| Apple is one of the most economically important perennial fruit crops globally and has high nutritional value.With the degradation of the environment,the production and the quality improvement of apple are limited by a range of biotic and abiotic stressful factors.To optimize their growth and survival,transcription factors(TFs)play crucial roles in plant response to those stresses by activating or repressing stress-related downstream genes expression.The bHLH(basic helix-loop-helix)superfamily is the second largest TF family in plants,its member has been found in all three eukaryotic kingdoms.As the conversed structures,bHLH proteins participate in a variety of physiological processes by homo-or heterologous dimers.Researches have shown that bHLH proteins were involved in many development processes,for example,regulating the development of flower,fruit,trichrome and root,the formation of porosity and shoot differentiation.As well as related to the signal and hormones regulating processes.As more and more bHLH proteins identified and functionally characterized,it showed an important role of these genes in regulating stress resistance in plant.In this study,we identified the apple bHLH genes and analyzed the evolutionary relationship by bioinformatics approaches.Then we detected the expression patterns of MdbHLHs in different development stages of structures/organs and after the treatment of salt or various phytohormones by PCR technology.The results are as follow:1.We identified 175 MdbHLH genes in the wide-genome of apple.Based on a multiple sequence alignment the proteins were named sequentially as MdbHLH001 to MdbHLH175,based on the equivalent classification in Arabidopsis and rice.2.The phylogenetic relationship between apple and Arabidopsis bHLH transcription factors showed that apple bHLH genes were devided into 23 subgroups,the members of each subgroup had similar motifs and exon/nitrox structures,supporting the phylogenetic relationships within bHLH gene family.3.Synteny analysis indicated that 66 of the 175(38%)apple bHLH genes were associated with either tandem or segmental duplication events,suggesting segmental and tandem duplications played a key role in the expansion of the apple bHLH family.4.We found that the selected genes belonging to subgroups III and IV exhibited diverse transcription profiles in all 10 organs/tissues examined,for example,MdbHLH012 was preferentially expressed in roots,MdbHLH038 and MdbHLH039 were highly expressed in young leaves,and MdbHLH021 was predominantly expressed in young fruit.MdbHLH015 and MdbHLH002 were preferentially expressed in flower buds,and MdbHLH047 and MdbHLH048 were preferentially expressed in mature leaves,indicating distinct functions in apple development.5.After the treatment of high salt,most of subgroups III and IV MdbHLHs were up-regulated,to varying degrees,except that MdbHLH020 showed clear down-regulation suggesting these genes play an important role in the process of resistance to salt stress.6.After the treatments of ABA,MeJA,SA,GAs and Eth,Among the 19 targeted genes,14 were up-regulated(defined as Log2-fold increase of at least 2.0 in all three biological replicates)by the ABA treatment;after MeJA treatment,13 genes were up-regulated;after SA treatment,one gene(MdbHLH050)was down-regulated,and 15 were up-regulated;after GA treatment,two genes(MdbHLH012 and MdbHLH020)were down-regulated,and 14 were up-regulated;after Eth treatment,four genes(MdbHLH003,MdbHLH004,MdbHLH015 and MdbHLH017)were down-regulated,and 12 were up-regulated,indicating that the regulatory role of bHLH proteins under different stresses is complicated. |
