Plant Mol Biol Rep (2011)29:525-532 D0110.1007/s11105-010-0256-3 Components of the Arabidopsis CBF Cold-Response Pathway Are Conserved in Non-heading Chinese Cabbage Fangling Jiang·Feng Wang·Zhen Wu.Ying Li· Gongjun Shi.Jingding Hu.Xilin Hou Published online:16 November 2010 C Springer-Verlag 2010 Abstract Many plants increase in freezing tolerance upon are similar to those of Arabidopsis.We conclude that exposure to low non-freezing temperatures,a phenomenon components of the CBF cold-response pathway are highly known as cold acclimation.Cold acclimation in Arabidopsis conserved in non-heading Chinese cabbage. involves rapid cold-induced expression of the inducer of C-repeat/dehydration-responsive element-binding factor Keywords Non-heading Chinese cabbage (CBF)expression (ICE)transcriptional activators followed Cold-responsive genes.ICE.CBF.COR by expression of the CBF;subsequently,CBF-targeted genes that increase freezing tolerance.Here,we present Abbreviations evidence for a CBF cold-response pathway in non-heading ICE Inducer of CBF expression Chinese cabbage (Brassica campestris ssp.chinensis L. CBF CRT-binding factors Makino).We show that non-heading Chinese cabbage COR Cold-regulated genes encodes ICEl-like gene BrICEI that bracket an open reading frame of 1,491 bp encoding a protein with a potential bHLH domain,which accumulates rapidly in response to low temperature followed closely by expres- Introduction sion of the BrCBF gene,an ortholog of the Arabidopsis CBF3-like gene,and then BrCOR/4 gene,an ortholog of Cold stress adversely affects plant growth and development the Arabidopsis CBF-targeted COR15b gene.An align- and thus limits crop productivity.Diverse plant species ment of the later two genes from Arabidopsis,Brassica tolerate cold stress to a varying degree,which depends on napus revealed the presence of conserved CANNTG core reprogramming gene expression to modify their physiology, element and AP2 domain in BrCBF and a CCG core metabolism,and growth.Cold signal in plants is transmit- element in BrCOR/4.In addition,BrCBF and BrCOR/4 ted to activate C-repeat/drought-responsive element-binding showed increased expression induced by low temperature factor (CBF)-dependent and CBF-independent transcrip- as well as salt and drought,but not by ABA stress which tional pathway,of which CBF-dependent pathway activates CBF regulon.CBF transcription factor genes are induced by the constitutively expressed inducer of CBF expression (ICE)1 by binding to the CBF promoter.ICE1-CBF cold- E.Jiang·E.Wang·Z.Wu.Y.Li·G.Shi·J.Hu·XHou(☒ response pathway is conserved in diverse plant species State Key Laboratory of Crop Genetics and Germplasm Enhancement, (Fowler et al.1996;Goulas et al.2003;Lee et al.2005; Nanjing 210095,China Chinnusamy et al.2007;Meng et al.2008;Wang et al. e-mail:hxl@njau.edu.cn 2009;Chinnusamy et al.2010).In this pathway,ICE's core sequence bHLH can recognize DNA with the consensus E.Jiang'E.Wang'Z.WuY.Li·G.Shi·J.Hu·XHou Key Laboratory of Southern Vegetable Crop Genetic sequence CANNTG (Chinnusamy et al.2003;Meshi and Improvement,Ministry of Agriculture, Iwabuchi 1995)involving in the CBF or DNA replication- Nanjing 210095,China related element-binding(DREB)proteins,while CBF or ②Springer
Components of the Arabidopsis CBF Cold-Response Pathway Are Conserved in Non-heading Chinese Cabbage Fangling Jiang & Feng Wang & Zhen Wu & Ying Li & Gongjun Shi & Jingding Hu & Xilin Hou Published online: 16 November 2010 # Springer-Verlag 2010 Abstract Many plants increase in freezing tolerance upon exposure to low non-freezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the inducer of C-repeat/dehydration-responsive element-binding factor (CBF) expression (ICE) transcriptional activators followed by expression of the CBF; subsequently, CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino). We show that non-heading Chinese cabbage encodes ICE1-like gene BrICE1 that bracket an open reading frame of 1,491 bp encoding a protein with a potential bHLH domain, which accumulates rapidly in response to low temperature followed closely by expression of the BrCBF gene, an ortholog of the Arabidopsis CBF3-like gene, and then BrCOR14 gene, an ortholog of the Arabidopsis CBF-targeted COR15b gene. An alignment of the later two genes from Arabidopsis, Brassica napus revealed the presence of conserved CANNTG core element and AP2 domain in BrCBF and a CCG core element in BrCOR14. In addition, BrCBF and BrCOR14 showed increased expression induced by low temperature as well as salt and drought, but not by ABA stress which are similar to those of Arabidopsis. We conclude that components of the CBF cold-response pathway are highly conserved in non-heading Chinese cabbage. Keywords Non-heading Chinese cabbage . Cold-responsive genes. ICE . CBF. COR Abbreviations ICE Inducer of CBF expression CBF CRT-binding factors COR Cold-regulated genes Introduction Cold stress adversely affects plant growth and development and thus limits crop productivity. Diverse plant species tolerate cold stress to a varying degree, which depends on reprogramming gene expression to modify their physiology, metabolism, and growth. Cold signal in plants is transmitted to activate C-repeat/drought-responsive element-binding factor (CBF)-dependent and CBF-independent transcriptional pathway, of which CBF-dependent pathway activates CBF regulon. CBF transcription factor genes are induced by the constitutively expressed inducer of CBF expression (ICE)1 by binding to the CBF promoter. ICE1-CBF coldresponse pathway is conserved in diverse plant species (Fowler et al. 1996; Goulas et al. 2003; Lee et al. 2005; Chinnusamy et al. 2007; Meng et al. 2008; Wang et al. 2009; Chinnusamy et al. 2010). In this pathway, ICE’s core sequence bHLH can recognize DNA with the consensus sequence CANNTG (Chinnusamy et al. 2003; Meshi and Iwabuchi 1995) involving in the CBF or DNA replicationrelated element-binding (DREB) proteins, while CBF or F. Jiang : F. Wang : Z. Wu : Y. Li : G. Shi : J. Hu : X. Hou (*) State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095, China e-mail: hxl@njau.edu.cn F. Jiang : F. Wang : Z. Wu : Y. Li : G. Shi : J. Hu : X. Hou Key Laboratory of Southern Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing 210095, China Plant Mol Biol Rep (2011) 29:525–532 DOI 10.1007/s11105-010-0256-3
526 Plant Mol Biol Rep (2011)29:525-532 DREB,a family of AP2-domain,are also transcriptional Isolation of cDNAs Encoding Cold-Response Proteins activators binded to the DRE/CRT element (GCC-box pathogenesis-regulated promoter element)and activated A non-heading Chinese cabbage cDNA fragment encoding transcription(Zhou et al.2007;Buittner and Singh 1997; an ICE-like polypeptide was isolated by reverse Stockinger et al.1997:Thomashow 1999)of the CBF transcription-polymerase chain reaction (RT-PCR)using regulon including genes,specifically corl5 or cor47,thus degenerate primers 5'-ATGGTTCTTGACGGAAACA increasing chilling and freezing tolerance of plants. ACGGTG-3'and 5'-AAAGGGCTTTAGTTCTTCTA Meanwhile,it should be noted that ICE/was expressed ACTCTGCTTC-3'based on the complete open reading constitutively,being only slightly up-regulated by cold, frame (ORF)of Arabidopsis cold-responsive gene ICEI but CBF expression was induced by cold treatment (Genbank accession number AY195621)and C.bursa- (Medina et al.1999;Gao et al.2002;Thomashow 2001; pastoris ICE53 (Genbank accession number AY506804). Chinnusamy et al.2003).Whereas the overexpression of The anticipate product size was 1,558 bp.Total RNA was CBF1 (Jaglo-Ottosen et al.1998)and DREBla/CBF3 isolated from seedlings incubated at 4C for 8 h using (Kasuga et al.1999)in Arabidopsis were shown to be able TaKaRa RNAiso Reagent(Takara,Japan).The first strand to drive expression of COR genes in the absence of low cDNA was reversed using TaKaRa RNA PCR Kit (AMV) temperature and impart constitutive salt and drought Ver.2.1 (TaKaRa,Japan).The PCR mixture contained tolerance,while not abscisic acid (ABA)stress,which 2.5 uL buffer (10xPCR),1.5 uL MgCL2 (25 mM), suggested that it is involved in the expression of cold-, 1.5 uL dNTPs (2.5 mM each),0.25 uL LA-Tag DNA salt-,and drought-regulated genes through an ABA- polymerase (5 UmL/L),10-pmol-specific primers each independent pathway (Kasuga et al.1999;Yamaguchi- 50 ng cDNA,and ddH2O up to 25 uL.Amplification Shinozaki and Shinozaki 1994). profile was 94C for 5 min,35 cycles of 94Cfor 30 s,65C Non-heading Chinese cabbage,like Arabidopsis,cold for 1 min,72C for I min 30 s,and a final extension of 72C acclimates and is a member of the Cruciferae family.We for 10 min.The products were resolved in 1.0%(whv)agarose speculate that non-heading Chinese cabbage may have gel and purified,then cloned into the pGEM-T vector similar cold acclimation process as Arabidopsis.Recently. (Tiangen,China)followed by sequencing. several cold-regulated genes have been cloned from Arabi- cDNAs encoding full-length CBF-like and COR-like dopsis,Capsella bursa-pastoris,and Brassica napus (Jaglo- proteins were isolated by RT-PCR and rapid amplification Ottosen et al.2001;Wang et al.2005).Up till now,there has of cDNA ends(RACE)previously (Jiang et al.2007a,b). been no report on the cloning of cold-regulated genes from The sequences for the entire cDNA insert were determined non-heading Chinese cabbage.In this paper,we reported that and deposited the molecular cloning of BrICEl,BrCBF,and BrCOR14 genes from non-heading Chinese cabbage,bioinformatics Bioinformatics Analysis analysis revealed that these three genes strongly resembled ICE,CBF,and COR genes from other species. Associated molecular information was analyzed using software Clustal W.and other databases listed below: NCBI (http://www.ncbi.nlm.nih.gov/),ProtParam (http:/ Materials and Methods us.expasy.org/tools/protparam.html),and TMHMMv2.0 (http://www.cbs.dtu.dk/services/TMHMM/).Alignment Plant Materials scores of the amino acid sequences of the identified cold- responsive genes with other known homologous proteins A non-heading Chinese cabbage (Brassica campestris ssp. were processed by PROSITE (http://www.Expasy.org/pro chinensis L.Makino)cold-resistant inbred line.043,from site/),InterProScan (http://www.ebi.ac.uk/Tools/InterPro non-heading Chinese cabbage project team in Nanjing Scan/)and WU-Blast2 (http://www.ebi.ac.uk/Tools/blast2 Agricultural University was used in the present study. index.html).Secondary structure analyses were carried out Healthy seeds were grown in controlled environmental by SOMPA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat. chambers at 20C to 22C under continuous cool-white plpage=npsa_sopm%20a.html). fluorescent illumination of 100 to 150 umolm2s light intensity as described by Gilmour et al.(1998)Stress Real-Time Fluorescence Quantitative PCR Analysis treatments were performed with seedlings at three-leaf stage.For cold acclimation,plants were incubated at 4C For cold acclimation.seedlings were transferred to 4C for under continuous cool-white fluorescent illumination at varying lengths of time (0 h,0.5 h,1 h,2 h,4 h,8 h,12 h, approximately 50 umolm2s light intensity for varying 24 h,4 days and 7 days),with Saran Wrap covered to slow lengths of time. evaporation.For ABA,salt and drought stresses,seedlings Springer
DREB, a family of AP2-domain, are also transcriptional activators binded to the DRE/CRT element (GCC-box pathogenesis-regulated promoter element) and activated transcription (Zhou et al. 2007; Büttner and Singh 1997; Stockinger et al. 1997; Thomashow 1999) of the CBF regulon including genes, specifically cor15 or cor47, thus increasing chilling and freezing tolerance of plants. Meanwhile, it should be noted that ICE1 was expressed constitutively, being only slightly up-regulated by cold, but CBF expression was induced by cold treatment (Medina et al. 1999; Gao et al. 2002; Thomashow 2001; Chinnusamy et al. 2003). Whereas the overexpression of CBF1 (Jaglo-Ottosen et al. 1998) and DREB1a/CBF3 (Kasuga et al. 1999) in Arabidopsis were shown to be able to drive expression of COR genes in the absence of low temperature and impart constitutive salt and drought tolerance, while not abscisic acid (ABA) stress, which suggested that it is involved in the expression of cold-, salt-, and drought-regulated genes through an ABAindependent pathway (Kasuga et al. 1999; YamaguchiShinozaki and Shinozaki 1994). Non-heading Chinese cabbage, like Arabidopsis, cold acclimates and is a member of the Cruciferae family. We speculate that non-heading Chinese cabbage may have similar cold acclimation process as Arabidopsis. Recently, several cold-regulated genes have been cloned from Arabidopsis, Capsella bursa-pastoris, and Brassica napus (JagloOttosen et al. 2001; Wang et al. 2005). Up till now, there has been no report on the cloning of cold-regulated genes from non-heading Chinese cabbage. In this paper, we reported that the molecular cloning of BrICE1, BrCBF, and BrCOR14 genes from non-heading Chinese cabbage, bioinformatics analysis revealed that these three genes strongly resembled ICE, CBF, and COR genes from other species. Materials and Methods Plant Materials A non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino) cold-resistant inbred line, 043, from non-heading Chinese cabbage project team in Nanjing Agricultural University was used in the present study. Healthy seeds were grown in controlled environmental chambers at 20°C to 22°C under continuous cool-white fluorescent illumination of 100 to 150 μmolm−2 s −1 light intensity as described by Gilmour et al. (1998) Stress treatments were performed with seedlings at three-leaf stage. For cold acclimation, plants were incubated at 4°C under continuous cool-white fluorescent illumination at approximately 50 μmolm−2 s −1 light intensity for varying lengths of time. Isolation of cDNAs Encoding Cold-Response Proteins A non-heading Chinese cabbage cDNA fragment encoding an ICE-like polypeptide was isolated by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers 5′-ATGGTTCTTGACGGAAACA ACGGTG-3′ and 5′-AAAGGGCTTTAGTTCTTCTA ACTCTGCTTC-3′ based on the complete open reading frame (ORF) of Arabidopsis cold-responsive gene ICE1 (Genbank accession number AY195621) and C. bursapastoris ICE53 (Genbank accession number AY506804). The anticipate product size was 1,558 bp. Total RNA was isolated from seedlings incubated at 4°C for 8 h using TaKaRa RNAiso Reagent (Takara, Japan). The first strand cDNA was reversed using TaKaRa RNA PCR Kit (AMV) Ver.2.1 (TaKaRa, Japan). The PCR mixture contained 2.5 μL buffer (10×PCR), 1.5 μL MgCL2 (25 mM), 1.5 μL dNTPs (2.5 mM each), 0.25 μL LA-Taq DNA polymerase (5 UmL/L), 10-pmol-specific primers each, 50 ng cDNA, and ddH2O up to 25 μL. Amplification profile was 94°C for 5 min, 35 cycles of 94°Cfor 30 s, 65°C for 1 min, 72°C for 1 min 30 s, and a final extension of 72°C for 10 min. The products were resolved in 1.0% (w/v) agarose gel and purified, then cloned into the pGEM-T vector (Tiangen, China) followed by sequencing. cDNAs encoding full-length CBF-like and COR-like proteins were isolated by RT-PCR and rapid amplification of cDNA ends (RACE) previously (Jiang et al. 2007a, b). The sequences for the entire cDNA insert were determined and deposited. Bioinformatics Analysis Associated molecular information was analyzed using software Clustal W, and other databases listed below: NCBI (http://www.ncbi.nlm.nih.gov/), ProtParam (http:// us.expasy.org/tools/protparam.html), and TMHMMv2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Alignment scores of the amino acid sequences of the identified coldresponsive genes with other known homologous proteins were processed by PROSITE (http://www.Expasy.org/pro site/), InterProScan (http://www.ebi.ac.uk/Tools/InterPro Scan/) and WU-Blast2 (http://www.ebi.ac.uk/Tools/blast2/ index.html). Secondary structure analyses were carried out by SOMPA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat. pl?page=npsa_sopm%20a.html). Real-Time Fluorescence Quantitative PCR Analysis For cold acclimation, seedlings were transferred to 4°C for varying lengths of time (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 4 days and 7 days), with SaranWrap covered to slow evaporation. For ABA, salt and drought stresses, seedlings 526 Plant Mol Biol Rep (2011) 29:525–532
Plant Mol Biol Rep (2011)29:525-532 527 were raised on the water saturated cotton in the growth illustrated that BrICEl was more likely an ortholog of chamber at 20C to 22C.ABA stress was carried out by ICEl.BrCBF was 73%identical to A.thaliana CBF3 and 200 uM Abscisic acid,Salt stress was induced by 200 mM 72%to A.thaliana CBF2 and CBFI which suggest that NaCl and drought stress was imposed by 300 mM mannitol BrCBF was an ortholog of CBF3,while BrCOR14 was for 0 h,0.5 h,2 h,4 h,8 h,24 h and 4 days,respectively. with 76%identity to A.thaliana COR15B and 69%identity Total RNAs were isolated separately.Gene-specific primers to A.thaliana COR15A which demonstrated that BrCOR14 for BrICEI,BrCBF,and BrCOR14 were 5'-ACAACAA was more like an orthology of COR15B.Alignment scores CGCAACACCCT-3'and 5-ACGACGCCAACA CCTCT-3'. of the amino acid sequences of the three genes with other 5'-GTGTGTGAAGTGAGGGAACCAAAC-3'and 5'-CC known cold-responsive proteins in Cruciferous family also AAGCCGAGTCCGCATAAT-3',5'-TTCTTCTT show high concordance (Table 2). TCCCCAGCG-3'and 5'-TTCCATCACCTTTCTCGG-3' The results also suggested that the BrICEl was highly designed based on non-heading Chinese cabbage BrICEl, conservative to the ICEl in possessing a bipartite nuclear BrCBF,and BrCOR/4 genes.Primers for Actins were localization signal (NLS)domain from K304 to K353 and a 5'-ACAACTCCATCATGAAGTGT-3'and 5'-GAGATC bHLH domain from R316 to L352,as well as potential CACATCTGTTGGAA-3'used as control.Real-time PCR recognition sites,for instance basic-leucine zipper (bZIP) was carried out by One Step SYBR PrimescriptTM that contains a basic region mediating sequence-specific RT-PCR Kit (Takara,Japan).Mixtures contained 12.5 ul DNA binding followed by a leucine zipper region(required SYBR,10 pmol adaptor primers each,50 ng cDNA,ddH2O for dimerization)transcription factors,HRI (shown to bind up to 25 ul.Amplification profile was 95C for 120 s,35 the small G protein rho or to activate PKN in its GTP-bound cycles of 95C for 10 s,55C for 20 s,and 72C for 20 s on form)and Pfam domain (Fig.2). Corbett Rotor-Gene 3000A.Each reaction was carried out Besides BrCBF gene contained a consensus sequence, in a separate PCR system with two replicates and was CANNTG (CACCTG).Alignment of the BrCBF proteins repeated three times.The data were analyzed using data indicated that it contained a highly conserved AP2 DNA- analysis software that comes with the machine. binding domain of 60 amino acid residues from /s2 to A109 as well as three potential strikingly conserved elements, YRG (consisted of 23 amino acids containing the con- Results served YRG amino acid motif and was functionally important for DNA binding),NLS domain (from amino Isolation of cDNAs Encoding Cold-Response Proteins acid R42 to Rs9)and ALA-RICH domain (from amino acid Ts4 to K128).Besides,the prediction also revealed that RNA quality was determined by 1%agarose gel electro- BrCBF contains a dehydration-responsive element(Fig.3). phoresis.28S,18S,and 5S RNA in total extracted RNA In addition,the DNA sequence at the presumed transcrip- had clean bands and proper proportion (Fig.1).BrICE/, tion site of BrCOR/4.CCG.is identified as a common core BrCBF,and BrCOR/4 were obtained by RT-PCR and sequence. RACE.Overall information of the putative proteins was shown in Table 1.The BrICEl protein was estimated to Secondary Structure Analysis have a half life of about 4.4 h and instability index of 44.13. thus being classified as unstable,while BrCBF and Secondary structure analyses indicated(Fig.4)that BrICEl BrCOR14 were classified as stable.Furthermore,alignment consisted of 158 a-helices,40 B-turns jointed by 83 scores of the amino acid sequences of the identified cold- extended strands,and 216 random coils which resembled responsive genes with other known homologous proteins the secondary structure of Arabidopsis ICE1 (NP 189309.2) predicted that BrICEl was 89%identity to Arabidopsis and CbICE53 (C.bursa-pastoris AAS79350). ICEl and 59%to Arabidopsis thaliana ICE2 which BrCBF consisted of 84 a-helices,nine B-turns jointed by 33 extended strands and 90 random coils which commendably Fig.1 RNA quality resembled the secondary structure of CBF3(AF074602)and determination CbCBF (AY391121.1).It was also note worthy that a-helices occurred predominantly in the structure of BrCBF Moreover,the BrCOR14 consisted of 78 a-helices,seven B-turns jointed by 17 extended strands,and 26 random coils, 28S 18S which did not resemble the secondary structure of COR15B (A.thaliana NM_129814)and CbCOR15B (AY437888.1) 5S that much.Subsequently,homology modeling analysis was carried out,while no suitable target was found due to the 鱼Springer
were raised on the water saturated cotton in the growth chamber at 20°C to 22°C. ABA stress was carried out by 200 μM Abscisic acid, Salt stress was induced by 200 mM NaCl and drought stress was imposed by 300 mM mannitol for 0 h, 0.5 h, 2 h, 4 h, 8 h, 24 h and 4 days, respectively. Total RNAs were isolated separately. Gene-specific primers for BrICE1, BrCBF, and BrCOR14 were 5′-ACAACAA CGCAACACCCT-3′ and 5′-ACGACGCCAACA CCTCT-3′, 5′-GTGTGTGAAGTGAGGGAACCAAAC-3′ and 5′-CC AAGCCGAGTCCGCATAAT- 3 ′, 5′-TTCTTCTT TCCCCAGCG-3′ and 5′-TTCCATCACCTTTCTCGG-3′ designed based on non-heading Chinese cabbage BrICE1, BrCBF, and BrCOR14 genes. Primers for Actins were 5′-ACAACTCCATCATGAAGTGT-3′ and 5′-GAGATC CACATCTGTTGGAA-3′ used as control. Real-time PCR was carried out by One Step SYBR® Primescript™ RT-PCR Kit (Takara, Japan). Mixtures contained 12.5 μl SYBR, 10 pmol adaptor primers each, 50 ng cDNA, ddH2O up to 25 μl. Amplification profile was 95°C for 120 s, 35 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s on Corbett Rotor-Gene 3000A. Each reaction was carried out in a separate PCR system with two replicates and was repeated three times. The data were analyzed using data analysis software that comes with the machine. Results Isolation of cDNAs Encoding Cold-Response Proteins RNA quality was determined by 1% agarose gel electrophoresis. 28S, 18S, and 5S RNA in total extracted RNA had clean bands and proper proportion (Fig. 1). BrICE1, BrCBF, and BrCOR14 were obtained by RT-PCR and RACE. Overall information of the putative proteins was shown in Table 1. The BrICE1 protein was estimated to have a half life of about 4.4 h and instability index of 44.13, thus being classified as unstable, while BrCBF and BrCOR14 were classified as stable. Furthermore, alignment scores of the amino acid sequences of the identified coldresponsive genes with other known homologous proteins predicted that BrICE1 was 89% identity to Arabidopsis ICE1 and 59% to Arabidopsis thaliana ICE2 which illustrated that BrICE1 was more likely an ortholog of ICE1, BrCBF was 73% identical to A. thaliana CBF3 and 72% to A. thaliana CBF2 and CBF1 which suggest that BrCBF was an ortholog of CBF3, while BrCOR14 was with 76% identity to A. thaliana COR15B and 69% identity to A. thaliana COR15A which demonstrated that BrCOR14 was more like an orthology of COR15B. Alignment scores of the amino acid sequences of the three genes with other known cold-responsive proteins in Cruciferous family also show high concordance (Table 2). The results also suggested that the BrICE1 was highly conservative to the ICE1 in possessing a bipartite nuclear localization signal (NLS) domain from K304 to K353 and a bHLH domain from R316 to L352, as well as potential recognition sites, for instance basic-leucine zipper (bZIP) that contains a basic region mediating sequence-specific DNA binding followed by a leucine zipper region (required for dimerization) transcription factors, HR1 (shown to bind the small G protein rho or to activate PKN in its GTP-bound form) and Pfam domain (Fig. 2). Besides BrCBF gene contained a consensus sequence, CANNTG (CACCTG). Alignment of the BrCBF proteins indicated that it contained a highly conserved AP2 DNAbinding domain of 60 amino acid residues from I52 to A109 as well as three potential strikingly conserved elements, YRG (consisted of 23 amino acids containing the conserved YRG amino acid motif and was functionally important for DNA binding), NLS domain (from amino acid R42 to R59) and ALA-RICH domain (from amino acid T84 to K128). Besides, the prediction also revealed that BrCBF contains a dehydration-responsive element (Fig. 3). In addition, the DNA sequence at the presumed transcription site of BrCOR14, CCG, is identified as a common core sequence. Secondary Structure Analysis Secondary structure analyses indicated (Fig. 4) that BrICE1 consisted of 158 α-helices, 40 β-turns jointed by 83 extended strands, and 216 random coils which resembled the secondary structure of Arabidopsis ICE1 (NP_189309.2) and CbICE53 (C. bursa-pastoris AAS79350). BrCBF consisted of 84 a-helices, nine β-turns jointed by 33 extended strands and 90 random coils which commendably resembled the secondary structure of CBF3 (AF074602) and CbCBF (AY391121.1). It was also note worthy that a-helices occurred predominantly in the structure of BrCBF. Moreover, the BrCOR14 consisted of 78 α-helices, seven β-turns jointed by 17 extended strands, and 26 random coils, which did not resemble the secondary structure of COR15B (A. thaliana NM_129814) and CbCOR15B (AY437888.1) that much. Subsequently, homology modeling analysis was carried out, while no suitable target was found due to the Fig. 1 RNA quality determination Plant Mol Biol Rep (2011) 29:525–532 527
528 Plant Mol Biol Rep (2011)29:525-532 Table 1 Overall information of Br/CEI,BrCBF,and BrCOR/4 genes Name Genbank cDNA Open reading Amino Isoelectric Molecular Half life (h) Instability accession No length (bp) frame (bp) acids point (pD) weight (kDa) index BrICEl EU374158 1.558 1,491 497 4.98 127.40 4.4 (unstable) 44.13 BrCBF DQ402470 1,003 648 216 5.32 24.05 30 (stable) 36.73 BrCOR14 D0192529 549 387 129 5.61 13.75 30(stable) 23.24 extremely low homology with the proteins deposited in all were 6.13-fold and 117.14-fold higher than the basal, databases respectively.But maximum accumulation of Br/CE/was at 24 h,whereas at 8 h BrCBF showed the highest Real-Time Fluorescence Quantitative PCR Analysis expression.In addition,BrICEl,BrCBF,and BrCOR14 genes'maximum expression appeared all at 4 h under Real-time PCR analysis showed that Br/CE/,BrCBF and drought stress carried out by Mannitol treatment with 2.2- BrCOR/4 generally displayed a trend of increased first and 6.2-13.7-fold higher than the basal(Fig.6). then decreased with the highest expression at 1,4,and 24 h, respectively,under 4C treatment.Comparison of the expression quantity of the three genes at different time Discussion points revealed that BrICEI's expression was more than 9.15-fold higher in 1 h after 4C treatment compared with Bioinformatics analysis revealed that BrICEI,BrCBF,and the basal expression and decreased immediately,followed BrCOR14 genes strongly resemble Arabidopsis ICEl, by accumulation of BrCBF at 4 h and which dramatically CBF3,and COR/5b separately.Simultaneously sequence decreased after that,and BrCOR/4 with the highest analysis showed that BrICEl contains the highly conserved expression at 24 h (49.9-fold more than the basal bHLH domain necessary in Arabidopsis ICE families,as expression)and hold the line till 7 days(Fig.5). well as bZIP and HR1 which strongly suggest that BrICEl While for ABA treatment only under 0.5 h did Br/CE/ is a DNA-binding protein.Whereas BrCBF has CANNTG show 4.3-fold higher expression than the basal.The other core element,AP2 domain as well as conserved amino acid two genes showed no significant difference at different time sequences,specifically YRG and NLS domain.While AP2 points.For salt(NaCl)treatment,the maximum accumula- and NLS have been evolutionarily conserved elements tion of BrICEI was 3.48-fold higher.BrCBF and BrCOR14 necessary for the structure or function of these CBF Table 2 Comparison of the non-heading Chinese cabbage amino acid sequences of BrICEl,BrCBF,and BrCOR14 with other cold-responsive protcins in the NCBI database Enzyme source Number of a.a Identity (% Positives (% Genbank accession No. Brassica campestris ssp.chinensis L.Makino (BrICEl) 498 EU374158 Arabidopsis thaliana (ICE1) 494 89 92 NP189309.2 A.thaliana (ICE2) 828 59 67 NP172746.1 Capsella bursa-pastoris (Cbice53) 492 89 AAS79350 B.campestris ssp.chinensis (BrCBF) 216 DQ402470 A.thaliana (CBF3) 216 73 85 ACI15599.1 A.thaliana (CBF2) 216 72 84 NP567719 A.thaliana (CBF1) 213 72 82 ABV27062.1 B.juncea (DREB1B) 214 84 91 ABX00639.1 B.napus(CBF) 214 6 91 AAD45623.1 B.campestris ssp.chinensis (BrCOR14) 129 DQ192529 A.thaliana (COR15B) 141 76 85 NP181781.1 A.thaliana (COR15A) 139 69 76 NP181782 B.rapa subsp.Pekinensis (COR) 129 97 98 ABF60663.1 B.napus (BN115) 142 78 83 AAA66068.1 Capsella bursa-pastoris (CBCOR15) 139 71 78 AAR99417.1 ≌Springer
extremely low homology with the proteins deposited in all databases. Real-Time Fluorescence Quantitative PCR Analysis Real-time PCR analysis showed that BrICE1, BrCBF and BrCOR14 generally displayed a trend of increased first and then decreased with the highest expression at 1, 4, and 24 h, respectively, under 4°C treatment. Comparison of the expression quantity of the three genes at different time points revealed that BrICE1’s expression was more than 9.15-fold higher in 1 h after 4°C treatment compared with the basal expression and decreased immediately, followed by accumulation of BrCBF at 4 h and which dramatically decreased after that, and BrCOR14 with the highest expression at 24 h (49.9-fold more than the basal expression) and hold the line till 7 days (Fig. 5). While for ABA treatment only under 0.5 h did BrICE1 show 4.3-fold higher expression than the basal. The other two genes showed no significant difference at different time points. For salt (NaCl) treatment, the maximum accumulation of BrICE1 was 3.48-fold higher. BrCBF and BrCOR14 were 6.13-fold and 117.14-fold higher than the basal, respectively. But maximum accumulation of BrICE1 was at 24 h, whereas at 8 h BrCBF showed the highest expression. In addition, BrICE1, BrCBF, and BrCOR14 genes’ maximum expression appeared all at 4 h under drought stress carried out by Mannitol treatment with 2.2– 6.2–13.7-fold higher than the basal (Fig. 6). Discussion Bioinformatics analysis revealed that BrICE1, BrCBF, and BrCOR14 genes strongly resemble Arabidopsis ICE1, CBF3, and COR15b separately. Simultaneously sequence analysis showed that BrICE1 contains the highly conserved bHLH domain necessary in Arabidopsis ICE families, as well as bZIP and HR1 which strongly suggest that BrICE1 is a DNA-binding protein. Whereas BrCBF has CANNTG core element, AP2 domain as well as conserved amino acid sequences, specifically YRG and NLS domain. While AP2 and NLS have been evolutionarily conserved elements necessary for the structure or function of these CBF Table 2 Comparison of the non-heading Chinese cabbage amino acid sequences of BrICE1, BrCBF, and BrCOR14 with other cold-responsive proteins in the NCBI database Enzyme source Number of a. a Identity (%) Positives (%) Genbank accession No. Brassica campestris ssp. chinensis L. Makino (BrICE1) 498 – – EU 374158 Arabidopsis thaliana (ICE1) 494 89 92 NP_189309.2 A. thaliana (ICE2) 828 59 67 NP_172746.1 Capsella bursa-pastoris (Cbice53) 492 86 89 AAS79350 B. campestris ssp. chinensis (BrCBF) 216 – – DQ402470 A. thaliana (CBF3) 216 73 85 ACI15599.1 A. thaliana (CBF2) 216 72 84 NP_567719 A. thaliana (CBF1) 213 72 82 ABV27062.1 B. juncea (DREB1B) 214 84 91 ABX00639.1 B. napus (CBF) 214 83 91 AAD45623.1 B. campestris ssp. chinensis (BrCOR14) 129 – – DQ192529 A. thaliana (COR15B) 141 76 85 NP_181781.1 A. thaliana (COR15A) 139 69 76 NP_181782 B. rapa subsp. Pekinensis (COR) 129 97 98 ABF60663.1 B. napus (BN115) 142 78 83 AAA66068.1 Capsella bursa-pastoris (CBCOR15) 139 71 78 AAR99417.1 Table 1 Overall information of BrICE1, BrCBF, and BrCOR14 genes Name Genbank accession No. cDNA length (bp) Open reading frame (bp) Amino acids Isoelectric point (pI) Molecular weight (kDa) Half life (h) Instability index BrICE1 EU374158 1,558 1,491 497 4.98 127.40 4.4 (unstable) 44.13 BrCBF DQ402470 1,003 648 216 5.32 24.05 30 (stable) 36.73 BrCOR14 DQ192529 549 387 129 5.61 13.75 30 (stable) 23.24 528 Plant Mol Biol Rep (2011) 29:525–532
Plant Mol Biol Rep (2011)29:525-532 529 Fig.2 The alignment of Consensus MgLDGnnGGgNLGgGGGGggg. .EEEnnEAgNgannEDgGQFKPNLEGGGDWFtSnQP 60 BrICEl with ICEl protein from BrICE1 MVLDGNNGGVWLGGGGGGGGGERVQEEENEEASWGRNQEDGGQFKPMLEGGGDWFTSNQP Arabidopsis thaliana.Nuclear ICEl MGLDGNNGGGVWLNGGGG.......EREENEEGSWGRNQEDGSSQFKPMLEGDWFSSNOP localization signal (NLS).bHLH Consensus HPQDLOMLQnQQDFRFLGGFGFnPnDnLLLLQNSNNSSSceSPSaAFSLDPSOnSFLaWa 120 domain are indicated BrICE1 HPQDLOMLOSQQDFRFLGGFGFNPNDNLLLLOHSMDSSSSCSPSQAFSLDPSOVSFLAAA ICEl HPQDLOMLONOPDFRYFGGE PFNPNDNLLLQHSIDSSSSCSPSQAFSLDPSOONOFLSTN Consensus nnKgCLLnVVPSaanPFnaAFEggSNgGFnnQIaAPVVgGggst TQggRNVPNFLNARSa 180 BrICE1 NNKSCLLNVVPSSANPFDNAFEFGSDSGFLNQIQAPVSMGFGSLTQLGSSVPDFLSARSL ICEI NNKGCLLNVPSSANPFDNAFEFGSESGFLNQIHAPISMGFGSLTQLGNRDLSSVPDFLSA Consensus LPPEannatnncKgGSGGFTaLELEGgGgPAangg.VGnRaKVLKPLEVLASSGAQPTLF 240 B立工C3 LPPENNNATPLCGGGGGGFTPLELEGFGSPASF...VGSRPKVLKPLEVLASSGAQPTLF ICEI RSLLAPESNNNNTMLCGGFTAPLELEGFGSPANGGFVGNRAKVLKPLEVLASSGAOPTLF Consensus QKRAAMRQSSGSKMGnSESSGMRRLSDDGDMDETGVEVSGLnYESDELnESGKAaESVQn 300 BrICE1 QKRAAMROSSGSKMGNSESSGMRRLSDDGDMDETGVEVSGLNYESDELNESGKASESVON ICE1 RQSSGSKMGNSESSGMRRFSDDGDMDETGTEVSGLNYESDEINESGKAA Consensus gGGGKGKKKGMPAKnLMAERRRRKKLnDRLYMLRSVVPKISKMDRASILGDAIDYLKELL 360 BrICE1 GGGKGKKKGMPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDRASILGDAIDYLKELL ICE1 GGGGKGKKKGMPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDRASILGDAIDYLKELL Consensus 420 BrICE1 QRINDLHNELESTPTGSLPPTSSSFHPLTPTPOTLSCRVKEELCPSSLPSPKGQQARVEV ICE1 Consensus 480 BrICE1 RLREGRAVSIHMFCGRRPGLLLATMKALDNLGLDVQQAVISCFNGFALDVFRAEOCQEGO ICE1 Consensus 501 BrICE1 EILPDQIKAVLFDTAGYAGMI ICE1 EILPDQIKAVLFDTAGYAGMI proteins in Arabidopsis,B.napus,and C.bursa-pastoris, common core sequence (Fig.7),which strongly suggested implying they might be indispensable to the function of that BrICEl,BrCBF and BrCOR14 play a critical role in BrCBF in controlling gene expression.Furthermore. cold-responsive pathway similar to Arabidopsis ICEl. BrCOR/4 contains C-repeat DRE sequences common core CBF3,and COR/5b genes. sequence CGCCGTC,closely resembling the C-repeat Secondary structure prediction is a key element in many DRE sequences in the promoters of the Arabidopsis genes different approaches to protein structure analysis,simplify- CORI5a.GGCCGAC.and COR78/RD29A.TACCGAC ing the 20-state amino acid sequence into typically three (Stockinger et al.1997).An intriguing hypothesis thus states (helix,strand or coil/loop)and is often used to raised is that BrICEl,the bHLH family,is members of a provide constraints for comparative modeling or as a superfamily of DNA-binding proteins that recognize starting point for fold recognition.Indeed,the recent work BrCBF,a family of CBF or DREB (DRE binding)proteins shows that accurate protein secondary structure information having,potentially,CANNTG as a common core sequence, is a useful baseline in fold recognition (Wilson et al.2002). while BrCBF,the AP2 domain protein is a superfamily of The secondary structure analysis showed that BrICEl and DNA-binding proteins that recognize BrCOR/4,a family of BrCBF highly resembled ICE and CBF genes in Arabi- cis-acting regulatory elements having,potentially,CCG as a dopsis and C.bursa-pastoris.The N-terminal of the BrCBF NLS Consensus MNtSSaSWFVEgFGnDBEVttVaGGDYIPtLAaSCPKKPAGRKKFRETRHPIYRGVRRRn 60 CBF3 MSSFSAFSEMFGSDYESSISSGGDYIPTLASSCPKKPAGRKKFRETRHPIYRGVRRRN BrCBE MNTFPASTEMVGSENESPVTTVAGGDYY PMLAASCPKKPAGRKKOTRHTYRGVRLRK AP2 domain Consensus SGKWVCEVREPnKKERIWLGTFQTAEMAARAHDVAALALRGRgACLOFADSAWRLRIPEt 120 CBE3 SGKWVCEVREPNKKTRIWLGTFOTAEMAARAHDVAALALRGRSACLNFADSAWRLRIPES BrCBF SGKWVCEVREPNKKSRIWLGTFKTAEMAARAHDVAALALRGRGACLNYADSAWRLRIPET Consensus TCaKDIQKAAAEAALAFQaENeDVTtDnGFnMEETLVEAIYTAEnnEnAFHNHDEaMFEM 180 CBF3 TCAKDIOKAAAEAALAFODEMCDVTTDHGFDMEETLVEAIYTAEOSENAFYMHDEAMFEM BrCBE TCHKDIQKAAAEAALAFEAEKSDVTMONGQNMEETIVEAIFTEENNDVFYMDEESMLEMP Consensus asLaanaAggMLLPLPVNQgnnnNENggaDNNVNLHSY 218 CBF3 PSLLANMAEGMLLPLPSVOWNHNHEVDGDDDDVSLWSY BrCBE Fig.3 The alignment of BrCBF with CBF3 protein from Arabidopsis thaliana.Nuclear localization signal (NLS),AP2domain,and other domains are indicated 么Springer
proteins in Arabidopsis, B. napus, and C. bursa-pastoris, implying they might be indispensable to the function of BrCBF in controlling gene expression. Furthermore, BrCOR14 contains C-repeat DRE sequences common core sequence CGCCGTC, closely resembling the C-repeat DRE sequences in the promoters of the Arabidopsis genes COR15a, GGCCGAC, and COR78/RD29A, TACCGAC (Stockinger et al. 1997). An intriguing hypothesis thus raised is that BrICE1, the bHLH family, is members of a superfamily of DNA-binding proteins that recognize BrCBF, a family of CBF or DREB (DRE binding) proteins having, potentially, CANNTG as a common core sequence, while BrCBF, the AP2 domain protein is a superfamily of DNA-binding proteins that recognize BrCOR14, a family of cis-acting regulatory elements having, potentially, CCG as a common core sequence (Fig. 7), which strongly suggested that BrICE1, BrCBF and BrCOR14 play a critical role in cold-responsive pathway similar to Arabidopsis ICE1, CBF3, and COR15b genes. Secondary structure prediction is a key element in many different approaches to protein structure analysis, simplifying the 20-state amino acid sequence into typically three states (helix, strand or coil/loop) and is often used to provide constraints for comparative modeling or as a starting point for fold recognition. Indeed, the recent work shows that accurate protein secondary structure information is a useful baseline in fold recognition (Wilson et al. 2002). The secondary structure analysis showed that BrICE1 and BrCBF highly resembled ICE and CBF genes in Arabidopsis and C. bursa-pastoris. The N-terminal of the BrCBF Fig. 3 The alignment of BrCBF with CBF3 protein from Arabidopsis thaliana. Nuclear localization signal (NLS), AP2domain, and other domains are indicated Fig. 2 The alignment of BrICE1 with ICE1 protein from Arabidopsis thaliana. Nuclear localization signal (NLS), bHLH domain are indicated Plant Mol Biol Rep (2011) 29:525–532 529
530 Plant Mol Biol Rep (2011)29:525-532 BrCE1 H+音HW ICE1 HMH+☐-4H 150 CblCE53 41e BrCBF 154 CBF3 1●线 CbCBF ●a i● BrCOR14 COR15B 4 CbCOR15B 40 Fig.4 Comparison of the secondary structure of Brassica campestris ssp.chinensis L.Makino,Arabidopsis thaliana and Capsella bursa-pastoris ICE.CBF,and COR genes helix,tum,strand,and coil were indicated in line with decreased length in tum,respectively was composed of some a-helices similar to CBF3 and tively,these structures might mediate protein-protein inter- CbCBF as protection of the protein from being destroyed actions which are important for CBF functions.These by some endoenzymes.A hypothesis for the function of interactions might involve the ability to form homo-or these a-helices structures was that they were involved in hetero-dimmers similar to that observed for the MADS box DNA binding likely through the interaction of their family of plant regulatory proteins (Huang et al.1996; hydrophobic face with the major groove of DNA.Altema- Riechmann and Meyerowitz 1997).The results also illus- trated that the main structure of BrCOR14 was a-helices 120 ■BrIcE1日BrC8 F BrC0R14 which suggested that BrCOR14 was not a globulin,and these a-helices might mediate protein-protein interactions 100 which are important for COR functions(Altus et al.1996). Besides using real-time PCR expression analysis,it was 80 revealed that expression of BrICEl increased immediately and showed the highest level of expression at 1 h,followed by dramatic accumulation of BrCBF at 4 h and sharply 0 decreased after that.While the highest level of BrCOR/4 expression was observed at 24 h and hold the line till 7 days.These results demonstrated that BrICEI was 自 图 expressed constitutively and might be involved in the cold 0.5h1h2h 4h 8h 12 24h 4d 7d acclimation process,thus,it might belong to the ice gene Time family.Furthermore,the finding that the expression levels of BrCBF increased at early stages of cold exposure and Fig.5 Real-time fluorescence quantitative PCR analysis of Br/CEl, BrCBF,and BrCOR/4 genes in non-heading Chinese cabbage in decreased thereafter indicated that BrCBF,in the course of response to cold(4C treatment) cold acclimation,might be regulated by some upstream ②Springer
was composed of some α-helices similar to CBF3 and CbCBF as protection of the protein from being destroyed by some endoenzymes. A hypothesis for the function of these a-helices structures was that they were involved in DNA binding likely through the interaction of their hydrophobic face with the major groove of DNA. Alternatively, these structures might mediate protein–protein interactions which are important for CBF functions. These interactions might involve the ability to form homo- or hetero-dimmers similar to that observed for the MADS box family of plant regulatory proteins (Huang et al. 1996; Riechmann and Meyerowitz 1997). The results also illustrated that the main structure of BrCOR14 was a-helices which suggested that BrCOR14 was not a globulin, and these a-helices might mediate protein–protein interactions which are important for COR functions (Altus et al. 1996). Besides using real-time PCR expression analysis, it was revealed that expression of BrICE1 increased immediately and showed the highest level of expression at 1 h, followed by dramatic accumulation of BrCBF at 4 h and sharply decreased after that. While the highest level of BrCOR14 expression was observed at 24 h and hold the line till 7 days. These results demonstrated that BrICE1 was expressed constitutively and might be involved in the cold acclimation process, thus, it might belong to the ice gene family. Furthermore, the finding that the expression levels of BrCBF increased at early stages of cold exposure and decreased thereafter indicated that BrCBF, in the course of cold acclimation, might be regulated by some upstream Fig. 5 Real-time fluorescence quantitative PCR analysis of BrICE1, BrCBF, and BrCOR14 genes in non-heading Chinese cabbage in response to cold (4°C treatment) Fig. 4 Comparison of the secondary structure of Brassica campestris ssp. chinensis L. Makino, Arabidopsis thaliana and Capsella bursa-pastoris ICE, CBF, and COR genes helix, turn, strand, and coil were indicated in line with decreased length in turn, respectively 530 Plant Mol Biol Rep (2011) 29:525–532
Plant Mol Biol Rep(2011)29:525-532 531 ■BrICE1口BrCBF BrCOR14 120 elements that had been found to be involved in ABA- 100 ABA regulated gene expression in a number of genes.The other 80 intriguing hypothesis thus raised is that BrCOR/4 was not o involved in the ABA-response pathway. 90 Further studies in ABA,salt and drought stresses also carried out (Yamaguchi-Shinozaki and Shinozaki 1994). 30 Results showed that with salt and drought treatments.the 120 three genes all showed ascending expression,while the NaCl highest expression of the three genes all appeared at 4 h under drought treatment,not coming from ICE,CBF,and % COR in that order.which might likely indicate that the pathway of salt and drought stresses did not completely 40 resembled that of the cold pathway.Only when treated with 20 ABA within 0.5 h did Br/CE/show higher expression than the basal while the other two genes showed no significant 120 difference at different time points.These results resembled 100 Mannitol those of COR genes involved in the expression of cold-, 80 salt-,and drought-regulated genes through an ABA- independent pathway in Arabidopsis at the same time % verifying the hypothesis that BrCOR/4 is not involved in % the ABA-response pathway.While BrICEl was slightly up- 0 Oh 0.5h 2h 4h 8h 24h regulated by drought stress which is different from 4d Time Arabidopsis that ICE was not induced by drought stress Fig.6 Real-time fluorescence quantitative PCR analysis of Br/CE/, (Yang et al.2005).The reason need to be determined BrCBF,and BrCOR/4 genes in non-heading Chinese cabbage in further. response to ABA,salt (NaCl)and drought(Mannitol)stresses Through the above-mentioned bioinformatics analysis and cold acclimation assay,it was found that BrICEl,BrCBF,and genes or proteins,for instance Br/CEl,as enhancers and BrCOR/4 have many common characteristics with Arabi- some downstream genes or their products,specifically dopsis ICEl,CBF3,and CORI5B genes.There is also a BrCOR14,as suppressors.BrICEl increased quickly and BrLOS2 gene being cloned from non-heading Chinese then decreased immediately,while BrCBF and BrCOR/4 cabbage.Therefore,it is highly presumed that non-heading can maintain the higher expression for a relatively long time, Chinese cabbage may have similar cold acclimation process which also verified the hypothesis that BrICEl protein is strongly resembling that of Arabidopsis (Jiang et al.2007c; unstable whereas BrCBF and BrCOR/4 are stable. Yang et al.2005).Results also suggest that BrCBF and In addition,BrCBF contains a dehydration-responsive BrCOR14 are involved in the expression of cold-,salt-and element (Liu et al.1998)which suggests that BrCBF might drought-regulated genes through an ABA-independent path- be responsible not only to cold stress but also to way.So these genes might be the potential breeding dehydration.While a potential ABA-responsive element resources through transformation to improve the cold,salt, (ABRE).CACGTG (Guiltinan et al.1990:Williams et al. as well as drought tolerance.It is important to bear in mind, 1992)is not found in BrCOR/4.ABREs are cis-acting however,that constitutive high-level overexpression of the CBF genes can result in undesirable agronomic traits.In Cold Arabidopsis,high-level CBF overexpression can cause a BrICE1 bHLH poly(A) "stunted"growth phenotype,a decrease in seed yield,and a delay in flowering (Liu et al.1998;Gilmour et al.2000), Binding whether strategies such as using stress-inducible promoters BrCBF- CACCTG AP2 poly(A) to drive BrCBF expression can be developed to attain the Potential MAC domain Binding potential positive effects of CBF regulon engineering without BrCOR14 CGCCGTC poly(A) incurring undesirable negative traits remains to be deter- mined(Kasuga et al.1999).Recently,studies on cold stress Potential GCC box signaling and tolerance also revealed that post-transcriptional Increased Freezing Tolerance regulation at pre-mRNA processing and export from nucleus Fig.7 The anticipated non-heading Chinese cabbage CBF cold- plays a role in cold acclimation.Cold stress-regulated response pathway miRNAs have been identified in Arabidopsis and rice 鱼Springer
genes or proteins, for instance BrICE1, as enhancers and some downstream genes or their products, specifically BrCOR14, as suppressors. BrICE1 increased quickly and then decreased immediately, while BrCBF and BrCOR14 can maintain the higher expression for a relatively long time, which also verified the hypothesis that BrICE1 protein is unstable whereas BrCBF and BrCOR14 are stable. In addition, BrCBF contains a dehydration-responsive element (Liu et al. 1998) which suggests that BrCBF might be responsible not only to cold stress but also to dehydration. While a potential ABA-responsive element (ABRE), CACGTG (Guiltinan et al. 1990; Williams et al. 1992) is not found in BrCOR14. ABREs are cis-acting elements that had been found to be involved in ABAregulated gene expression in a number of genes. The other intriguing hypothesis thus raised is that BrCOR14 was not involved in the ABA-response pathway. Further studies in ABA, salt and drought stresses also carried out (Yamaguchi-Shinozaki and Shinozaki 1994). Results showed that with salt and drought treatments, the three genes all showed ascending expression, while the highest expression of the three genes all appeared at 4 h under drought treatment, not coming from ICE, CBF, and COR in that order, which might likely indicate that the pathway of salt and drought stresses did not completely resembled that of the cold pathway. Only when treated with ABA within 0.5 h did BrICE1 show higher expression than the basal while the other two genes showed no significant difference at different time points. These results resembled those of COR genes involved in the expression of cold-, salt-, and drought-regulated genes through an ABAindependent pathway in Arabidopsis at the same time verifying the hypothesis that BrCOR14 is not involved in the ABA-response pathway. While BrICE1 was slightly upregulated by drought stress which is different from Arabidopsis that ICE was not induced by drought stress (Yang et al. 2005). The reason need to be determined further. Through the above-mentioned bioinformatics analysis and cold acclimation assay, it was found that BrICE1, BrCBF, and BrCOR14 have many common characteristics with Arabidopsis ICE1, CBF3, and COR15B genes. There is also a BrLOS2 gene being cloned from non-heading Chinese cabbage. Therefore, it is highly presumed that non-heading Chinese cabbage may have similar cold acclimation process strongly resembling that of Arabidopsis (Jiang et al. 2007c; Yang et al. 2005). Results also suggest that BrCBF and BrCOR14 are involved in the expression of cold-, salt- and drought-regulated genes through an ABA-independent pathway. So these genes might be the potential breeding resources through transformation to improve the cold, salt, as well as drought tolerance. It is important to bear in mind, however, that constitutive high-level overexpression of the CBF genes can result in undesirable agronomic traits. In Arabidopsis, high-level CBF overexpression can cause a “stunted” growth phenotype, a decrease in seed yield, and a delay in flowering (Liu et al. 1998; Gilmour et al. 2000), whether strategies such as using stress-inducible promoters to drive BrCBF expression can be developed to attain the potential positive effects of CBF regulon engineering without incurring undesirable negative traits remains to be determined (Kasuga et al. 1999). Recently, studies on cold stress signaling and tolerance also revealed that post-transcriptional regulation at pre-mRNA processing and export from nucleus plays a role in cold acclimation. Cold stress-regulated miRNAs have been identified in Arabidopsis and rice Fig. 7 The anticipated non-heading Chinese cabbage CBF coldresponse pathway Fig. 6 Real-time fluorescence quantitative PCR analysis of BrICE1, BrCBF, and BrCOR14 genes in non-heading Chinese cabbage in response to ABA, salt (NaCl) and drought (Mannitol) stresses Plant Mol Biol Rep (2011) 29:525–532 531
532 Plant Mol Biol Rep (2011)29:525-532 (Chinnusamy et al.2010).Does non-heading Chinese Jiang FL,Hou XL,Shi GJ,Cui XM (2007b)Cloning and cabbage have pre-mRNA processing in cold acclimation? characterization of full length cDNA of BrCOR/4 gene from Further studies also need to be carried out. Brassica campestris ssp.chinensis.Jiangsu J of Agr Sci 23:34- 38 (in Chinese) Jiang FL,Hou XL,Shi GJ,Cui XM(2007c)Cloning and characterization Acknowledgements This research was partially supported by the of full length cDNA of BrLOS2 gene from Brassica campestris ssp Natural Science Foundation of Jiangsu Province (BK2009311)and chinensis.J Nanjing Agri Uni 30:27-32 (in Chinese) National Science and Technology Support Program(2009BADB8B03-1). Kasuga M,Liu Q,Miura S,Yamaguchi-Shinozaki K,Shinozaki K (1999)Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor.Nat References Biotechnol 17:287-291 Lee B,Henderson DA,Zhu JK (2005)The arabidopsis cold responsive transcriptome and its regulation by ICEl.Plant Cell Altus NN,Uemura M,Steponkus PL,Gilmour SJ,Lin C,Thomashow 17:3155-3175 MF(1996)Constiutive expression of the cold-regulated Arabidopsis Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K, thaliana COR15a gene affects both chloroplast and protoplast Shinozaki K(1998)Two transcription factors,DREBI and DREB2 freezing tolerance.Plant Biol 93:13404-13409 with an EREBP/AP2 DNA binding domain separate two cellular Buttner M.Singh KB (1997)Arabidopsis thaliana ethylene respon- signal transduction pathways in drought-and low-temperature- sive element binding protein (AtEBP),an ethylene inducible responsive gene expression,respectively,in Arabidopsis.Plant Cell GCC box DNA-binding protein interacts with an ocs element 10:1391-1406 binding protein.Proc Natl Acad Sci USA 94:5961-5966 Medina J,Bargues M,Terol J,Perez-Alonso M,Salinas J(1999)The Chinnusamy V,Ohta M,Kanrar S,Lee BH,Hong XH,Agarwal M, Arabidopsis CBF gene family is composed of three genes Zhu JK (2003)ICEl:a regulator of cold-induced transcriptome encoding AP2 domain-containing proteins whose expression is and freezing tolerance in Arabidopsis.Genes Dev 17:1043-1054 regulated by low temperature but not by abscisic acid or Chinnusamy V.Zhu JH,Zhu JK(2007)Cold stress regulation of gene dehydration.Plant Physiol 119:463-470 expression in plants.Trends Plant Sci 12:444-451 Meng SS,Dane F,Si Y,Ebel R,Zhang CK (2008)Gene expression Chinnusamy V,Zhu JK,Sunkar R(2010)Gene regulation during cold analysis of cold treated versus cold acclimated Poncirus stress acclimation in plants.Methods Mol Biol 639:39-55 trifoliata.Euphytica 164(1):209-219 Fowler DB.Limin AE,Wang S.Ward RW (1996)Relationship Meshi T.Iwabuchi M (1995)Plant transcription factors.Plant Cell between low-temperature tolerance and vernalization response in Physiol36:1405-1420 wheat and rye.Can J Plant Sci 76:37-42 Riechmann JL.Meyerowitz EM (1997)Domain proteins in plant Gao MJ.Allard G.Byass L.Flanagan AM.Singh J(2002)Regulation development.Biol Chem 10:1079-1101 and characterization of four CBF transcription factors from Stockinger EJ,Gilmour SJ,Thomashow MF(1997)Arabidopsis thaliana Brassica napus.Plant Mol Biol 49:459-471 CBFI encodes an AP2 domain-containing transcriptional activator Gilmour SJ,Zarka DG,Stockinger EJ,Salazar MP.Houghton JM. that binds to the C-repeat/DRE,a cis-acting DNA regulatory Thomashow MF (1998)Low temperature regulation of the element that stimulates transcription in response to low temperature Arabidopsis CBF family of AP2 transcriptional activators as an and water deficit.Proc Natl Acad Sci USA 94:1035-1040 early step in cold-induced COR gene expression.Plant J 16:433- Thomashow MF (1999)Plant cold acclimation:freezing tolerance 442 genes and regulatory mechanisms.Annu Rev Plant Physiol Plant Gilmour SJ,Sebolt AM,Salazar MP,Everard JD,Thomashow MF Mol Biol 50:571-599 (2000)Overexpression of the Arabidopsis CBF3 transcriptional Thomashow MF(2001)So what's new in the field of plant cold activator mimics multiple biochemical changes associated with acclimation？Lots！Plant Physiol125:89-93 cold acclimation.Plant Physiol 124:1854-1865 Wang XL,Sun XQ,Liu SX,Liu L,Liu XJ,Sun XF,Tang KX(2005) Goulas E,Dily FL,Ozouf J,Ourry A (2003)Effects of a cold Molecular cloning and characterization of a novel ice gene from treatment of the root system on white clover(Trifolium repens L.) Capsella bursa-pastoris.Mol Biol 39:18-25 morphogenesis and nitrogen reserve accumulation.J Plant Wang L,Li XW,Zhao Q.Jing SL,Chen SF,Yuan HY (2009) Physiol160:893-902 Identification of genes induced in response to low-temperature Guiltinan MJ,Marcotte WR,Quatrano RS (1990)A plant leucine treatment in tea leaves.Plant Mol Biol Rep 27:257-265 zipper protein that recognizes an abscisic acid response element. Williams ME,Foster R,Chua NH(1992)Sequences flanking the Science250:267-271 hexameric G-box core CACGTG affect the specificity of protein Huang H,Tudor M,Su T,Zhang Y,Hu Y,Ma H(1996)DNA binding binding.Plant Cell 4:485-496 properties of two Arabidopsis MADS domain proteins:binding Wilson CL,Hubbard SJ,Doig AJ(2002)A critical assessment of the consensus and dimer formation.Plant Cell 8:81-94 secondary structure a-helices and their termini in proteins Jaglo-Ottosen KR,Gilmour SJ,Zarka DG,Schabenberger O, Protein Eng 15(7):545-554 Thomashow MF (1998)Arabidopsis CBFI overexpression induces Yamaguchi-Shinozaki K,Shinozaki K(1994)A novel cis-acting element COR genes and enhances freezing tolerance.Science 280:104-106 in an Arabidopsis gene is involved in responsiveness to drought. Jaglo-Ottosen KR,Kleff S,Amundsen KL,Zhang X,Haake V,Zhang low-temperature,or high-salt stress.Plant Cell 6:251-264 JZ,Deits T.Thomashow MF (2001)Components of the Yang TW,Zhang LJ,Zhang TG,Zhang H,Xu SJ,An LZ (2005) Arabidopsis C-repeat/dehydration-responsive element binding Transcriptional regulation network of cold-responsive genes in factor cold-response pathway are conserved in Brassica napus higher plants.Plant Sci 169:987-995 and other plant species.Plant Physiol 127:910-917 Zhou N,Robinson SJ,Huebert T,Bate NJ,Parkin IAP (2007) Jiang FL,Hou XL,Shi GJ,Cui XM(2007a)Cloning and characterization Comparative genome organization reveals a single copy of CBF in of full length cDNA of BrCBF gene from Brassica campestris ssp. the freezing tolerant crucifer Thlaspi arvense.Plant Mol Biol chinensis.J Nanjing Agri Uni 30:18-22 (in chinensis) 65:693-705 么Springer
(Chinnusamy et al. 2010). Does non-heading Chinese cabbage have pre-mRNA processing in cold acclimation? Further studies also need to be carried out. Acknowledgements This research was partially supported by the Natural Science Foundation of Jiangsu Province (BK2009311) and National Science and Technology Support Program (2009BADB8B03-1). References Altus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin C, Thomashow MF (1996) Constiutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Plant Biol 93:13404–13409 Büttner M, Singh KB (1997) Arabidopsis thaliana ethylene responsive element binding protein (AtEBP), an ethylene inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. Proc Natl Acad Sci USA 94:5961–5966 Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong XH, Agarwal M, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17:1043–1054 Chinnusamy V, Zhu JH, Zhu JK (2007) Cold stress regulation of gene expression in plants. Trends Plant Sci 12:444–451 Chinnusamy V, Zhu JK, Sunkar R (2010) Gene regulation during cold stress acclimation in plants. Methods Mol Biol 639:39–55 Fowler DB, Limin AE, Wang S, Ward RW (1996) Relationship between low-temperature tolerance and vernalization response in wheat and rye. Can J Plant Sci 76:37–42 Gao MJ, Allard G, Byass L, Flanagan AM, Singh J (2002) Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Mol Biol 49:459–471 Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16:433– 442 Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol 124:1854–1865 Goulas E, Dily FL, Ozouf J, Ourry A (2003) Effects of a cold treatment of the root system on white clover (Trifolium repens L.) morphogenesis and nitrogen reserve accumulation. J Plant Physiol 160:893–902 Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250:267–271 Huang H, Tudor M, Su T, Zhang Y, Hu Y, Ma H (1996) DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation. Plant Cell 8:81–94 Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280:104–106 Jaglo-Ottosen KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, Thomashow MF (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol 127:910–917 Jiang FL, Hou XL, Shi GJ, Cui XM (2007a) Cloning and characterization of full length cDNA of BrCBF gene from Brassica campestris ssp. chinensis. J Nanjing Agri Uni 30:18–22 (in chinensis) Jiang FL, Hou XL, Shi GJ, Cui XM (2007b) Cloning and characterization of full length cDNA of BrCOR14 gene from Brassica campestris ssp. chinensis. Jiangsu J of Agr Sci 23:34– 38 (in Chinese) Jiang FL, Hou XL, Shi GJ, Cui XM (2007c) Cloning and characterization of full length cDNA of BrLOS2 gene from Brassica campestris ssp. chinensis. J Nanjing Agri Uni 30:27–32 (in Chinese) Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291 Lee B, Henderson DA, Zhu JK (2005) The arabidopsis coldresponsive transcriptome and its regulation by ICE1. Plant Cell 17:3155–3175 Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperatureresponsive gene expression, respectively, in Arabidopsis. Plant Cell 10:1391–1406 Medina J, Bargues M, Terol J, Pérez-Alonso M, Salinas J (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol 119:463–470 Meng SS, Dane F, Si Y, Ebel R, Zhang CK (2008) Gene expression analysis of cold treated versus cold acclimated Poncirus trifoliata. Euphytica 164(1):209–219 Meshi T, Iwabuchi M (1995) Plant transcription factors. Plant Cell Physiol 36:1405–1420 Riechmann JL, Meyerowitz EM (1997) Domain proteins in plant development. Biol Chem 10:1079–1101 Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599 Thomashow MF (2001) So what’s new in the field of plant cold acclimation? Lots! Plant Physiol 125:89–93 Wang XL, Sun XQ, Liu SX, Liu L, Liu XJ, Sun XF, Tang KX (2005) Molecular cloning and characterization of a novel ice gene from Capsella bursa-pastoris. Mol Biol 39:18–25 Wang L, Li XW, Zhao Q, Jing SL, Chen SF, Yuan HY (2009) Identification of genes induced in response to low-temperature treatment in tea leaves. Plant Mol Biol Rep 27:257–265 Williams ME, Foster R, Chua NH (1992) Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4:485–496 Wilson CL, Hubbard SJ, Doig AJ (2002) A critical assessment of the secondary structure α-helices and their termini in proteins. Protein Eng 15(7):545–554 Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264 Yang TW, Zhang LJ, Zhang TG, Zhang H, Xu SJ, An LZ (2005) Transcriptional regulation network of cold-responsive genes in higher plants. Plant Sci 169:987–995 Zhou N, Robinson SJ, Huebert T, Bate NJ, Parkin IAP (2007) Comparative genome organization reveals a single copy of CBF in the freezing tolerant crucifer Thlaspi arvense. Plant Mol Biol 65:693–705 532 Plant Mol Biol Rep (2011) 29:525–532