect altered expression of cold-regulated genes, however, expression analysis of CBFs and CBF regulon genes indicated that the CBF pathway is functioning normally in atcsp3-2 (Fig. 4). On the other hand, several genes were identified that were down-regulated in atcsp3-2. Most of these down-regulated genes in atcsp3-2 have been linked to stress responses (supplemental Table S1 and Fig. 5); however, it is not yet clear how they are related or their potential mechanistic role in freezing tolerance. It is interesting to note that six of the down-regulated genes in atcsp3-2 are known to be up-regulated in the ada2b-1 mutant (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar). ADA2 is a histone acetyltransferase with a putative transcriptional adaptor function (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar, 44.Stockinger E.J. Mao Y. Regier M.K. Triezenberg S.J. Thomashow M.F. Nucleic Acids Res. 2001; 29: 1524-1533Crossref PubMed Scopus (214) Google Scholar). The ada2b-1 mutant is constitutively more freezing tolerant than wild-type plants without overexpressing COR genes (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar), suggesting there may be a cross-talk between the AtCSP3 and ADA2. Current information suggests that AtCSP3 controls the expression of genes that are necessary for freezing tolerance but are not CBF-regulated genes. Molecular genetic analyses of several mutants such as esk1 and hos9 have indicated a significant role for such CBF-independent pathways in freezing tolerance in Arabidopsis (5.Xin Z. Browse J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7799-7804Crossref PubMed Scopus (332) Google Scholar, 6.Zhu J. Shi H. Lee B.H. Damsz B. Cheng S. Stirm V. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9873-9878Crossref PubMed Scopus (201) Google Scholar, 45.Xin Z. Mandaokar A. Chen J. Last R.L. Browse J. Plant J. 2007; 49: 786-799Crossref PubMed Scopus (113) Google Scholar).The biochemical activity of AtCSP3 is very similar to that of WCSP1, unwinding dsDNA, and binding RNA and DNA (supplemental Fig. S1). In addition, AtCSP3 complemented the E. coli csp mutant (Fig. 1B). These data suggested that AtCSP3 functions as RNA chaperone in vivo. AtCSP3 may thus act to enhance translation of bulk or specific mRNA important for freezing tolerance by destabilizing RNA duplex produced under low temperature conditions. Another possibility is that AtCSP3 regulates mRNA stability by mediating RNA duplex formation, which can stabilize mRNA from exonucleolytic degradation. In bacterial systems, RNA chaperones regulate gene expression at both the transcription and post-transcription levels. Our data, together with the recent finding that bacterial CSPs can confer stress tolerance in plants (46.Castiglioni P. Warner D. Bensen R.J. Anstrom D.C. Harrison J. Stoecker M. Abad M. Kumar G. Salvador S. D'Ordine R. Navarro S. Back S. Fernandes M. Targolli J. Dasgupta S. Bonin C. Luethy M.H. Heard J.E. Plant Physiol. 2008; 147: 446-455Crossref PubMed Scopus (323) Google Scholar), support a functional conservation of plant and bacterial CSD proteins in acquiring stress tolerance. Many plant species acquire substantial freezing tolerance after exposure to low but non-freezing temperatures, a process known as cold acclimation (1.Sakai A. Larcher W. Frost Survival of Plants: Responses and Adaptations to Freezing Stress. Springer-Verlag, New York1987Crossref Google Scholar). Cold acclimation (CA) 2The abbreviations used are: CAcold acclimationCORcold-regulatedCAcold acclimationCSPcold shock proteinCSDcold shock domainGUSβ-glucuronidaseMHCmajor histocompatibility complexmRNPmessenger ribonucleoprotein complexNAnon-acclimationWTwild typeX-gluc5-bromo-4-chloro-3-indolyl-β-d-glucuronideGSTglutathione S-transferaseGFPgreen fluorescent protein.2The abbreviations used are: CAcold acclimationCORcold-regulatedCAcold acclimationCSPcold shock proteinCSDcold shock domainGUSβ-glucuronidaseMHCmajor histocompatibility complexmRNPmessenger ribonucleoprotein complexNAnon-acclimationWTwild typeX-gluc5-bromo-4-chloro-3-indolyl-β-d-glucuronideGSTglutathione S-transferaseGFPgreen fluorescent protein. induces cellular and physiological changes including alteration in gene expression (2.Guy C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 187-223Crossref Scopus (1139) Google Scholar). Cold-regulated (COR) genes are highly expressed during cold acclimation and contribute substantially to acquiring freezing tolerance. The C-repeat-binding factors (CBF) or dehydration responsive element-binding protein 1 (DREB1) have been identified as transcription activators for COR gene expression (3.Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2342) Google Scholar, 4.Jaglo-Ottosen K.R. Gilmour S.J. Zarka D.G. Schabenberger O. Thomashow M.F. Science. 1998; 280: 104-106Crossref PubMed Scopus (1355) Google Scholar). They play a key role in the signal transduction pathway for cold acclimation and ectopic expression of CBF genes in plants confers tolerance against freezing and other related stresses (3.Liu Q. Kasuga M. Sakuma Y. Abe H. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2342) Google Scholar, 4.Jaglo-Ottosen K.R. Gilmour S.J. Zarka D.G. Schabenberger O. Thomashow M.F. Science. 1998; 280: 104-106Crossref PubMed Scopus (1355) Google Scholar). In addition to the major CBF-dependent pathway, CBF-independent pathways are also thought to be necessary for cold acclimation (5.Xin Z. Browse J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7799-7804Crossref PubMed Scopus (332) Google Scholar, 6.Zhu J. Shi H. Lee B.H. Damsz B. Cheng S. Stirm V. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9873-9878Crossref PubMed Scopus (201) Google Scholar, 7.Zhu J. Verslues P.E. Zheng X. Lee B.H. Zhan X. Manabe Y. Sokolchik I. Zhu Y. Dong C.H. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9966-9971Crossref PubMed Scopus (155) Google Scholar). Elucidating the role of these non-CBF pathways is thus required to fully understand cold acclimation mechanisms in plants. cold acclimation cold-regulated cold acclimation cold shock protein cold shock domain β-glucuronidase major histocompatibility complex messenger ribonucleoprotein complex non-acclimation wild type 5-bromo-4-chloro-3-indolyl-β-d-glucuronide glutathione S-transferase green fluorescent protein. cold acclimation cold-regulated cold acclimation cold shock protein cold shock domain β-glucuronidase major histocompatibility complex messenger ribonucleoprotein complex non-acclimation wild type 5-bromo-4-chloro-3-indolyl-β-d-glucuronide glutathione S-transferase green fluorescent protein. Cold acclimation is also observed in diverse organisms including bacteria, where it is known as the cold shock response. The major cold shock protein (CSP) in Escherichia coli, CspA, is dramatically induced immediately following a temperature downshift and accumulates to represent up to 10% of total soluble protein (8.Phadtare S. Tyagi S. Inouye M. Severinov K. J. Biol. Chem. 2002; 277: 46706-46711Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9.Phadtare S. Alsina J. Inouye M. Curr. Opin Microbiol. 1999; 2: 199-215Crossref Scopus (271) Google Scholar). Nine members of the CSP gene family (cspA to cspI) have been identified in E. coli, and four of these have been shown to be induced by cold shock (10.Yamanaka K. Fang L. Inouye M. Mol. Microbiol. 1998; 27: 247-255Crossref PubMed Scopus (273) Google Scholar). The three-dimensional structure of CSP proteins consists of a closed five-stranded anti-parallel β-barrel capped by a long flexible loop and contains two consensus RNA-binding motifs (RNP1 and RNP2) that contribute to bind nucleic acids (8.Phadtare S. Tyagi S. Inouye M. Severinov K. J. Biol. Chem. 2002; 277: 46706-46711Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11.Weber M.H. Fricke I. Doll N. Marahiel M.A. Nucleic Acids Res. 2002; 30: 375-378Crossref PubMed Scopus (16) Google Scholar, 12.Kloks C.P. Spronk C.A. Lasonder E. Hoffmann A. Vuister G.W. Grzesiek S. Hilbers C.W. J. Mol. Biol. 2002; 316: 317-326Crossref PubMed Scopus (105) Google Scholar). CSPs can destabilize the secondary structures in RNA and thus function as RNA chaperones to regulate transcription and translation in bacteria (11.Weber M.H. Fricke I. Doll N. Marahiel M.A. Nucleic Acids Res. 2002; 30: 375-378Crossref PubMed Scopus (16) Google Scholar, 13.Graumann P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Bacterial CSPs are considered to be the most ancient form of the RNA binding protein, which is also found in eukaryote proteins as a RNA-binding domain called the cold shock domain (CSD) (13.Graumann P.L. Marahiel M.A. Trends Biochem. Sci. 1998; 23: 286-290Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Among the most widely studied eukaryotic CSD proteins is the Y-Box protein family. All vertebrate Y-box proteins contain a variable N-terminal domain, cold shock domain, and a C-terminal auxiliary domain. Y-box protein was originally identified as a protein binding to the Y-box sequence (CTGATTGG) of the major histocompatibility complex (MHC) class II gene promoter (14.Didier D.K. Schiffenbauer J. Woulfe S.L. Zacheis M. Schwartz B.D. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 7322-7326Crossref PubMed Scopus (356) Google Scholar). Subsequent studies have shown that Y-box proteins are transcription factors that can negatively or positively regulate gene expression in several genes (15.Higashi K. Inagaki Y. Suzuki N. Mitsui S. Mauviel A. Kaneko H. Nakatsuka I. J. Biol. Chem. 2003; 278: 5156-5162Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16.Lasham A. Moloney S. Hale T. Homer C. Zhang Y.F. Murison J.G. Braithwaite A.W. Watson J. J. Biol. Chem. 2003; 278: 35516-35523Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Despite the function as a transcription factor, the majority of the human Y-box protein YB-1 is found in the cytosol as part of the messenger ribonucleoprotein complex (mRNP). YB-1 stimulates or inhibits translation depending on the YB-1/mRNA ratio (17.Nekrasov M.P. Ivshina M.P. Chernov K.G. Kovrigina E.A. Evdokimova V.M. Thomas A.A. Hershey J.W. Ovchinnikov L.P. J. Biol. Chem. 2003; 278: 13936-13943Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18.Pisarev A.V. Skabkin M.A. Thomas A.A. Merrick W.C. Ovchinnikov L.P. Shatsky I.N. J. Biol. Chem. 2002; 277: 15445-15451Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Another class of eukaryotic CSD protein is the newly emerging LIN28 family. Initially identified as a heterochronic gene in Caenorhabditis elegans (19.Moss E.G. Lee R.C. Ambros V. Cell. 1997; 88: 637-646Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar), LIN28 is now known to function in the enhancement of translation (20.Polesskaya A. Cuvellier S. Naguibneva I. Duquet A. Moss E.G. Harel-Bellan A. Genes Dev. 2007; 21: 1125-1138Crossref PubMed Scopus (237) Google Scholar), biogenesis of miRNA (21.Viswanathan S.R. Daley G.Q. Gregory R.I. Science. 2008; 320: 97-100Crossref PubMed Scopus (1172) Google Scholar), and generation of induced pluripotent stem cells (22.Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar). Highly conserved CSD is also found in a diverse genera of lower and higher plants (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). In wheat, the wheat cold shock protein 1 (WCSP1) accumulates in crown tissue during cold acclimation (24.Karlson D. Nakaminami K. Toyomasu T. Imai R. J. Biol. Chem. 2002; 277: 35248-35256Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and is composed of a CSD and a glycine-rich domain containing three CCHC zinc fingers (Fig. 1A). WCSP1 displays activity to bind ssDNA, dsDNA, and RNA, and unwinds nucleic acid duplexes (24.Karlson D. Nakaminami K. Toyomasu T. Imai R. J. Biol. Chem. 2002; 277: 35248-35256Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 25.Nakaminami K. Sasaki K. Kajita S. Takeda H. Karlson D. Ohgi K. Imai R. FEBS Lett. 2005; 579: 4887-4891Crossref PubMed Scopus (34) Google Scholar, 26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar). Heterologous expression of WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold sensitive phenotype. WCSP1 was also demonstrated to have transcriptional anti-termination activity in E. coli (26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar). These studies indicated that WCSP1 functions as a RNA chaperone to destabilize RNA secondary structures. However, the detailed functions of WCSP1 in planta remain to be elucidated. Arabidopsis thaliana has four CSD proteins that displayed differential regulation in response to low temperature (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). Two of these proteins (AtGRP2/AtCSP2/At4g38680 and AtGRP2b/AtCSP4/At2g21060) contain two CCHC zinc fingers and the other two (AtCSP1/At4g36020 and AtCSP3/At2g17870) contain seven CCHC zinc fingers within the glycine-rich region (23.Karlson D. Imai R. Plant Physiol. 2003; 131: 12-15Crossref PubMed Scopus (108) Google Scholar). AtCSP2 has been subject to further characterization (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar, 29.Kim J.S. Park S.J. Kwak K.J. Kim Y.O. Kim J.Y. Song J. Jang B. Jung C.H. Kang H. Nucleic Acids Res. 2007; 35: 506-516Crossref PubMed Scopus (196) Google Scholar) and shown to unwind a nucleic acid duplex and partially complement the E. coli cspA, cspB, cspE, cspG quadruple deletion mutant (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). AtCSP2 is regulated by developmental cues, as well as low temperature (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar), and is possibly involved in flowering time control (28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar). AtCSP2 mRNA (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar, 28.Fusaro A.F. Bocca S.N. Ramos R.L. Barrôco R.M. Magioli C. Jorge V.C. Coutinho T.C. Rangel-Lima C.M. De Rycke R. Inzé D. Engler G. Sachetto-Martins G. Planta. 2007; 225: 1339-1351Crossref PubMed Scopus (89) Google Scholar) and protein levels (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar) increased during cold acclimation, and localization of AtCSP2::GFP was shown to be in the nucleolus and cytoplasm (27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). To elucidate the regulatory mechanism of Arabidopsis CSD proteins during cold acclimation, we have chosen the Arabidopsis AtCSP3 (At2g17870) protein for further characterization. AtCSP3 was shown to function as an RNA chaperone, sharing this biochemical function with bacterial CSPs and wheat WCSP1. In vivo functional analyses with overexpressors and a knock-out mutant as well as expression analyses indicate that AtCSP3 regulates freezing tolerance in Arabidopsis during cold acclimation independent of the CBF/DREB1 pathway. DiscussionCold shock domain proteins have been identified in a variety of organisms ranging from bacteria to mammals. In higher plants, cold shock domain proteins are involved in the cold response and share conserved functions with bacterial CSPs (26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar, 27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). In the current study, we have shown that the Arabidopsis AtCSP3 functions as a RNA chaperone and is involved in the acquisition of freezing tolerance.Expression analysis indicated that the level of AtCSP3 transcripts increased in response to cold in both shoots and roots (Fig. 2, A and B), with a maximum level of expression at around 12 h of cold treatment. The fact that AtCSP3 expression was not modulated by drought, NaCl, or ABA (data not shown), suggested that AtCSP3 function is mainly associated with cold stress. The spatial expression pattern of AtCSP3 was characterized using a reporter gene fusion construct (Fig. 2B and supplemental Fig. S2). AtCSP3 promoter-GUS expression was limited to shoot and root apical regions of vegetative plants, which are considered to be tissues that primarily sense environmental cues. The shoot apical meristem was reported to sense low temperature signals during vernalization (36.Schwabe W.W. J. Exp. Bot. 1954; 5: 389-400Crossref Scopus (20) Google Scholar, 37.Curtis O.F. Chang H.T. Amer. J. Bot. 1930; 17: 1047-1048Google Scholar), whereas root tips are also known to sense environmental signals such as gravity and water availability (38.Takahashi H. J. Plant Res. 1997; 110: 163-169Crossref PubMed Google Scholar). In response to cold, GUS and GFP expression was extended to a broader region of root (Fig. 2B, and supplemental Fig. S2K). Because root growth was inhibited during cold treatment, this extension was not due to cell division of GUS-expressing cells. It is thus plausible that a signal from root tip is transduced toward the basal part of root to induce expression of AtCSP3.Transgenic expression of AtCSP3-GFP driven by the native AtCSP3 promoter revealed that AtCSP3 was localized to both the nucleus and cytoplasm (Fig. 2C and supplemental Fig. S2K). The nuclear and cytoplasmic localization is consistent with a role for AtCSP3 involving interaction with mRNA. Arabidopsis LOS4 encodes a DEAD-box RNA helicase and is required for efficient export of RNA from the nucleus to the cytoplasm (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar). LOS4 also localizes to the nucleus and cytoplasm, and might be important for nuclear pore remodeling under cold temperatures (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar, 40.Gong Z. Dong C.H. Lee H. Zhu J. Xiong L. Gong D. Stevenson B. Zhu J.K. Plant Cell. 2005; 17: 256-267Crossref PubMed Scopus (272) Google Scholar). RNA helicases and RNA chaperones are involved in various steps of RNA metabolism (41.Sommerville J. Bioessays. 1999; 21: 319-325Crossref PubMed Scopus (144) Google Scholar). In Bacillus subtilis, CspB physically interacts with cold-induced DEAD-box RNA helicases, CshA and CshB (42.Hunger K. Beckering C.L. Wiegeshoff F. Graumann P.L. Marahiel M.A. J. Bacteriol. 2006; 188: 240-248Crossref PubMed Scopus (92) Google Scholar). It will be interesting to determine if plant CSD proteins interact with RNA helicases.The atcsp3-2 mutant plant was more sensitive to freezing than wild type under both NA and CA conditions (Fig. 3, D–F). A change in freezing tolerance may reflect altered expression of cold-regulated genes, however, expression analysis of CBFs and CBF regulon genes indicated that the CBF pathway is functioning normally in atcsp3-2 (Fig. 4). On the other hand, several genes were identified that were down-regulated in atcsp3-2. Most of these down-regulated genes in atcsp3-2 have been linked to stress responses (supplemental Table S1 and Fig. 5); however, it is not yet clear how they are related or their potential mechanistic role in freezing tolerance. It is interesting to note that six of the down-regulated genes in atcsp3-2 are known to be up-regulated in the ada2b-1 mutant (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar). ADA2 is a histone acetyltransferase with a putative transcriptional adaptor function (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar, 44.Stockinger E.J. Mao Y. Regier M.K. Triezenberg S.J. Thomashow M.F. Nucleic Acids Res. 2001; 29: 1524-1533Crossref PubMed Scopus (214) Google Scholar). The ada2b-1 mutant is constitutively more freezing tolerant than wild-type plants without overexpressing COR genes (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar), suggesting there may be a cross-talk between the AtCSP3 and ADA2. Current information suggests that AtCSP3 controls the expression of genes that are necessary for freezing tolerance but are not CBF-regulated genes. Molecular genetic analyses of several mutants such as esk1 and hos9 have indicated a significant role for such CBF-independent pathways in freezing tolerance in Arabidopsis (5.Xin Z. Browse J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7799-7804Crossref PubMed Scopus (332) Google Scholar, 6.Zhu J. Shi H. Lee B.H. Damsz B. Cheng S. Stirm V. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9873-9878Crossref PubMed Scopus (201) Google Scholar, 45.Xin Z. Mandaokar A. Chen J. Last R.L. Browse J. Plant J. 2007; 49: 786-799Crossref PubMed Scopus (113) Google Scholar).The biochemical activity of AtCSP3 is very similar to that of WCSP1, unwinding dsDNA, and binding RNA and DNA (supplemental Fig. S1). In addition, AtCSP3 complemented the E. coli csp mutant (Fig. 1B). These data suggested that AtCSP3 functions as RNA chaperone in vivo. AtCSP3 may thus act to enhance translation of bulk or specific mRNA important for freezing tolerance by destabilizing RNA duplex produced under low temperature conditions. Another possibility is that AtCSP3 regulates mRNA stability by mediating RNA duplex formation, which can stabilize mRNA from exonucleolytic degradation. In bacterial systems, RNA chaperones regulate gene expression at both the transcription and post-transcription levels. Our data, together with the recent finding that bacterial CSPs can confer stress tolerance in plants (46.Castiglioni P. Warner D. Bensen R.J. Anstrom D.C. Harrison J. Stoecker M. Abad M. Kumar G. Salvador S. D'Ordine R. Navarro S. Back S. Fernandes M. Targolli J. Dasgupta S. Bonin C. Luethy M.H. Heard J.E. Plant Physiol. 2008; 147: 446-455Crossref PubMed Scopus (323) Google Scholar), support a functional conservation of plant and bacterial CSD proteins in acquiring stress tolerance. Cold shock domain proteins have been identified in a variety of organisms ranging from bacteria to mammals. In higher plants, cold shock domain proteins are involved in the cold response and share conserved functions with bacterial CSPs (26.Nakaminami K. Karlson D.T. Imai R. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10122-10127Crossref PubMed Scopus (139) Google Scholar, 27.Sasaki K. Kim M.H. Imai R. Biochem. Biophys. Res. Commun. 2007; 364: 633-638Crossref PubMed Scopus (86) Google Scholar). In the current study, we have shown that the Arabidopsis AtCSP3 functions as a RNA chaperone and is involved in the acquisition of freezing tolerance. Expression analysis indicated that the level of AtCSP3 transcripts increased in response to cold in both shoots and roots (Fig. 2, A and B), with a maximum level of expression at around 12 h of cold treatment. The fact that AtCSP3 expression was not modulated by drought, NaCl, or ABA (data not shown), suggested that AtCSP3 function is mainly associated with cold stress. The spatial expression pattern of AtCSP3 was characterized using a reporter gene fusion construct (Fig. 2B and supplemental Fig. S2). AtCSP3 promoter-GUS expression was limited to shoot and root apical regions of vegetative plants, which are considered to be tissues that primarily sense environmental cues. The shoot apical meristem was reported to sense low temperature signals during vernalization (36.Schwabe W.W. J. Exp. Bot. 1954; 5: 389-400Crossref Scopus (20) Google Scholar, 37.Curtis O.F. Chang H.T. Amer. J. Bot. 1930; 17: 1047-1048Google Scholar), whereas root tips are also known to sense environmental signals such as gravity and water availability (38.Takahashi H. J. Plant Res. 1997; 110: 163-169Crossref PubMed Google Scholar). In response to cold, GUS and GFP expression was extended to a broader region of root (Fig. 2B, and supplemental Fig. S2K). Because root growth was inhibited during cold treatment, this extension was not due to cell division of GUS-expressing cells. It is thus plausible that a signal from root tip is transduced toward the basal part of root to induce expression of AtCSP3. Transgenic expression of AtCSP3-GFP driven by the native AtCSP3 promoter revealed that AtCSP3 was localized to both the nucleus and cytoplasm (Fig. 2C and supplemental Fig. S2K). The nuclear and cytoplasmic localization is consistent with a role for AtCSP3 involving interaction with mRNA. Arabidopsis LOS4 encodes a DEAD-box RNA helicase and is required for efficient export of RNA from the nucleus to the cytoplasm (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar). LOS4 also localizes to the nucleus and cytoplasm, and might be important for nuclear pore remodeling under cold temperatures (39.Gong Z. Lee H. Xiong L. Jagendorf A. Stevenson B. Zhu J.K. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11507-11512Crossref PubMed Scopus (213) Google Scholar, 40.Gong Z. Dong C.H. Lee H. Zhu J. Xiong L. Gong D. Stevenson B. Zhu J.K. Plant Cell. 2005; 17: 256-267Crossref PubMed Scopus (272) Google Scholar). RNA helicases and RNA chaperones are involved in various steps of RNA metabolism (41.Sommerville J. Bioessays. 1999; 21: 319-325Crossref PubMed Scopus (144) Google Scholar). In Bacillus subtilis, CspB physically interacts with cold-induced DEAD-box RNA helicases, CshA and CshB (42.Hunger K. Beckering C.L. Wiegeshoff F. Graumann P.L. Marahiel M.A. J. Bacteriol. 2006; 188: 240-248Crossref PubMed Scopus (92) Google Scholar). It will be interesting to determine if plant CSD proteins interact with RNA helicases. The atcsp3-2 mutant plant was more sensitive to freezing than wild type under both NA and CA conditions (Fig. 3, D–F). A change in freezing tolerance may reflect altered expression of cold-regulated genes, however, expression analysis of CBFs and CBF regulon genes indicated that the CBF pathway is functioning normally in atcsp3-2 (Fig. 4). On the other hand, several genes were identified that were down-regulated in atcsp3-2. Most of these down-regulated genes in atcsp3-2 have been linked to stress responses (supplemental Table S1 and Fig. 5); however, it is not yet clear how they are related or their potential mechanistic role in freezing tolerance. It is interesting to note that six of the down-regulated genes in atcsp3-2 are known to be up-regulated in the ada2b-1 mutant (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar). ADA2 is a histone acetyltransferase with a putative transcriptional adaptor function (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar, 44.Stockinger E.J. Mao Y. Regier M.K. Triezenberg S.J. Thomashow M.F. Nucleic Acids Res. 2001; 29: 1524-1533Crossref PubMed Scopus (214) Google Scholar). The ada2b-1 mutant is constitutively more freezing tolerant than wild-type plants without overexpressing COR genes (43.Vlachonasios K.E. Thomashow M.F. Triezenberg S.J. Plant Cell. 2003; 15: 626-638Crossref PubMed Scopus (246) Google Scholar), suggesting there may be a cross-talk between the AtCSP3 and ADA2. Current information suggests that AtCSP3 controls the expression of genes that are necessary for freezing tolerance but are not CBF-regulated genes. Molecular genetic analyses of several mutants such as esk1 and hos9 have indicated a significant role for such CBF-independent pathways in freezing tolerance in Arabidopsis (5.Xin Z. Browse J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 7799-7804Crossref PubMed Scopus (332) Google Scholar, 6.Zhu J. Shi H. Lee B.H. Damsz B. Cheng S. Stirm V. Zhu J.K. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9873-9878Crossref PubMed Scopus (201) Google Scholar, 45.Xin Z. Mandaokar A. Chen J. Last R.L. Browse J. Plant J. 2007; 49: 786-799Crossref PubMed Scopus (113) Google Scholar). The biochemical activity of AtCSP3 is very similar to that of WCSP1, unwinding dsDNA, and binding RNA and DNA (supplemental Fig. S1). In addition, AtCSP3 complemented the E. coli csp mutant (Fig. 1B). These data suggested that AtCSP3 functions as RNA chaperone in vivo. AtCSP3 may thus act to enhance translation of bulk or specific mRNA important for freezing tolerance by destabilizing RNA duplex produced under low temperature conditions. Another possibility is that AtCSP3 regulates mRNA stability by mediating RNA duplex formation, which can stabilize mRNA from exonucleolytic degradation. In bacterial systems, RNA chaperones regulate gene expression at both the transcription and post-transcription levels. Our data, together with the recent finding that bacterial CSPs can confer stress tolerance in plants (46.Castiglioni P. Warner D. Bensen R.J. Anstrom D.C. Harrison J. Stoecker M. Abad M. Kumar G. Salvador S. D'Ordine R. Navarro S. Back S. Fernandes M. Targolli J. Dasgupta S. Bonin C. Luethy M.H. Heard J.E. Plant Physiol. 2008; 147: 446-455Crossref PubMed Scopus (323) Google Scholar), support a functional conservation of plant and bacterial CSD proteins in acquiring stress tolerance. We thank Drs. Masayori Inouye and Tsuyoshi Nakagawa for providing the E. coli BX04 strain and the pGWB4 binary vector, respectively. We also thank ABRC for supplying the mutant Arabidopsis line and Dr. Derek Goto for critical reading of the manuscript. Supplementary Material Download .pdf (.12 MB) Help with pdf files Download .pdf (.12 MB) Help with pdf files
Referência(s)