Investigation On B-box Protein(BBX) Family In Apple And MdBBX10 In Response To Abiotic Stress

The B-box proteins?BBXs?are a family of proteins containing one/two B-box domain?s?,which belongs to super family of zinc finger proteins.Some plant BBXs have been demonstrated to act as transcription factors in the regulation of flowering and photomorphogenesis.The Arabidopsis BBX5 is recently reported to be involved in signaling pathways induced by abiotic and biotic stresses.A B-box protein encoding gene from apple was found to be up-regulated from gene expression profile under cold stress.In this study,all apple BBXs?MdBBXs?were identified through a genome-wide survey.Their chromosomal distributions,gene structures and expression patterns in different organs were investigated.The function domains of BBXs and crucial amino acid residues were also explored by segmental deletions and site-directed mutagenisis.By overexpression MdBBX10 in Arabidopsis,the function of Md BBXs in plants was studied.The main results obtained were showed as the following:?1?64 MdBBXs members were identified in apple genome.To identify the genes encoding BBXs in the apple genome,two different methods were used.Each potential candidate acquired by the above two methods was further examined the presence of B-box,by submitting them to InterPro and SMART databases,respectively.Finally,64 B-box containing proteins were identified and the encoding genes were named from MdBBX1 to MdBBX64,based on their chromosomal locations.Most MdBBX genes were tandem duplication genes or were located in homologous regions,suggesting that more BBX members in apple than those of other plants were due to gene tandem duplication,segmental duplication and genome-wide duplication in apple.?2?Phylogenetic tree was constructed to analyze the evolutionary relationship of MdBBXs.All 64 MdBBXs were divided into 5 groups and named as group I to V.Members in the three former groups?group I,II and III?contained B-box domain?s?and an additional domain,CCT domain,with two B-boxes in group I and II,and one B-box in group III.The majority of members in group IV and group V had no CCT domain with two and one B-box domain?s?,respectively.The motif logos of the B-box domain of apple BBXs were generated by submitting the sequences to the MEME website.The distribution of conservative amino acid residues in these two domains was similar but not identical.There were five conservative Cys residues related to zinc finger with four of them in the Cys-X-X-Cys motifs.?3?Stress-related cis-elements were widely distributed in the promoter of MdBBXs.To identify putative cis-acting elements related to stresses,the genomic sequence?1,500 bp?upstream of the MdBBXs 5'-UTRs were analyzed by submitting these promoter sequences to the PlantCARE database.Total of 48 promoters of 64 MdBBXs were found in the apple genome database and more than 30 promoters contained cis-acting elements related to stresses,including the MYB binding site?MBS?,heat stress response elements?HSE?,stress response elements?TC-rich repeats?,anaerobic response elements?ARE?and salicylic acid response elements?TCA-element?.The expressions of MdBBXs were up-regulated to some extent in the roots and leaves by qRT-PCR assay of 12 MdBBXs members from different groups.Six of them were sensitive to PEG,NaCl,cold and exogenous abscisic acid treatments,with their expressions enhanced more than 20-fold.The results suggested that MdBBXs may take part in response to abiotic stress.?4?Expression of MdBBX10 enhanced the resistance of E.coli cells to salts and osmolyte.The E.coli with recombinant MdBBX10 could survive when inoculated in solid or liquid medium with salt and osmolyte,respectively.Deficiency of B-box domain of MdBBX10 led the loss of anti-stress functions.Five conservative cysteines in B-box domain played crucial roles in stress resistance,which are involved in two of metal iron binding sites of zinc finger motifs in BBXs.However,the mutations of nonconservative-cysteines had similar activities of wild-type MdBBX10.Our results showed conservative cysteines are important for coordinating the zinc ions to keep the correct spatial structure of B-box domain.?5?The expression pattern of MdBBX10 in plants was analyzed.The promoter sequence of MdBBX10 was cloned to the vector?pBI121?before the reporter gene GUS,letting the expression of GUS regulated by the promoter of MdBBX10.The homozygous transgenic Arabidopsis of MdBBX10-pro were obtained.The results showed that the GUS signals mainly existed in the cells of rapidly growing tissues,like tips of young roots and leaves.Deeper staining of GUS was observed under H₂O₂,NaCl,PEG or ABA treatments,suggesting that MdBBX10 was responded to stresses in plants.?6?Overexpression of MdBBX10 in Arabidopsis enhanced plant tolerance to stresses.Homozygous plants of overexpressing Md BBX10 in Arabidopsis were obtained.Under normal conditions,no significant differences were observed in the growth among the wild-type?WT?and transgenic plants.However,on the medium with NaCl or mannitol,the OE lines germinated much earlier and showed significantly higher germination rates than the WT.When the plants were watered with 200mM NaCl at the seedling stage,the survival rate of OE plants was higher than that of WT plants.The expression of ABA-related gene was assayed as the key genes in ABA signal pathway.The expression of ABA1 and ABA2 in OE strains was higher than that of WT under stress treatmentes,which was consistent with the more sensitivity of OE strain to exogenous ABA.Similarly,the expressions of the active oxygen scavenging enzyme genes?SOD,POD and GST?were significantly higher than that of WT,while the contents of active oxygen species were significantly lower than that of WT.The results showed that MdBBX10 may participate in stress response through ABA signaling.