Gene cloning, sequence analysis and MdCIbHLH1 subcellular localization
Within the publicly available apple GenBank (http://www.ncbi.nlm.nih.gov
), there are a large number of ESTs exhibiting high similarity to Arabidopsis
bHLH TF (transcription factors) from BLAST searches. Differential expression analysis was conducted with semi-quantitative RT-PCR to identify bHLH ESTs associated with cold induction in apple, and a differentially expressed EST was isolated using this methodology (Figure ). Subsequently, the full-length cDNA was cloned using the RACE technique following EST-based in silico
cloning. Hereafter, the gene is referred to as MdCIbHLH1
; GenBank accession number ABS50521
Figure 1 Gene expression, sequence analysis and subcellular localization of MdCIbHLH1.(A) Semi-quantitative RT-PCR analysis of MdCIbHLH1 at 25°C and 4°C. The apple tissue cultures were treated for 4 h. (B) Genomic structure of MdCIbHLH1, AtICE1 (more ...)
Sequence analysis showed that three introns were present in the open reading frame (ORF) of MdCIbHLH1 along with four exons of the following lengths: 1113 bp, 230 bp, 169 bp and 84 bp (Figure ). The MdCIbHLH1 ORF was found to be 1596 bp in length and to encode a predicted protein containing 531 amino acid residues with a molecular weight of 57.4 kD.
The MEGA 4.0 program was used to construct phylogenetic trees for the bHLH proteins. For this analysis, all Arabidopsis bHLH proteins and MdCIbHLH1 were included and aligned. The MdCIbHLH1 protein clustered within the same clade as AtbHLH116 (AtICE1) and AtbHLH033 (AtICE2) (Figure ). In addition, genomic comparisons revealed that the MdCIbHLH1, AtICE1 and AtICE2 genes all contained four exons, indicating a similar genomic composition (Figure ).
The predicted MdCIbHLH1 protein was found to contain a conserved bHLH domain and a nuclear localization signal http://www.bioinfo.tsinghua.edu.cn/SubLoc/
. The bHLH domain of MdCIbHLH1 exhibited high similarity to the bHLH domains of ICE proteins in other plant species (Figure ).
The presumed nuclear localization signal in MdCIbHLH1 was sufficient to direct a GFP fusion protein to the nucleus. The subcellular localization of MdCIbHLH1 was investigated by introducing a translational fusion between MdCIbHLH1 and GFP into onion epidermal cells using Agrobacterium-mediated transformation. Cells expressing the MdCIbHLH1:GFP fusion gene showed GFP fluorescence only in the nucleus (Figure ), while cells expressing GFP alone showed GFP fluorescence throughout the entire cell (Figure ). This result suggests that the MdCIbHLH1 protein subcellularly localizes to the nucleus.
Expression of MdCIbHLH1 in different tissues and in response to cold stress
MdCIbHLH1 transcript levels were analyzed in different tissues by semi-quantitative RT-PCR. The results showed that MdCIbHLH1 transcripts were constitutively present at different levels in the various tissues tested, including the spring shoot, dormant bud, floral bud, flower, young leaf, mature leaf, young fruit, fruit skin and callus (Figure ). To determine whether MdCIbHLH1 is induced by cold stress, its expression was analyzed by semi-quantitative RT-PCR in apple tissue cultures exposed to 4°C for different amounts of time. The results showed that MdCIbHLH1 expression was positively induced by the chilling treatment at 4°C (Figure ).
Figure 2 Expression profiles of the MdCIbHLH1 gene in apple. (A) Expression analysis of MdCIbHLH1 in SS (spring shoot), DB (dormant bud), FB (floral buds), F (flower), YL (young leaf), ML (mature leaf), YF (young fruit), FS (fruit skin) and C (callus). (B) Expression (more ...)
, AtCBFs are crucial components acting downstream of AtICE1 in the cold signaling pathway ([9
]). In the apple genome, there are five CBF genes: MdCBF1
(NM_124578) and MdCBF5
(NM_101131). Phylogenetic analysis indicated that the five MdCBFs are highly similar to CBFs in other plant species, including grapevine, poplar, tomato, Arabidopsis
and rice (Figure ). Interestingly, the expression of all five MdCBFs
was positively induced by chilling treatment at 4°C following the increased expression of MdCIbHLH1
(Figure ), suggesting that MdCIbHLH1
may act upstream of the MdCBFs
MdCIbHLH1 regulates AtCBFs by binding to their promoters in Arabidopsis
The similarity of MdCIbHLH1 to AtICE1 and AtICE2 indicates that it may function in a similar way to the AtICE proteins. To examine if the MdCIbHLH1 protein binds to MYC recognition sites associated with cold signaling, as AtICE1 does, EMSA was conducted to assess the DNA-binding capacity of MdCIbHLH1 to four CANNTG motifs, MYC1, MYC2, MYC3(5) and MYC4, in the AtCBF3
]. Shifted bands were observed for MYC4 (Figure ). When 100-fold and 400-fold excess of non-labeled competitor DNA probe for MYC4 was added to the reaction, the presence of shifted bands was reduced (Figure ). In contrast, a mutated competitor had less of an effect on the binding between MdCIbHLH1 and the MYC4 fragment, compared to the wild-type competitor (Figure ). Therefore, the excess quantity of the non-labeled probe interfered with the specific binding of the labeled MYC4 promoter fragment. The non-specific competitor did not have this effect, indicating that the MdCIbHLH1 protein specifically binds to the MYC4 recognition site in the AtCBF3
Figure 3 MdCIbHLH1 binds to the AtCBF3 promoter and increased chilling tolerance in Arabidopsis. (A) Interaction between the MdCIbHLH1 protein and labeled MYC1, MYC2, MYC3(5) and MYC4 DNA fragments. (B) MdCIbHLH1 binding to the MYC4 DNA fragment. The labeled probes (more ...)
Band shifts were also observed for the reactions between MdCIbHLH1 and the MYC1, MYC2 and MYC3(5) probes (Figure ). However, the binding was not abolished by the addition of unlabeled wild-type MYC1, MYC2 and MYC3(5) competitor, respectively (Figure ), indicating that the binding of MdCIbHLH1 to MYC1, MYC2 and MYC3(5) was not as specific as the binding to MYC4. This is not the case for AtICE1, which specifically binds to all four recognition sites [9
To further characterize the functions of the MdCIbHLH1 gene, a construct containing MdCIbHLH1 driven by the CaMV 35S promoter was genetically transformed into Arabidopsis. Three homozygous transgenic lines, L1, L2 and L3, were used for further investigation. Semi-quantitative RT-PCR showed higher levels of MdCIbHLH1 transcripts in the three lines than in the WT (wild type) control. In addition, the expression of AtCBFs increased remarkably in the 3 transgenic lines compared to the control (Figure ). Transgenic seedlings were used to examine chilling tolerance. Relative root lengths were measured after the chilling treatment. The results showed that at 20°C the ectopic expression of MdCIbHLH1 inhibited the root growth of the transgenic Arabidiopsis seedlings (Figure ). In contrast, at 4°C the relative root lengths of L1, L2 and L3 transgenic seedlings were 1.1, 1.04 and 1.19, respectively, while that of WT control was 0.52 (Figure ). Therefore, heterologous expression of MdCIbHLH1 conferred enhanced chilling tolerance in transgenic Arabidopsis seedlings.
MdCIbHLH1 regulates cold tolerance via the CBF pathway in apple
EMSA was used to verify the specific binding of MdCIbHLH1 to the MYC sequence in the AtCBF3 promoter. The promoters of the five MdCBFs also contain MYC recognition sites, and ChIP-PCR was conducted to determine whether MdCIbHLH1 protein binds to the promoters of the five CBF genes in apple. An MdCIbHLH1-GFP fusion construct driven by the CaMV 35S promoter was introduced into apple tissue cultures and calluses using Agrobacterium-mediated transformation. ChIP assays were performed using GFP antibodies. The ChIP product of transgenic callus transformed with an empty vector was used as a nonspecific control. After isolation of cross-linked chromatin, immunoprecipitated DNA was analyzed by PCR (Figure ). The results showed that MdCIbHLH1 specifically bound to the MdCBF2 promoter at three regions (Figure ). However, it failed to bind to the regions tested in the MdCBF1, MdCBF3, MdCBF4 and MdCBF5 promoters. The specific binding of MdCIbHLH1 to the MdCBF2 promoter was also observed in transgenic apple calluses (Figure ).
Figure 4 ChIP analysis of MdCIbHLH1 in transgenic apple callus containing either the empty vector (pBIN) or the MdCIbHLH1 gene (TGC) . (A-E) PCR products amplified from the promoters of MdCBF1 (A), MdCBF2 (B), MdCBF3 (C), MdCBF4 (C) and MdCBF5 (D) were size-fractionated (more ...)
For the functional characterization of MdCIbHLH1 in apple callus, the transgenic callus line expressing the MdCIbHLH1 gene was referred to as the TGC (transgenic callus) line, and the transgenic callus line containing the empty vector pBIN was referred to as the pBIN control. Semi-quantitative RT-PCR showed higher transcript levels of MdCIbHLH1 in the TGC line than in the pBIN control, suggesting that the MdCIbHLH1 gene was overexpressed in the TGC line. In addition, the expression of MdCBF1, MdCBF2, MdCBF3, MdCBF4 and MdCBF5 was upregulated in the TGC line compared to the pBIN control (Figure ), further suggesting that MdCIbHLH1 functions upstream of the MdCBFs and upregulates their expression in part by directly binding to the MdCBF2 promoter. These results indicate that MdCIbHLH1 overexpression conferred enhanced chilling tolerance in the TGC line (Figure ).
Figure 5 Stress tolerance assay of MdCIbHLH1 in transgenic apple. (A) Semi-quantitative RT-PCR analysis of MdCIbHLH1, MdCBF1, MdCBF2, MdCBF3, MdCBF4 and MdCBF5 in transgenic apple calluses containing either an empty vector (pBIN) or the MdCIbHLH1 gene (TGC). Differences (more ...)
The function of MdCIbHLH1 was also characterized in transgenic apple tissue cultures. Semi-quantitative RT-PCR showed high transcript levels of MdCIbHLH1 in 2 transgenic lines, L1 and L2, indicating that the MdCIbHLH1 gene was overexpressed in these transgenic lines. In addition, MdCIbHLH1 was found to upregulate the expression of MdCBF1, MdCBF2, MdCBF3, MdCBF4 and MdCBF5 (Figure ), further confirming that MdCIbHLH1 works upstream of the MdCBFs. WT and MdCIbHLH1 transgenic lines grown on MS media were exposed to 0°C temperature for 7 days. Following a 7-day recovery, the WT control was nearly dead, while the 2 transgenic lines were still alive (Figure ). MdCIbHLH1 overexpression therefore improved the tolerance to cold stress in transgenic apple lines.
Overexpression of MdCIbHLH1 in tobacco
To examine the function of MdCIbHLH1 in another crop, transgenic tobacco plants heterologously expressing the MdCIbHLH1 gene were obtained. Semi-quantitative RT-PCR showed that the MdCIbHLH1 gene was heterologously expressed in three transgenic tobacco lines, L1, L2 and L3, at different levels (Figure ). Fifteen-day-old seedlings were used to examine tolerance to chilling stress. After maintaining the seedlings at 4°C for 30 days, their survival rate was determined. The results showed that the survival ratios of L1, L2 and L3 seedlings were 26.2 ± 1.2%, 61 ± 2.9% and 81.4 ± 2.3%, respectively, while that of the WT control was 6.9 ± 2% (Figure ). In addition, the three transgenic lines produced much more protectant proline but exhibited reduced injury, as indicated by the reduced MDA content and electrolyte leakage under chilling conditions compared to WT (Figure ). Heterologous MdCIbHLH1 expression therefore conferred enhanced chilling tolerance to transgenic tobacco plants. Fifteen-day-old seedlings were also used to assess freezing tolerance. The results showed that ectopic expression of MdCIbHLH1 noticeably enhanced the freezing tolerance of L1, L2 and L3 seedlings (Figure ). The survival ratios of the three transgenic lines were positively correlated with the expression levels of MdCIbHLH1, indicating that their enhanced tolerance was derived from the ectopic expression of MdCIbHLH1.
Figure 6 Accumulation of MdCIbHLH1 and the effects of MdCIbHLH1 on chilling tolerance in tobacco. (A) The transcript levels of MdCIbHLH1 in transgenic tobacco. The NtACTIN gene was used as a loading control. WT, wild-type; L1, L2 and L3, transgenic tobacco lines. (more ...)
Adult plants were also used to assess chilling stress tolerance. The results showed that L1, L2 and L3 plants exhibited enhanced tolerance to chilling compared to WT plants (Figure ). After 4°C treatment for 10 h, WT plants started to wilt, while transgenic plants were near normal in appearance. Following a 2-h recovery under normal conditions, WT plants showed necrosis at the leaf margin, while the three transgenic lines completely recovered (Figure ). In addition, transgenic plants produced much less MDA than WT plants and therefore had less injury in their membrane systems, as indicated by the reduced electrolyte leakage relative to the WT control under chilling stress (Figure ).
Taken together, these data indicate that MdCIbHLH1 is involved in chilling tolerance in various plant species.
MdCIbHLH1 protein is modified by ubiquitination and sumoylation
To determine the stability of the MdCIbHLH1-GFP fusion protein under cold stress, immunoblots were conducted using transgenic apple calluses. The results showed that MdCIbHLH1 protein gradually degraded to very low levels upon exposure to cold stress for 6 h. However, MG132, a 26S proteasome-specific protease inhibitor, remarkably suppressed the cold-induced protein degradation (Figure ), suggesting that the ubiquitination-associated proteasome is involved in this process. Therefore, in addition to cold-induced transcription, cold temperatures may also affect MdCIbHLH1 activity by modulating its protein abundance at a posttranslational level.
Figure 7 Cold-induced degradation and post-translational modification of MdCIbHLH1. (A) Detection of MdCIbHLH1-GFP protein by Western blot analysis. TGC calluses were maintained at 15°C for the cold treatment. MdCIbHLH1-GFP protein levels were analyzed (more ...)
It has been reported that AtICE1 protein degrades at low temperatures through a ubiquitin-proteasome pathway [30
]. In addition, sumoylation may function as a ubiquitin antagonist [31
]. To determine if MdCIbHLH1 protein is modified by ubiquitination or sumoylation, MdCIbHLH1-GFP protein was immunoprecipitated using anti-GFP antibody in the TGC line (Figure ). The precipitated proteins were detected with anti-ubiquitin and anti-SUMO antibodies to determine the occurrence of ubiquitination and sumoylation modifications, respectively. Positive signals were detected for both antibodies (Figure ), suggesting that ubiquitination and sumoylation of the MdCIbHLH1 protein occur in vivo in TGC calluses.