To Investigate the Relationship between Substrates of the N-end Rule Pathway and Genes Regulated by EBP Binding Sites (GCCGCC) in Arabidopsis thaliana

AbstractThe N-end rule pathway is a highly conserved mechanism present in all eukaryotes. It targets specific proteins based on the recognition of the substrate's N-terminal amino acid by E3-ligase for degradation via ubiquitin-proteasome pathway in the cell. In A.thaliana, the Group VII ERFs transcription factors are substrates of N-end rule pathway that begin with ‘Met-Cys. In presence of oxygen and nitric oxide, cysteine is oxidized and arginylated and recognized by the E3-ligase PROTEOLYSIS 6 (PRT6). Previous studies on prt6 mutant endosperm containing promABI5::GUS showed that the ABI5 5’UTR region that contains 2 EBP boxes (GCCGCC) mediate regulation via Group VII ERFs. Whereas, expression was inhibited when two EBP boxes were absent. Hence, suggesting that genes containing EBP boxes as similar as ABI5 might also be regulated through the N-end rule pathway.In this project fourteen genes containing two copies of GCCGCC in the 5’UTR/promoter were identified as potential candidate genes to be regulated by Group VII ERFs, similarly to ABI5. Successful amplification of the promoter region of five of these genes [AT4G01026 (PYL7), AT1G14810, AT3G54510 (ERD4), At3G13440 and AT5G44420 (PDF1.2)] was followed by cloning into a GUS-reporter plasmid and transformation into Arabidopsis wild type (Col-0) and prt6-1 plants. Leaves and flowers of transgenic plants were analysed by GUS staining to reveal promoter activity. This analysis has shown that these promoters enhanced GUS expression in prt6 in comparison to Col-0, suggesting that genes are regulated by the Group VII ERFs.Analysis of in-vivo binding of ERFs to selected promoters was done by Chromatin Immuno- precipitation (ChIP). To confirm the hypothesis that promoters carrying GCCGCC binding sites are bound in-vivo by Group VII ERFs. Future work involves analysis of mutant combinations of prt6 with genes such as PYL7 and ERD4 for ABA assay