The in vitro tissue cultures of apple (Malus domestic cv. 'Royal Gala') were grown at 25 ± 1°C under a 16 h photoperiod for gene cloning and genetic transformation. 'Orin' apple calluses were cultured in the dark at 25 ± 1°C and used for genetic transformation. Arabidopsis thaliana (ecotype 'Columbia 0') was grown at 20 ± 1°C under a 16 h photoperiod and used for genetic transformation. Tobacco (Nicotiana tobaccum L. 'NC89') was grown at 25 ± 1°C under a 12 h photoperiod and used for genetic transformation. In vitro tissue cultures of 'Royal Gala' were used for expression analysis after cold treatment (4 ± 1°C).
Semi-quantitative RT-PCR assays for gene expression
Total RNAs were extracted from apple and Arabidopsis samples using the RNAplant plus Reagent (Tiangen, China) and TRIzol Reagent (Invitrogen, USA), respectively, according to the manufacturers' instructions. First-strand cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit transcriptase (TaKaRa, Japan) according to the manufacturer's instructions.
The PCR reaction mixture contained 200 ng cDNA, 1 μl of each primer (10 mM), 2.5 μl 10× Taq DNA polymerase buffer containing Mg2+ (20 mM), 2 μl deoxyribonucleotide (dNTP) (2.5 mM) and 0.25 μl 5 U/μl transTaq DNA polymerase (Trans, China) in a 25 μl reaction volume. For the semi-quantitative RT-PCR reactions, the numbers of reaction cycle were 25-30. MdACTIN was used to normalize cDNA loading. The following primers were used for the PCR reaction: MdCIbHLH1S: 5'-ATGGACGACAGGGAGGAC-3', MdCIbHLHlA: 5'-GGAGGAGGAAGAGTCCAC-3'; MdCBF1S: 5'-CGGTGTTTCGGGGTGTAAG-3'; MdCBF1A: 5'-AAGTGCCCAGCCAAATCC-3'; MdCBF2S: 5'-ATCCGACGGCCGAGATGGCA-3'; MdCBF2A: 5'-CCAAACTCCGCTGGCCGGAA-3'; MdCBF3S: 5'-AAGCGGCGATGATGAGAA-3'; MdCBF3A: 5'-CGTCATCGTTGCTTTCCAT-3'; MdCBF4S: 5'-GTCATTTTGGCATCCAGCAC-3'; MdCBF4A: 5'-TTGTTCCTCCTCACTCCTC-3'; MdCBF5S: 5'-GGAAGCAGCAGAACAGTTT-3'; MdCBF5A: 5'-CACGACATCATCGGCATTG-3'; MdACTIN was amplified using previously reported primers .
Subcellular localization of MdCIbHLH1
The full-length coding region of MdCIbHLH1 was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter. Transient expression of the 35S::MdCIbHLH1:GFP fusion construct and the GFP control vector was introduced into onion epidermal cells using the Agrobacterium-mediated genetic transformation method. Transformed cells were cultured on MS media for 16-24 h in the dark and observed using a laser confocal microscope (Zeiss LSM510, Germany).
Electrophoretic mobility shift assay (EMSA)
The primers used to amplify the MdCIbHLH1 ORF were MdCIbHLH1-zS, 5'-GCGAATTCATGGACGACAGGGAGGAC-3', and MdCIbHLH1-zA, 5'-GCGCGGCCGCCATCATGCCATGGAACCCG-3', containing EcoRI and NotI restriction sites, respectively. The amplified fragment was then inserted into the expression vector pPICZαA (Invitrogen, USA) following digestion with EcoRI and NotI. The resultant expression vector was transformed into the yeast strain GS115 following digestion with BstXI. The MdCIbHLH1 protein was prepared according to the manufacturer's instruction manual. The EMSA was carried out according to the manufacturer's instructions (Thermo, USA). The double-stranded oligonucleotides MYC1 (CATTTTACAATTGCTTCGCT), MYC2 (CTCTGGACACATGGCAGATC), MYC3(5) (ACCCCACCATTTGTTAATGC) and MYC4 (ACAATTACAACTGCATGCTT)  were used as probes and competitors for the EMSAs. MYC4-mut (ACAATTAACACGTCATGCTT) was used as the mutated competitor.
ChIP analysis was performed using the Chromatin Immunoprecipitation Assay Kit (Millipore, USA) and a modified version of the manufacturer's instructions. Protein and DNA were cross-linked by adding formaldehyde directly to the culture medium to a final concentration of 1%, and the reaction was stopped 10 min later by adding 2 M glycine to a final concentration of 0.125 M. Plant extracts were incubated with GFP antibody (Beyotime, China). The antibody-bound complex was precipitated with protein A-Sepharose beads. The DNA fragments in the immunoprecipitated complex were released by reversing the cross-linking at 65°C for 5 h. Purified immunoprecipitated DNA was first analyzed by PCR and visualized by gel electrophoresis. The primers used for the regular PCR are shown in Additional file 1: Table S1. The following PCR conditions were used: incubation at 95°C for 5 min to activate the polymerase; 35 amplification cycles at 95°C for 20 s, 60°C for 15 s and 72°C for 20 s; and a final extension at 72°C for 10 min. The results are expressed as the ratio to input DNA.
Genetic transformation of MdCIbHLH1
The primers used to amplify MdCIbHLH1 cDNA were MdCIbHLH1-fS, 5'-GGATGGACGACAGGGAGGA-3', and MdCIbHLH1-fA, 5'-TGCAATCTACATCATGCCATGG-3'. Subsequently, the PCR product was cloned into the pMD18-T vector (TaKaRa, Japan). MdCIbHLH1 was double-digested with XbaI and SalI and then ligated into the pBI121 vector under the control of the CaMV 35S promoter. The binary construct 35S::MdCIbHLH1 was introduced into Agrobacterium tumefaciens GV3101, and Arabidopsis plants were transformed using the flower dipping method described by .
A. tumefaciens strain LBA4404 containing either the 35S::MdCIbHLH1:GFP or 35S::GFP binary construct was used to transform apple calluses. 'Orin' apple calluses were immersed into Agrobacterium suspension cultures for 10 min. The calluses were then co-cultivated in MS media with 1.5 mg l-1 2,4-D and 0.4 mg l-1 BA at 25 ± 1°C in the dark for two days. After co-cultivation, the callus was transferred to MS screening media containing 1.5 mg l-1 2,4-D, 0.4 mg l-1 BA, 100 mg l-1 kanamycin and 250 mg l-1 carbenicillin. The calluses were subcultured four times at 15-day intervals to obtain transgenic calluses. The same binary construct was used for the apple transformation.
Apple leaf segments were excised from shoots grown in vitro 4 weeks after subculture. Leaf strips were immersed into Agrobacterium suspension cultures for 10 min and then transferred onto MS media for co-cultivation at 25 ± 1°C in the dark for 2 days. The leaf strips were subsequently transferred to MS media containing 0.15 mg l-1 NAA, 5 mg l-1 6-BA, 10 mg l-1 kanamycin and 250 mg l-1 carbenicillin for regeneration and screening. After adventitious shoots regenerated, they were transferred to MS media containing 0.5 mg l-1 6-BA, 0.1 mg l-1 NAA, 10 mg l-1 kanamycin and 250 mg l-1 carbenicillin for subculturing. The plantlets were used for further investigation.
Young leaves of vigorously growing tobacco were excised into approximately 1 cm × 1 cm strips. Leaf strips were immersed into Agrobacterium suspension cultures for 10 min and then transferred onto MS media for co-cultivation at 25 ± 1°C in the dark for 2 days. Subsequently, leaf strips were transferred to MS media containing 0.2 mg l-1 NAA, 3 mg l-1 6-BA, 100 mg l-1 kanamycin and 250 mg l-1 carbenicillin for regeneration and screening. After adventitious shoots regenerated, they were transferred to MS medium containing 1.5 mg l-1 NAA, 150 mg l-1 kanamycin and 250 mg l-1 carbenicillin for rooting. Rooted plantlets were transplanted to soil. After self-pollination, the homozygous transgenic lines were used for further investigation.
Tolerance analysis of transgenic plants
For the growth measurements of Arabidopsis, 7-day-old seedlings were maintained at 4 ± 1°C with a light intensity of 100-150 μmol m-2 sec-1 for 30 days, while at 20 ± 1°C under the same conditions for 7 days as control. The treatment plates were placed vertically with seedlings in the upright position. Three replicates were used for each treatment. Increases in relative root length were measured with a ruler. The relative root length was calculated as the elongation of root length/original root length.
Fifteen-day-old TGC (35S::MdCIbHLH1:GFP) and pBIN (35S::GFP) calluses (0.5 g) were treated with cold stress. For the cold treatment, the calluses were incubated at 15°C in dark. The control calluses were transferred to MS media and grown for an additional 23 days in dark. The weight increment was measured after this period.
Apple shoot tissue cultures were subcultured on MS nutrient media with 3% sucrose and 0.8% agar for 4 weeks, and the shoot tissue cultures were used to examine cold tolerance. Cold stress was imposed by incubating the cultures at 0 ± 1°C for 7 days with a light intensity of 150 μmol m-2 sec-1. The cultures were allowed to recover for 7 days under normal conditions.
Tobacco seedlings were germinated on MS nutrient media with 3% sucrose and 0.8% agar for 15 days, and the seedlings were used to examine chilling and freezing tolerance. Chilling stress was imposed by incubating the seedlings at 4 ± 1°C with a light intensity of 100-150 μmol m-2 sec-1. Seedling survival was scored visually after 30 days. For the freezing tolerance assay, the seedlings were cold-acclimated at 4 ± 1°C for 3 days and then frozen at -10°C for 1 to 5 h in dark. The frozen seedlings were thawed overnight at 4°C and transferred back to normal conditions for recovery. Seedling survival was scored visually after 3 days.
Adult tobacco plants grown in potted soil were also used to assess chilling tolerance. Plants were cold-acclimated for 48 h at 15 ± 1°C under a 16-h photoperiod and then exposed to 4°C for 10 h in dark. The plants that underwent the chilling treatment were allowed to recover under normal conditions for 2 h.
For the chilling assay, the proline and MDA (malondialdehyde) contents were measured along with the relative electrolyte leakage content, as previously described .
Western blot protocol and analysis of sumoylation and ubiquitination
For the western blot analysis, TGC calluses were incubated at 15°C in the dark. A total of 2 g of callus material was ground for each sample in a buffer containing 20 mM Tris (pH 7.4), 100 mM NaCl, 0.5% Nonidet P-40, 0.5 mM EDTA, 0.5 mM PMSF and 0.5% protease inhibitor mixture (Sigma, Germany). MdCIbHLH1 protein levels were determined using a protein gel blot with an anti-GFP antibody, as previously described .
For the ubiquitination and sumoylation assays, 15-day-old calluses were pretreated with 50 μM MG132 proteasome inhibitor in the dark. Total proteins were extracted as described above. The protein complexes were immunoprecipitated using the Pierce Classic Protein A IP Kit (Thermo, USA) and anti-GFP (Beyotime, China). The resultant proteins were separated with SDS-PAGE and blotted onto a PVDF membrane (Roche, USA). The gel blot was probed with anti-Ub (Sigma, Germany) and anti- SUMO1 (Cell Signaling Technology, USA) antibodies and visualized by chemiluminescence using the ECL plus kit (Trans, China) according to the manufacturer's instructions.
The data are presented as the average of three replicates. SAS (Statistical Analysis System) software (SAS Corporation, Cory, NC, USA) was used for the analysis.