A R2R3 Type MYB Transcription Factor Is Involved in the Cold Regulation of CBF Genes and in Acquired Freezing Tolerance

Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress. Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress. Cold temperatures have a huge impact on the survivability and distribution of living organisms. Plants, being sessile, have evolved efficient mechanisms to sense and adapt to low temperature stress. Plant responses to adverse low temperature are manifested at physiological, molecular and biochemical levels. Many temperate plants have the potential to increase their freezing tolerance after a prior exposure to nonfreezing temperatures, a process known as cold acclimation (1Guy C.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 187-223Crossref Scopus (1159) Google Scholar, 3Browse J. Xin Z. Curr. Opin. Plant Biol. 2001; 4: 241-246Crossref PubMed Scopus (187) Google Scholar). At the molecular level, a specific set of proteins is induced in response to low temperature, which helps plants cope with chilling and freezing stress (4Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2713) Google Scholar, 8Hsieh T.H. Lee J.T. Yang P.T. Chiu L.H. Charng Y.Y. Wang Y.C. Chan M.T. Plant Physiol. 2002; 129: 1086-1094Crossref PubMed Scopus (385) Google Scholar). Proteins induced during cold acclimation include enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids, and antioxidants, molecular chaperones, antifreeze proteins, and many others with a presumed function in tolerance to cellular dehydration caused by apoplastic freezing (1Guy C.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 187-223Crossref Scopus (1159) Google Scholar, 4Thomashow M.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 571-599Crossref PubMed Scopus (2713) Google Scholar, 9Mohapatra S.S. Wolfraim L. Poole R.J. Dhindsa R.S. Plant Physiol. 1989; 89: 375-380Crossref PubMed Scopus (140) Google Scholar). Promoters of many of the cold-responsive genes have the DRE/CRT/LTRE (dehydration responsive element/C-repeat/low temperature responsive element) sequence, a cis element necessary and sufficient for gene transcription under cold stress (10Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1994; 6: 251-264Crossref PubMed Scopus (1571) Google Scholar, 12Jiang C. Iu B. Singh J. Plant Mol. Biol. 1996; 30: 679-684Crossref PubMed Google Scholar). The CBF/DREB family of transcription factors binds to this sequence and activates cold-responsive genes (11Stockinger E.J. Gilmour S.J. Thomashow M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1035-1040Crossref PubMed Scopus (1428) Google Scholar, 13Liu Q. Sakuma Y. Abe H. Kasuga M. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2424) Google Scholar). The CBF transcription factor genes are also induced by cold, and their induction is regulated by components upstream in the cold response pathways (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar, 17Teige M. Scheikl E. Eulgem T. Doczi R. Ichimura K. Shinozaki K. Dangl J.L. Hirt H. T. Mol. Cell. 2004; 15: 141-152Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar). In addition, it has been shown that a loss-of-function mutation in CBF2 results in increased expression of CBF1 and CBF3, implying that CBF2 negatively regulates the expression of CBF1 and CBF3 (18Novillo F. Alonso J.M. Ecker J.R. Salinas J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3985-3990Crossref PubMed Scopus (430) Google Scholar). In addition to the CBF pathway, recent studies have revealed the presence of parallel pathways associated with cold acclimation (19Seki M. Narusaka M. Abe H. Kasuga M. Yamaguchi-Shinozaki K. Carninci P. Hayashizaki Y. Shinozaki K. Plant Cell. 2001; 13: 61-72Crossref PubMed Scopus (941) Google Scholar, 21Kreps J.A. Wu Y. Chang H.S. Zhu T. Wang X. Harper J.F. Plant Physiol. 2002; 130: 2129-2141Crossref PubMed Scopus (1225) Google Scholar). Some important components mediating cold tolerance through CBF-independent pathways include homeodomain and MYB-type transcription factors (22Zhu 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 (208) Google Scholar, 23Zhu J. Verslues P.E. Zheng X. Lee B.H. Zhan X. Manabe Y. 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 (161) Google Scholar). Support for the existence of CBF-independent pathways has also come from the analysis of the eskimo1 mutants of Arabidopsis (24Xin Z. Browse J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7799-7804Crossref PubMed Scopus (335) Google Scholar), which are constitutively freezing tolerant, without any apparent effect on the CBF regulon. Apart from large changes in gene transcript levels, extensive reconfiguration of the metabolome also takes place in response to cold temperatures (25Cook D. Fowler S. Fiehn O. Thomashow M.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15243-15248Crossref PubMed Scopus (552) Google Scholar, 26Kaplan F. Kopka J. Haskell D.W. Zhao W. Schiller K.C. Gatzke N. Sung D.Y. Guy C.L. Plant Physiol. 2004; 136: 4159-4168Crossref PubMed Scopus (800) Google Scholar). A critical component in the activation of CBF3 and a number of other cold-responsive transcription factor genes in Arabidopsis is ICE1 (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar). ICE1 is a constitutively expressed transcription factor of the bHLH 2The abbreviations used are: bHLH, basic helix-loop-helix; GST, glutathione S-transferase; GFP, green fluorescent protein; Luc, luciferase; RNAi, RNA interference; EMSA, electrophoretic mobility shift assay; WT, wild type. family that can bind to the Myc recognition elements in the CBF3 promoter. A dominant mutation in ICE1 blocks the cold induction of CBF3 and many other transcription factors, and reduces the expression of their downstream genes (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar, 27Lee B.H. Henderson D.A. Zhu J.K. Plant Cell. 2005; 17: 3155-3175Crossref PubMed Scopus (596) Google Scholar). Apart from Myc recognition sequences, many putative Myb binding sequences are present in the promoters of CBF genes (28Shinwari Z.K. Nakashima K. Miura S. Kasuga M. Seki M. Yamaguchi-Shinozaki K. Shinozaki K. Biochem. Biophys. Res. Commun. 1998; 250: 161-170Crossref PubMed Scopus (291) Google Scholar) indicating that MYB-like transcription factors may also play a role in controlling CBF gene expression. Furthermore, some reports suggest that the interplay of MYC-like bHLH transcription factors and MYB co-transcription factors and/or WD repeat containing factors is required for transcriptional activation of target genes (29Spelt C. Quattrocchio F. Mol J.N.M. Koes R. Plant Cell. 2000; 12: 1619-1631Crossref PubMed Scopus (380) Google Scholar, 30Walker A.R. Davison P.A. Bolognesi-Winfield A.C. James C.M. Srinivasan N. Blundell T.L. Esch J.J. Marks M.D. Gray J.C. Plant Cell. 1999; 11: 1337-1349Crossref PubMed Scopus (737) Google Scholar). In the present study, we have identified a MYB-like transcription factor involved in the cold regulation of CBF genes. This transcription factor, referred to as MYB15, interacts physically with ICE1. MYB15 binds to sequences in the promoters of CBF1, 2, and 3 genes. Transgenic plants overexpressing MYB15 show reduced levels of CBF3, CBF2 and CBF1 transcripts in the cold. MYB15 loss-of-function mutant plants show increased levels of CBF3 as well as CBF1 and CBF2. Overexpression of MYB15 results in decreased tolerance to freezing stress, whereas its knock-out mutant exhibits increased freezing tolerance. These results suggest that MYB15 is involved in the cold-regulation of CBF genes and in cold stress tolerance. Gene Expression Analysis—For RNA analysis, 10-day-old seedlings of wild-type and ice1 mutant plants grown on separate halves of the same Murashige-Skoog (MS) nutrient agar plate were used. Total RNA extracted from control and stressed plants was analyzed by RNA blotting as described by Liu and Zhu (31Liu J. Zhu J.K. Plant Physiol. 1997; 114: 591-596Crossref PubMed Scopus (293) Google Scholar). RNA isolated from the plants overexpressing MYB15, the of MYB15 and the myb15 mutant was extracted and to The was with MYB15 to the of CBF3, and gene was used as a the MYB15 expression analysis in RNA was extracted from and of RNA was used to The was used to MYB15 gene and gene was as an control the and was with and and in the and of the The to of ICE1 was from in as a with and and in the and of the of the of ICE1 were with and in and of the and were in the of and were on U. M. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). were for Expression and of in MYB15 in was with the and The was in the and of the and was with and and in and of The protein was to the MYB15 and were E. were grown at to of and for protein expression was induced by for at The were by and the were in and and on for and were The was by at for at was in the and by the addition of at the were and with proteins were with glutathione binding with CBF were from the CBF promoters of the are shown in with were from with of the were with and A of of the was with of protein at temperature for protein was with of for at temperature prior to their with the The complex was on in and by Expression was in and of the expression M. M. H. Plant J. 2000; PubMed Google Scholar). The of the (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar) and a responsive M. M. H. Plant J. 2000; PubMed Google Scholar), were Arabidopsis by Y. L. M. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). In were to the of MYB15 and ICE1. MYB15 in and in and of were used for in transcription and ICE1 and were in and of A of of the was in with of the RNA and of the transcript of MYB15 and was in with of the in the presence of and transcripts were in the of and their proteins were with of the to the and on the were used to In a separate was and used for with were as described U. M. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Expression and of of the MYB15 a upstream of the of MYB15 was with and from the Arabidopsis and in and of The was in and in Arabidopsis plants by N. Mol. Biol. 1998; Google Scholar). The plants were on containing of Transgenic seedlings were with at as described in R.A. J. 6: PubMed Scopus Google Scholar) and under an of the MYB15 was with and and in and of containing a gene D.W. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). seedlings on with were on and were under a with a and a Transgenic and was with and and in of The containing the was from the and in the of the The was the of Arabidopsis plants was by The plants were by 3 at after from were and used in the plants were and of overexpression plants were used for the of the of MYB15 was with and The was in and of the The was Arabidopsis and were on with of mutant of MYB15 in were used to the of gene knock-out was by of MYB15 with RNA was extracted from the and analyzed for CBF expression. the freezing tolerance of the MYB15 overexpression and wild-type plants were in after freezing was as described by M. L. Lee H. B. Zhu J.K. Plant Cell. 1998; 10: PubMed Scopus Google Scholar). for was in a containing of and the was in a freezing set at temperature were The temperature of the was to to at the temperature was were and on to The were to containing of and by of The with the were to temperature, of the were The of was as the of the that after The was with in results from of the are freezing was as described (24Xin Z. Browse J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7799-7804Crossref PubMed Scopus (335) Google Scholar). wild-type and myb15 were in separate halves of the same agar plate with and were used for of freezing of at the were at under seedlings were at and for in were on in a freezing set to for were on the plants the was to at The were after being at the temperatures for at for in the and to under of the seedlings was after MYB15 Expression in and ice1 MYB transcription factors that may function with ICE1 in cold response we from a of the of wild and ice1 mutant plants with cold for (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar). We that the expression of as MYB15 was in ice1 mutant that in the wild type. RNA analysis that MYB15 expression is up-regulated by cold stress in the wild and ice1 mutant plants with the MYB15 expression is in ice1 after of cold The expression is also after 3 of cold the in ice1 after of cold as well as in that MYB15 is expressed constitutively at low levels in Transgenic plants the gene under the control of the MYB15 were analyzed to the distribution of was in and indicating that MYB15 is MYB15 with analysis was used to MYB15 with ICE1. of ICE1 protein were used as and MYB15 was used as to their the ICE1 protein activation of the gene we used of ICE1 as MYB15 with of ICE1 with the of ICE1 The of MYB15 and ICE1 was the to activate the gene The of ICE1 was by and used as to the MYB15 in ICE1. The to of ICE1 was to with MYB15 We used protein to the ICE1 and was to ICE1 ICE1 was to MYB15 was was to ICE1 MYB15 proteins, These results suggest that MYB15 interacts with ICE1. MYB15 to Myb in the Promoters of CBF mobility shift were to MYB15 bind to elements in CBF of the CBF promoters were and used for complex was with and of the CBF1 whereas other of the CBF1 binding with MYB15 CBF2 were binding was with and whereas binding was with MYB15 was to bind to of the CBF3 promoter. These were by the addition of cold with the same sequences. factors to the Myb family have binding to Myb recognition sequences and A. J.M. A. J. Plant J. 1998; PubMed Scopus Google Scholar) Myb recognition sequences. MYB15 binds to and and binds to a to Myb recognition sequence A. J.M. A. J. Plant J. 1998; PubMed Scopus Google Scholar). The CBF used in this was to have many sequences to and Myb recognition A of the sequences present in the of promoters and the binding of MYB15 is shown in The results that MYB15 can bind to the CBF and the binding is by the Myb recognition of and Myb recognition sequences in the promoters of CBF1, and The used for mobility shift are in the and the binding is in the to the of the binding in a MYB15 and a in the of the MYB15 MYB15 was to the of the green fluorescent protein by a was expressed in Arabidopsis plants. of in the plants that the protein is present in the that MYB15 is under Cold stress the of MYB15 expression were to MYB15 a transcriptional was by MYB15 with the binding of the transcriptional under the control of a the and a were Arabidopsis by the increased to the control with without an containing the binding These results that MYB15 a transcriptional MYB15 Overexpression the Expression of CBF Genes under Cold was under the control of the in wild-type Arabidopsis plants the were analyzed in seedlings of the MYB15 overexpression in the wild was in MYB15 overexpression without cold cold was expressed at a in wild-type plants A and MYB15 overexpression show expression after the cold A and analysis that MYB15 was expressed to levels in the overexpression of cold stress, MYB15 transcript levels increased to levels in the overexpression We also the expression of and genes to These genes were up-regulated by cold stress transcript levels to reduced in MYB15 overexpression We analyzed of the overexpression for expression of CBFs and their downstream genes. control transcript of of the CBF genes was in the wild MYB15 overexpression 3 of cold stress, CBF3 as well as CBF1 and CBF2 transcript was present in the wild-type plants as with the MYB15 overexpression At the other was in the expression of CBF genes the wild and MYB15 overexpression in and downstream genes of was the MYB15 Expression of CBF Genes under Cold MYB15 in the to the effect of MYB15 In the expression was induced by cold to levels with the wild-type A and MYB15 gene in the was by analysis We the expression of CBFs and their downstream genes in of the MYB15 At 3 of cold stress was in expression of CBF1 and CBF2 genes in wild and CBF3 expression in the at this At and of cold stress CBF1, and CBF3 genes transcript levels in the as with the wild type. the downstream genes and in their expression wild and the the we identified a mutant in MYB15 analysis that the a of MYB15 transcript whereas the wild and a MYB15 expression The mutant was analyzed for the expression of the CBFs and their downstream genes. the mutant and plants expression of CBF1, and after 3 of cold genes to have levels of expression in the mutant as with the wild-type seedlings The levels of expression of the CBF genes in the mutant were at of cold stress after of cold the expression of the CBF genes was the mutant and wild-type plants expression in the mutant as with wild after of cold stress. expression in the myb15 mutant as with the wild at the and during cold stress. These results that MYB15 may play a role in controlling the expression of the CBF genes in We the expression of and and that their expression was in the myb15 mutant MYB15 Plant the effect of and of MYB15 on freezing an was Overexpression of MYB15 in levels of indicating a decreased freezing tolerance A and The for the wild-type and MYB15 overexpression plants were and under the for the wild-type plants increased to was for the MYB15 overexpression plants. In the myb15 mutant was to freezing stress under acclimation and and The for the wild-type and mutant plants were and The myb15 mutant was also to a freezing to the role of MYB15 in after freezing The myb15 mutant increased as with wild-type seedlings under freezing temperatures of the mutant seedlings of wild-type seedlings a freezing results show that MYB15 overexpression results in decreased freezing whereas its knock-out results in increased freezing tolerance. Cold acclimation involves a of transcriptional constitutively expressed transcription factors are in response to cold, and turn on the transcription of transcription factors as the CBF proteins can activate the expression of downstream cold-responsive genes that proteins with ICE1 has been identified as a transcription factor upstream of CBF3 (14Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1241) Google Scholar). In the present study, transcription factor, MYB15, was to play a role in the regulation of CBF genes under cold stress. MYB15 physically interacts with ICE1. transcription factors have been shown to important for the regulation of downstream genes (29Spelt C. Quattrocchio F. Mol J.N.M. Koes R. Plant Cell. 2000; 12: 1619-1631Crossref PubMed Scopus (380) Google Scholar, 30Walker A.R. Davison P.A. Bolognesi-Winfield A.C. James C.M. Srinivasan N. Blundell T.L. Esch J.J. Marks M.D. Gray J.C. Plant Cell. 1999; 11: 1337-1349Crossref PubMed Scopus (737) Google Scholar, E. L. J.M. B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). the of on the expression of the CBF genes is at the present the of an with other a role for MYB15 in gene expression. The proteins and bHLH proteins is known to through a sequence in MYB proteins and in of bHLH proteins B. J.F. Plant J. 2004; PubMed Scopus Google Scholar). ICE1 MYB15 have sequences, the is by MYB15 is expressed at a low in and is in the cold stress, MYB15 expression is the of this is in the ice1 ICE1 may binding to MYB15 through its downstream MYB15 expression in response to cold. MYB15 to the family of transcription factors and can bind to the of CBF with the presence of Myb recognition sequences in The role of MYB15 in is by that MYB15 and the expression of CBF genes and freezing tolerance. from the that MYB15 has transcriptional activation from MYB15 overexpression and mutant plants that MYB15 is a of CBF expression. The effect of MYB15 on CBF1 and is as on The CBF genes show increased expression in the knock-out mutant plants and decreased expression in the overexpression MYB15 with ICE1 and binding to CBF elements suggest a effect of MYB15 on CBF gene we an of by the complex network of transcriptional regulation of genes. in studies that CBF2 is a of downstream cold-responsive genes and its overexpression in plants leads to increased expression of downstream cold-responsive genes and freezing tolerance Q. Sakuma Y. Abe H. Kasuga M. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1998; 10: 1391-1406Crossref PubMed Scopus (2424) Google Scholar, M. Liu Q. Miura S. Yamaguchi-Shinozaki K. Shinozaki K. 1999; 17: PubMed Scopus Google Scholar, S.J. Fowler Thomashow M.F. Plant Mol. Biol. 2004; PubMed Scopus Google Scholar). in knock-out mutant downstream cold-responsive genes and freezing tolerance are also increased (18Novillo F. Alonso J.M. Ecker J.R. Salinas J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3985-3990Crossref PubMed Scopus (430) Google Scholar). This may by a regulation of CBF2 on CBF1 and 3 and the of CBF1 and 3 expression in mutant leads to the of Myb genes to MYB15 revealed that and expression was reduced in MYB15 overexpression their expression was by myb15 MYB15 and are other Myb and Myc family of transcription factors involved in the regulation of CBF genes. These proteins may with and/or with other physically and and a complex of transcription have a of MYB15 function in cold responsive gene of transcription factors to identified and in the It is that MYB15 may function as an and on the target sequences and proteins, as have been for some transcription factors in Y. N. 2002; PubMed Scopus Google Scholar, Biochem. Biophys. 2000; PubMed Scopus Google Scholar). is a in the role of MYB15 in gene expression. the CBF genes are negatively by MYB15 levels, the CBF downstream genes and are in the MYB15 overexpression plants. studies have that the CBFs are the transcription factors involved in the regulation of downstream genes (22Zhu 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 (208) Google Scholar, 23Zhu J. Verslues P.E. Zheng X. Lee B.H. Zhan X. Manabe Y. 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 (161) Google Scholar). negatively MYB15 may at the same other transcriptional of the downstream genes. It is also that MYB15 may negatively transcriptional of the downstream genes. These to a complex of transcriptional regulation of cold responsive gene expression. the function of MYB15 in this transcriptional results show that MYB15 a role in freezing tolerance and in the regulation of CBF genes under cold stress.