Gene Cloning Of AtNAC091 Membrane-Associated Transcription Factor And Its Biological Function Under Stress

Adversity may cause a large number of unfolded or misfolded proteins to accumulate in endoplasmic reticulum(ER),which leads to endoplasmic reticulum stress(ER stress).Cells may alleviate the ER stress by activating the unfolded protein response(UPR).In tHis process,the membrane-associated transcription factor(MTF)plays a critical role.It has been found that 062,089 and 103 of NAC family are involved in ER stress pathway in Arabidopsis thaliana.These genes may affect plant life activities by regulating downstream genes.In tHis study,we identified a plasma membrane-associated transcription factor NAC091(also known as TIP),which plays an important role in ER stress and other abiotic pathways.In this work,1)qRT-PCR was used to verify that the expression level of NAC091 was up-regulated by ER stress;2)The up-regulated expression of NAC091 was depend on bZIP60.Meanwhile,a classical ERSE-I element was found on the promoter sequence of NAC091,which further suggested that NAC091 was involved in the ER stress pathway;3)Yeast two-hybridization and BiFC technology were used to detect the transcriptional activation activity of NAC091D(without the transmembrane domain);4)Confocal laser microscopy was used to observe the migration of NAC091 from plasma membrane to nucleus under ER stress.At the same time,the intracellular activation of NAC091 protein was detected by western blot;5)Under ER stress,over-expression of NAC091 D could improve the stress resistance of bzip28bzip60 double mutant;6)By RNA-Seq analysis,induction of NAC091 D expression can cause up-regulation of downstream transcription factors and enzymes related to ER stress pathway(PIF4,NAC094 and bHLH,etc);7)Removing NAC091 via CRISPR/Cas9 mutation will cause the down-regulation of some relevant transcription factors and enzymes;8)NAC091 was also up-regulated by NaCl,low temperature(4?)and ABA,indicating that NAC091 was also involved in other abiotic stress pathways.The results of tHis study showed that the membrane-associated transcription factor NAC091 in arabidopsis thaliana could transmit ER stress signal from plasma membrane to nucleus and play an important role in alleviating ER stress and promoting cell survival.