Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. It is quickly followed by activation of the cold repeat binding factor (CBF) regulon, the set of genes that are induced in response to the CBF/ dehydration-responsive element binding (DREB) transcriptional activators. The immediate target genes of the CBF/DREB proteins have C-repeat(CRT)/ dehydration responsive element (DRE) elements in their promoters, the DNA regulatory sequence to which the CBF/DREB transcriptional activators bind (Allen et al., 1998; Jaglo et al., 1998). Expression of the CBF regulon results in an increase in freezing tolerance due, in part, to the induction of genes encoding cryoprotective proteins and enzymes involved in the synthesis of low-molecular weight cryoprotectants (Gilmour et al., 1998; Jaglo et al., 1998; Kasuga et al., 1999; Liu et al., 1998). Expression of the CBF regulon also results in an increase in tolerance to drought and high salinity (Kasuga et al., 1999) and appears to have a role in acclimation to chilling temperatures (Gong et al., 2002). CBF-like proteins containing the signature sequences are present in a wide range of plants including those that cold acclimate, such as Brassica napus (Jaglo et al., 2001) and barley (Hordeum vulgare) (Choi et al., 2002), as well as in plants that do not cold acclimate, such as tomato (Lycopersicon esculentum) (Jaglo et al., 2001; Zhang et al., 2004) and rice (Oryza sativa) (Choi et al., 2002; Dubouzet et al., 2003).

The meristematic tissues of temperate woody perennials must acclimate to freezing temperatures to survive the winter and resume growth the following year. The central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus. The induction kinetics and tissue specificity of four CBF paralogues (PtCBFs) were identified from the Populus trichocarpa genome sequence (Benedict et al., 2006). Is there any difference between Populus trichocarpa and Populus tomentosa? PtCBF5 gene was cloned and its expression in adverse circumstances was analyzed in Populus tomentosa.

1 Materials and methods 1.1 Plant materials

For the preparation of cDNA libraries, seedling was then placed in a 4 ℃ chamber for 4 h (cold). The plant samples were collected, immediately frozen in liquid nitrogen and stored at －70 ℃ for use. The tissue culture plantlets were germinated and grown in Murashige & Skoog basal medium supplemented with 6-BA 1 mg·L^-1 + NAA 0.2 mg·L^-1 in a growth chamber for 30 days, the growth conditions were:temperature, 25 ℃; light:dark, 16 h:8 h.

1.2 RT-PCR molecular cloning of P. tomentosa PtCBF5

RNA isolation and cDNA first strand synthesis of P. tomentosa were described (Zhou et al., 2007). Primers were designed based on putative poplar PtCBF nucleotide sequence, which were synthesized by Invitrogen. PCR Products are fractionated on agarose gel. For DNA sequencing, the band of interest was excised from the gel, purified using the Wizard PCR prep DNA purification kit (Promega), quantified by gel electrophoresis in comparison with DNA low mass standards (Tiangen) and ligated into plasmid pGEM-T (Promega). The plasmid was transformed into Escherichia coli DH5α. Clones containing the PCR products were selected by blue/white screening, conjugated plasmid was named pGEM-T-PtCBF5 which was purified by the Wizard Plus Plasmid DNA purification kit (sbsbio) and detected through digested by EcoR I. The PCR product was sequenced from the plasmid in both directions using the T7 and SP6 primers at the Invitrogen Biotechnology Company.

1.3 Analysis of PtCBF5 sequence

DNA sequence and its coding sequence and peptide were analyzed by DNAstar, Primer Premier 5.00, Match code biosoftwares. The multisequence alignment was performed by the CLUSTAL X program. Phylogenic tree of the amino acid sequence was constructed between PtCBF5 and other plants' CBF genes using Treeview.

1.4 RT-PCR analysis of the PtCBF5 transcripts in different tissues and in leaves under various stress conditions

The expression pattern of the PtCBF5 gene was studied by semi-quantitative RT-PCR (semi-QRT-PCR) in different tissues and under various stress conditions in leaves, in which actin gene was used as a control. The actin fragment was amplified by primers:5′-GGGTAGACCAAGACACACTGG-3′(forward); 5′-GGATGGCATGTGGAAGGGCAT-3′(reverse). The tissue culture plantlets were germinated and grown in Murashige & Skoog basal medium supplemented with 6-BA 1 mg·L^-1 + NAA 0.2 mg·L^-1 in a growth chamber for 30 days, and the growth conditions were:temperature, 25 ℃; light:dark, 16 h:8 h. For analysis of PtCBF5 expression in different tissues, the leaves, stems and roots were collected at normal growth condition. For timecourse analysis of PtCBF5 expression, poplar seedlings were exposed to four different treatments and the leaves were collected respectively at 0, 10, 30, 60 min and 2, 6, 12, and 24 h. The treatments were achieved by transferring into 200 g·L^-1 PEG8000 in liquid MS for drought, chilling at 4 ℃ in a growth chamber for coldness, transferring into 250 mmol·L^-1 NaCl in liquid MS for salt, and transferring into 100 μmol.L^-1 ABA in liquid MS for ABA treatment.

2 Results 2.1 Full-length coden region cDNA of PtCBF5

The full-length cDNA sequence of PtCBF5 was obtained through RT-PCR reaction using the primers designed for the amplification of full-length cDNA sequence of PtCBF5.PtCBF5 primers are:Fr 5′-CGGACCCATAAACCCTATAC-3′; Re 5′-CCCATGA CATTAATTCCTCCG-3′. In a 50 μL PCR reaction, 5 ng cDNA was used as template along with 50 mmol·L^-1 each primer, 2 units Pfu Taq polymerase (Promega). Amplification by PCR was performed with 1 μL of the reaction mixture after reverse transcription. After an initial preincubation step 4 min at 94 ℃, 30 reaction cycles was as follows:30 s at 95 ℃, 30 s at 59 ℃ and 30 s at 72 ℃. A final elongation step was allowed to proceed for 5 min at 72 ℃. A 837 bp fragment was obtained and fractionated on a 1% agarose gel, and then ligated into plasmid pGEM-T. The conjugated pGEM-T-PtCBF plasmid was detected through digested by EcoR I (Fig. 1). The full-length cDNA of PtCBF5 (Genbank No.:DQ354395) contains a 696 bp open reading frame (ORF) encoding a 231 amino acids protein (Fig. 2), 42 bp 5′uncoding translated region (5′UTR) end and 98 bp3′UTR, which were analyzed by DNAstar, Primer Premier 5.00, and Match code biosoftwares.

2.2 Sequence analysis of P. tomentosa PtCBF5

When PtCBF5is blasted in NCBI using tBLASTn program, the hit genes of high scores are all CBF family genes of other species, PtCBF5 belongs to the CBF family. The amino acid sequence of PtCBF5and CBFs from other plants are aligned. CBFs are the following genes:Arabidopsis thaliana AtCBF1, AtCBF2 and AtCBF3 (NM_118679, NM_118681, NM_118680);Oryza sativa OsDREB1A, OsDREB2A and OsDREB1B (AF300970, AF300971, AF300972);Populus balsamifera subsp. trichocarpa CBF1 (JGI666968), PtCBF2 (JGI346104), PtCBF3 (JGI548519), PtCBF4 (JGI63608);Poncirus trifoliata CBF1 (DQ790889);Citrus sinensis CBF2 (CD576150) (Champ et al., 2006); Populus euphratica PeDREB2(EF137176);Populus tomentosa CBF6 (DQ354394). The alignment of fourteen predicted protein sequences indicated that they are highly conserved (Fig. 3).

2.3 PtCBF5 transcripts in different tissues and accumulation of PtCBF5 transcripts in response to abiotic stresses in leaves

The expression pattern of the PtCBF5 gene in tissues and under various stress conditions in leaves through semi-quantitative RT-PCR (semi-QRT-PCR), using actin gene as inner expressing control. The result showed that the transcripts of PtCBF5 are the most abundant mRNA products in leaves, and a low-abundance was detected in roots and stems (Fig. 4).

When the shoots were then exposed for 0, 10, 30, 60 min, 2, 6, 12, or 24 h to the following treatments:cold, chilling at 4 ℃ in a growth chamber; dry, solutions containing 200 g·L^-1 PEG8000;NaCl, saline solutions containing 250 mmol·L^-1 NaCl; ABA, solutions containing 100 μmol·L^-1 ABA. PtCBF5 gene expression was induced within 30 min after freezing began, and PtCBF5 was strongly expressed after 2 h. PtCBF5 mRNA accumulated within 1 h after drought treatment, and it was strongly expressed after 6 h. However, there was no significant PtCBF5 mRNA accumulation within 24 h. Whereas the PtCBF5 mRNA did not accumulate in the treatments of NaCl and ABA (Fig. 5).

3 Discussion

The central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus (Benedict et al., 2006; Chen et al., 2009).PtCBF5 gene isolated from P. tomentosa is a new member of PtCBFs family.

In Populus trichocarpa, all four PtCBFs are cold-inducible in leaf tissue, only PtCBF1 and PtCBF3 are significantly induced in the stem (Benedict et al., 2006). However, the differential expression of the PtCBFs and differing clusters of CBF-responsive genes in annual (leaf) and perennial (stem) tissues suggest that the perennial-driven evolution of winter dormancy may have given rise to specific roles for these 'master-switches' in the different annual and perennial tissues of woody species.

PeDREB2 expression was up-regulated by salt, cold, and dehydration but not ABA application in the leaves of Populus euphratica. Under cold treatment, PeDREB2 expression was induced immediately and the level of product peaked at 6 h. Under dehydration treatment, PeDREB2 transcript accumulated rapidly and reached its maximum at 12 h. For high-salt treatment, expression of PeDREB2 became stronger within 24 h (Chen et al., 2009).PtCBF5 was expressed in all organs, with the highest levels in leaf. However, PeDREB2 was expressed in all organs, with the highest levels in root (Chen et al., 2009). PtCBF5 gene expression was induced within 30 min after freezing began, PtCBF5 is induced slower in poplar than CBF genes are induced in Arabidopsis, and it may be the difference between wood plants and herbage. Expression of PtCBF5can be detected in roots, stems and leaves under normal conditions, indicating its function in normal plant growth and development. Higher expression of PtCBF5 was induced by dehydration and cold treatments, indicating that it may function under osmotic stress like DREB1A/CBF3 members of other species.

Expression of the CBF regulon results in an increase in freezing tolerance due, in part, to the induction of genes encoding cryoprotective proteins and enzymes involved in the synthesis of low-molecular weight cryoprotectants (Gilmour et al., 1998; Jaglo et al., 1998; Kasuga et al., 1999; Liu et al., 1998; Pennycooke et al., 2008). Expression of the CBF regulon also results in an increase in tolerance to drought and high salinity (Kasuga et al., 1999) and appears to have a role in acclimation to chilling temperatures (Gong et al., 2002; Novillo et al., 2007). Identification of downstream target genes of PtCBF5 in poplar is required to determine its detailed functions. Further study using transgenic poplar (antisense RNA and overexpression) will elucidate its involvement in cold and dehydration stresses tolerance of Populus tomentosa.