Cloning And Functional Characterization Of An Apple BHLH Gene MdCIbHLH1

Apple (Malus×domestica B.) is one of the most important fruit tree crops in the world. Increasing in apple yield could significantly ease the pressure of agricultural products and bring more economical benefits to farmers. Low temperature is one of the major environmental factors that affect apple productivity in some commercial apple-growing regions. MdCIbHLH1 gene was isolated depending on its differential expression in cold response in apple (Malus×domestic cv.'Royal Gala'). To characterize the function of MdCIbHLH1 gene, MdCIbHLH1 gene was introduced into apple callus, tobacco, Arabidopsis and apple with Agrobacterium-mediated transformation.1. Cloning of MdCIbHLH1Within the publicly available apple GenBank (http://www.ncbi.nlm.nih.gov), there are a large number of ESTs showing high similarity in BLAST searches to Arabidopsis bHLH TFs. Differential expression analysis was conducted with semi-quantitative RT-PCR to search for bHLH ESTs associated with cold induction in apple. As a result, a differentially expressed EST was screened out. Subsequently, its full length cDNA was cloned and hereafter named MdCIbHLH1 (Cold-Induced bHLH1; GenBank accession number ABS50521).2. Sequence analysis of MdCIbHLH1MdCIbHLH1 ORF was 1596 bp in length, which encoded a predicated protein containing 531 amino acid residues with a molecular weight of 57.4 kD. Sequence analysis showed that 3 introns were inserted into the open reading frame (ORF) of MdCIbHLH1.Programs MEGA 4.0 were used to construct phylogenetic tree for the bHLH proteins. All arabidopsis bHLH proteins and MdCIbHLH1 were included and aligned. The result showed that MdCIbHLH1 protein was clustered within the same clade featured with AtbHLH116 (AtICE1: INDUCER OF CBF3 EXPRESSION1) and AtbHLH033 (AtICE2). In addition, comparison of genomic structure showed that all MdCIbHLH1, AtICE1 and AtICE2 genes contained four exons, suggesting their similar genomic arrangement.3. Expression of MdCIbHLH1 in different tissues and in response to various stressesThe MdCIbHLH1 transcript levels were analyzed in different tissues by quantitative real-time RT-PCR and semi-quantitative RT-PCR. The result showed that MdCIbHLH1 transcripts constitutively expressed at different levels in various tissues tested such as spring shoot, dormant bud, floral bud, flower, young leaf, mature leaf, young fruit, fruit skin and callus.The response of MdCIbHLH1 expression to cold was examined in balloon-stage flower bud, blossoming flower, young shoot and fruit. The result showed that the cold treatment positively induced the expression of MdCIbHLH1 in these tissues. In addition, it was found that two CBF-like genes MbDREB1 and MdCBF1 exhibited increased expression in these tissues upon exposed to cold signal as the same way as MdCIbHLH1.To examine if MdCIbHLH1 is induced by other stresses, its expression was analyzed with semi-quantitative RT-PCRs under various abiotic stresses. The results showed that MdCIbHLH1 expression was positively induced by low temperature, high salinity, PEG and ABA treatments. Interestingly, the expression of MbDREB1 and MdCBF1 were also positively induced by these stimuli, following the increased expression of MdCIbHLH1.4. MdCIbHLH1 binds to MYC recognition sites in the AtCBF3 promoterTo examine if MdCIbHLH1 protein binds to MYC recognition sites, EMSA was conducted to assess its DNA-binding capacity to four CANNTG motifs, i.e. MYC1, MYC2, MYC3(5) and MYC4, in AtCBF3 promoter. The results showed that the shifted bands were detected in the reaction between MYC4 and MdCIbHLH1 protein. When non-labeled competitor DNA probes of MYC4 were added in the reaction, the shifted bands tended to abolish. In contrast, the complex formed by MdCIbHLH1 and MYC4 fragment was less affected by a mutated competitor than by the wild-type competitor. MdCIbHLH1 protein specifically binds to MYC4 recognition site in AtCBF3 promoter. In addition, band shift was also observed with the reaction between MdCIbHLH1 protein and each of MYC1, MYC2 and MYC3(5) probes. However, the binding was not abolished by the addition of unlabeled wild-type competitor MYC1, MYC2 and MYC3(5), respectively, indicating that the binding of MdCIbHLH1 protein to MYC1, MYC2 and MYC3(5) are not as specific as that to MYC4.5. Overexpression of MdCIbHLH1 in apple callusTo characterize the function in vivo, MdCIbHLH1-GFP fusion gene driven by CaMV 35S promoter was introduced into apple callus with Agrobacterium-mediated transformation. Semi-quantitative RT-PCR showed a higher level of MdCIbHLH1 transcripts in transgenic callus line than control, suggesting that transgenic callus line overly expressed MdCIbHLH1 gene. In addition, the expression of MbDREB1 and MdCBF1 was upregulated in transgenic callus line compared with the control, suggesting that MdCIbHLH1 works upstream of MbDREB1 and MdCBF1, and thereby upregulates their expression. Furthermore, the influence of MdCIbHLH1 overexpression on the growth of transgenic callus was determined. The result demonstrated that the weight increments of transgenic callus line were much more than the control after a 23-day growth under cold and high salinity conditions, indicating the enhanced tolerance of transgenic callus line to cold and salt stresses.6. Degradation and modifications of MdCIbHLH1 ProteinTo determine MdCIbHLH1-GFP protein expression upon cold treatment, TGC callus was treated to analyze MdCIbHLH1-GFP protein by the immunoblot method. The result showed that MdCIbHLH1 protein remarkably degraded upon treated with a 0.5-h cold treatment, suggesting that, besides cold-induced transcription, cold regulates MdCIbHLH1 activity probably through another way, i.e. to modulate its abundance at posttranslational level. To examine if MdCIbHLH1 protein is modified with ubiquitin and SUMO, MdCIbHLH1-GFP protein was immunoprecipitated by anti-GFP antibody from TGC callus. The precipitated proteins were detected with anti-ubiquitin and anti-SUMO antibodies for ubiquitination and SUMOylation modifications, respectively. The result showed that both antibodies detected positive signals, suggesting that ubiquitination and SUMOylation modifications really occurred in MdCIbHLH1 protein of TGC callus in vivo.7. Overexpression of MdCIbHLH1 in tobacco, Arabidopsis and appleTo further characterize the functions in planta, the construct containing MdCIbHLH1 driven by 35S promoter was genetically transformed into tobacco, Arabidopsis and apple. MdCIbHLH1 overexpression enhanced the tolerance to chilling in transgenic tobacco, Arabidopsis and apple, suggesting its involvement in cold stress tolerance.