Cloning and functional analysis of PeNAC045 from Populus euphratica

NAC (NAM, ATAF1/2 and CUC2) domain proteins constitute one of the largest plant-specific transcription factors (TFs) and play an important role in regulating senescence, cell division, wood formation and biotic and abiotic stresses. In present study, we successfully isolated a stress responsive gene from Populus euphratica, i.e., PeNAC045. Sequencing results indicated that the length of PeNAC045 is 915 bp encoding 304 amino acids, and PeNAC045 shares 96.05% homology in amino acid sequence with PtrNAC045. The expression of PeNAC045 in response to NaCl and drought stress was characterized. PeNAC045 mRNA expression was strongly induced by high-salinity and drought treatment. The expression vector pBI121-PeNAC045-GFP was constructed using the full-length PeNAC045 cDNA cloned into the pBI121-GFP vector. After sequencing confirmation, the construct and positive control (empty vector) were transformed into Arabidopsis. Subcellular localization experiments in Arabidopsis indicated that the PeNAC045-GFP fusion protein was localized in the nucleus. Arabidopsis was stained with the DNA dye 4,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus. The expression vector pCAMBIA1301-PeNAC045 was constructed and transformed into Arabidopsis thaliana wild type (Col-0) and ataf2 mutant using floral dip method. Then, we obtained the PeNAC045 overexpression lines of Arabidopsis and ataf2/PeNAC045. To test the function of PeNAC045, each line was treated with NaCl. The germination percentage of PeNAC045 overexpression lines of Arabidopsis was reduced and the root length was shorter under 150 mmol/L NaCl stress, compared to ataf2 mutant and wild-type plants. Furthermore, the sensitivity of transgenic PeNAC045 overexpression lines to NaCl stress was increased at the seedling stage and the height of the seedlings was significantly lower than others. Our results indicated that PeNAC045, as a transcriptional regulator, negatively regulates the expression of stress responsive genes under NaCl stress.