Construction Of The TF-centered Y1H System And Functional Characterization Of A NAC Gene From Tamarix Hispida

The interaction between a protein and DNA is involved in almost all cellular functions and cellular processes.In the present,two complementary approaches are used to detect the interactions between a transcription factor(TF)and DNA,i.e.the TF-centered or protein-DNA approach,and the gene-centered or DNA-protein approach.We developed a protein-DNA method based on the yeast one-hybrid(Y1H)system to identify the motifs recognized by a defined TF,termed TF-centered Y1H.In this system,a random short DNA sequence insertion library was generated as the prey DNA sequences to interact with a defined TF as the bait.We identified six motifs that were specifically bound by AtbZIP53 using this system,including a novel motif BRS1(bZIP recognized site 1)and five known motifs(Dof,G-box,I-box,BS1,and MY3).The different subfamily bZIP members also recognize these six motifs,further confirming the reliability of the TF-centered Y1H results.Taken together,these results demonstrated that TF-centered Y1H could identify quickly the motifs bound by a defined TF,representing a reliable and efficient approach with the advantages of Y1H.Therefore,this TF-centered Y1H may have a wide application in protein-DNA interaction studies.NAC(NAM,ATAF1/2 and CUC2)domain proteins are plant-specific transcriptional regulators that play roles in diverse developmental processes,defense responses and signal transduction,and are important for drought and stress tolerances.We previously built seven transcriptomes from T.hispida and search the functional annotation of non-redundant unigenes.A total of 89 unigenes were identified as NAC family genes,including 21 NAC genes with full open reading frames(designed as ThNAC1-ThNAC21).Sequential analysis showed that most of these NACs contained a highly conserved N-terminal DNA-binding domain and a highly variable C-terminal transcriptional regulation domain.We speculated on the putative functions of the ThNAC proteins based on the evolutionary relationships and the molecular functions of their Arabidopsis closest homologues,especially the stress-responsive NAC proteins.Real-time RT-PCR showed that these ThNAC genes are all differentially expressed in the roots,stems,and leaves of T.hispida in response to salinity,drought,heavy metal stresses and abscisic acid(ABA)treatment,indicating that they play regulatory roles in the abiotic stress response and are involved in ABA-dependent stress-signaling pathways.According to the results of the molecular characterization and the transcription profilings of the 21 ThNAC genes under different abiotic stresses,we speculated on the ThNAC 13 gene may be important for the abiotic stress tolerance in T.hispida plants.Therefore,we firstly cloned the ThNAC1 3 gene and found that ThNAC 13 protein was localized in the nucleus.Transactivation assays demonstrated that ThNAC 13 functioned as a transcriptional activator using yeast two-hybrid system.Furthermore,domain deletion analysis revealed that the ThNAC13 transactivation activity was conferred by its C-terminal domain,located at 227-373 aa.Y1H assays showed that ThNAC13 can bind to the NAC recognition site(NACRS)elements with the core DNA sequence "CGT[G/A]",and we identified eight motifs that were specifically bound by ThNAC13 using the TF-centered Y1H system.Overexpression of ThNAC13 in Arabidopsis showed increased seed germination rate,fresh weight and root growth compared with wild-type(WT)plants under salt,drought and ABA stress conditions.Histochemistry stain and physiological analyses suggested that overexpression of ThNACl3 in Arabidopsis and transgenic T.hispida plants showed significantly increased the reactive oxygen species(ROS)scavenging capability,decreased cell membrane damage,which dramatically improved the abiotic stress tolerance in plants.