Functional Characterization Of F-box-containing Gene AtPP2-B11 And Genome-wide Analysis Of The RING Finger Proteins In Apple

The ubiquitin (Ub)/26S pathway is a major system for protein degradation that is conserved in all eukaryotes cells. The specificity of ubiquitination is largely determined by the many isoforms of E3 that recruits specific target protein(s). In Arabidopsis thaliana more than 1400 genes (~5%of the proteome) encode subunits of the E3 ubiquitin ligases, which confer substrate specificity to the pathway. F-box and RING finger genes are the most common family genes in Arabidopsis. The fuction and structure analysis of F-box in Arabidopsis and RING finger proteins in apple provide valuable information for understanding the classification and putative functions of the E3 gene family in higher plants. The obtained results are as follows:(1) First, an F-box-containing gene was analyzed. Using bioinformatics approaches, 31 E3s containing DRE (dehydration responsive element) motif were isolated from 1488 E3s in Arabidopsis, in which an F-box gene (AtPP2-B11) were extremely induced by drought treatment.(2) GUS activity assay showed that the DRE motif in the promoter region of AtPP2-B11 played partial roles in drought response. AtPP2-B11 also showed slightly increased expression treated by ABA, indicating that AtPP2-B11 may respons to drought both in ABA-independent and ABA-dependent pathways.(3) Transgenic plants overexpressing AtPP2-B11 exhibited decreased drought tolerance. Quantitative RT-PCR showed that the expression levels of a number of key ABA and drought stress marker genes such as LEA14,COR15a,RD29A in transgeneic plants were altered in drought treatment.(4) Interactive proteins were screened from drought-treatment cDNA library with the full-length of AtPP2-B11 as bait protein by Yeast Two-Hybrid System. More than 80 positive clones were identified by PCR colony screening and the representative clones were sequenced. Among them 20 clones were selected which interacted with full-length AtPP2-B11. Further Yeast Two-Hybrid assay and Bimolecular Fluorescenc Complementation (BiFC) assay showed that AtPP2-B11 protein interacted with LEA14 protein, indicating that AtPP2-B11 gene may fuction as a novel drought tolerance repressor by regulating the level of LEA14.(5) In addition, we analyzed the RING finger proteins in apple genome which were recently secquenced. In our study, 688 RING domains in 663 predicted proteins were identified in apple. Based on the characteristics of RING domains, nine RING type were identified: 367 RING-H2, 208 RING-HC, 10 RING-C2, 35 RING-v, 1 RING-D, 11 RING-S/T, 2 RING-G, 10 RING-mH2 and 44 RING-mHC, in which the first seven types were decribed previously in Arabidopsis, while the latter two types were newly identified in apple. Proteins containing RING finger motifs were further classified into 57 groups according to the different known or unknown domains outside the RING domains.