Isolation And Functional Characterization Of CBF And ICE Genes Response To Low Temperature In Dimocarpus Longan

Longan(Dimocarpus longan Lour.)is one of the most important economic fruit trees in South China.As a typical south subtropical fruit tree,longan is sensitive to low temperatures.Due to the main longan cultivar are very single,the mature stage of longan is very short in south China,and fresh fruit is vulnerable to storage at low temperatures,which often leads to seasonal oversupply and fruit overstock.Furthermore,frequently occurred cold extremes and frost weather in recent years affect longan yield and fruit quality,and even threaten the survival of longan trees.The above two problems have caused great economic losses in the development of longan industry.Therefore,there is an urgent need to develop cold-tolerant and high quality longan cultivars to reduce the damage to longan trees caused by low temperature,and extending the planting area to northward regions to prolong the mature stage and sale duration of longan fruits.However,due to limited understanding of the molecular mechanisms underlying the response of longan to low temperatures,little progress has been made in the breeding of cold-tolerant longan.Accumulating evidence have revealed that the ICE1-CBF regulatory pathway is the most important cold signaling pathway in plants.In this study,the main longan cultivar ‘shixia’ were used as plant material,the functional characteristics and transcriptional regulation mechanism of CBF and ICE1-like transcription factors in longan response to low temperature were discussed.The research results provide theoretical basis for understanding molecular mechanism of longan response to low temperature and benefit for longan cold-tolerant breeding.The main results are as follows:1.Three CBF(C-repeat-binding factor)genes were identified in D.longan by homologous searching and gene cloning,nominated DlCBF1,DlCBF2 and DlCBF3,respectively.The ORF(Open Reading Frame)of DlCBF1/2/3 was 687,726 and 636 bp.The sequence homology at the amino acid level of DlCBF1/2/3 were 38.59%～54.73%.Multiple sequence alignments showed that DlCBF1/2 containd AP2/ERF conserved domain and CBF characteristic sequences PKKP/RAGRx KFx ETRHP and DSAWR,as well as C-terminal LWSY/F conserved sequence,which indicated DlCBF1/2 are typical CBF transcription factors.However,the sequences located in DlCBF3 PKKP/R and DSAWR conserved domain were QKRK and EAASA sequences.Phylogenetic analysis showed that DlCBF1/2/3 is most closely related to Ct CBF of Citrus trifoliata,Pt CBF of Populus trichocarpa and Vr CBF of Vitis riparia,respectively.These results suggested that DlCBF1/2/3 might be involved in the cold stress response of longan.2.Expression analysis showed that DlCBF1/2 were expressed highest in young leaves and lowest in apical buds,while the expression of DlCBF3 was lower in all the tissues tested.The mature leaves were treatment in 4 °C for 3h,the expression of DlCBF1/2 in were significantly induced,then gradually increased to the peak value at 9h and then decreased.However,the expression of DlCBF3 was lower and the expression trend did not change significantly.DlCBF1/2/3 were mainly located in the nucleus,but a small amount of DlCBF1 also distributed in the cytoplasm.Transcriptional activity analysis indicated that DlCBF1/2/3 may potentially act as transcriptional activators.Studies on Arabidopsis genetic transformation demonstrated that overexpression of DlCBF1/2/3 enhance cold and freezing tolerance of transgenic plants by increasing proline content,decreasing ion leakage,reducing MDA and ROS accumulation and promoting the expression of COR(cold-responsive)genes(At RD29 A,At COR15 A,At COR47 and At KIN1).However,the freezing tolerance of DlCBF3-OE lines is lower than the DlCBF1/2-OE lines.These results indicated that DlCBF1/2/3 act as positive regulators in longan response to low temperature,and DlCBF1/2 may play more important role in longan response to cold stress.3.Two ICE(inducer of CBF expression)homologous genes DlICE1 and DlICE2 were identified in longan.The ORF of DlICE1/2 was 1602 and 1446 bp,respectively.The amino acid sequences homology of DlICE1/2 was 47.4%.Conserved domain analysis showed that DlICE1/2 contained a typical b HLH domain.Phylogenetic analysis indicated that DlICE1/2was closely related to Ct ICE1 of Citrus trifoliata and Va ICE1/2 of Vitis amurensis.DlICE1/2 were expressed at a lower level in all tissues tested,and the expression trend in the mature leaves did not change significantly after cold treatment at 4℃,suggesting that the post-transcriptional regulation may be more important for them function.DlICE1/2were located in the nucleus and have transcriptional activation capability.Overexpression of DlICE1 in Arabidopsis increased the proline content,decreased ion leakage,reduced MDA and ROS accumulation,and significantly induced the expression of CBF1/2/3 and downstream COR genes,thereby contributing to enhanced cold tolerance of transgenic plants.These findings indicate that DlICE1 positively regulates cold tolerance in longan.4.Promoters isolated and sequences analysis revealed that the DlCBF1/2/3 promoter including stress and hormone response related core cis-elements such as LTR and MYC motifs(response to cold),MYB and DRE motifs(response to drought),and ABRE motifs(response to ABA)and so on.The MYC motifs analysis showed that there are 2,1 and 1MYC motifs in the1000 bp promoter sequence of DlCBF1/2/3,respectively.However,the number of MYC motifs inn DlCBF3 promoter was significantly less than that of At CBF3.Additionally,the DlCBF1/2 promoter contains cold response elements LTR,while the LTR motifs was not exist in the DlCBF3 promoter,which are consistent with the expression trends of them response to cold stress.5.The results of the dual luciferase and Y1H(Yeast One-Hybrid)assays showed that DlICE1 bind to the DlCBF3 promoter and subsequently activate the reporter gene expressed,but not bind to the DlCBF1/2 promoter,indicating that DlICE1 specifically regulates the expression of DlCBF3.DlICE2 not bind to the DlCBF1/2/3 promoter,indicating that DlICE2 could not regulate the expression of DlCBF1/2/3 directly.In addition,the cis-acting element analysis showed that there is a CRT/DRE motif in DlCBF2 promoter.Dual luciferase and Y1 H assays proved that DlCBF1 bind to DlCBF2 promoter,indicating that DlCBF1 could regulate the expression of DlCBF2 in some degree.6.Sequences analysis of 16 longan CBF-regulated gene promoter sequences showed that there are 13 genes contain CRT/DRE motifs in the promoters of them,such as DlGols3,DlRD29 A and DlCOR47 and so on,but the number of CRT/DRE motifs in 10 genes among of them are significantly less than that of Arabidopsis homologous genes.These results indicated that these genes may be regulated by DlCBF1/2/3.Additionally,there aren’t CRT/DRE motifs in the promoters of DlKIN1,DlIP5 PII and DlMAP65-8 3 genes,indicating that they may not be regulated by DlCBF1/2/3.Therefore,it is speculated that the number of longan CBF-regulated genes are relatively reduced,and the CRT/DRE elements in promoters are also reduced,which leading to the regulatory function of DlCBF1/2/3 was weaken in longan response to low temperature.