The unfolded protein response (UPR) is activated to sustain cell survival by reducing misfolded protein accumulation in the endoplasmic reticulum (ER). The UPR also promotes programmed cell death (PCD) when the ER stress is severe; however, the underlying molecular mechanisms are less understood, especially in plants. Previously, two membrane-associated transcriptions factors (MTFs), bZIP28 and bZIP60, were identified as the key regulators for cell survival in the plant ER stress response. Here, we report the identification of another MTF, NAC089, as an important PCD regulator in Arabidopsis (Arabidopsis thaliana) plants. NAC089 relocates from the ER membrane to the nucleus under ER stress conditions. Inducible expression of a truncated form of NAC089, in which the transmembrane domain is deleted, induces PCD with increased caspase 3/7-like activity and DNA fragmentation. Knock-down NAC089 in Arabidopsis confers ER stress tolerance and impairs ER-stress-induced caspase-like activity. Transcriptional regulation analysis and ChIP-qPCR reveal that NAC089 plays important role in regulating downstream genes involved in PCD, such as NAC094, MC5 and BAG6. Furthermore, NAC089 is up-regulated by ER stress, which is directly controlled by bZIP28 and bZIP60. These results show that nuclear relocation of NAC089 promotes ER-stress-induced PCD, and both pro-survival and pro-death signals are elicited by bZIP28 and bZIP60 during plant ER stress response.

SUMMARYThe unfolded protein response (UPR) plays important roles in plant development and plant-pathogen interactions, as well as in plant adaptation to adverse environmental stresses. Previously bZIP28 and bZIP60 have been identified as important UPR regulators for mitigating the endoplasmic reticulum (ER) stress in Arabidopsis thaliana. Here we report the biological function of NAC103 in a novel transcriptional regulatory cascade, connecting bZIP60 to the UPR downstream genes in Arabidopsis. Expression of NAC103 was induced by ER stress, and was completely abolished in the bZIP60 null mutant. A new ER stress-responsive cis-element UPRE-III (TCATCG) on the NAC103 promoter was identified, and trans-activation of UPRE-III by bZIP60 was confirmed in both yeast cells and Arabidopsis protoplasts. The direct binding of bZIP60 to UPRE-III-containing DNA was also demonstrated in an electrophoretic mobility shift assay. NAC103 formed homodimers in yeast two-hybrid and bimolecular fluorescence complementation assays. It had transcriptional activation activity and was localized in the nucleus. Over-expression of NAC103 had pleiotropic effects on plant growth, and induced expression of several UPR downstream genes in Arabidopsis under normal growth conditions. The activation of UPR gene promoters by NAC103 was also confirmed in effector/reporter protoplast assays. Thus, our study demonstrates a transcriptional regulatory cascade in which NAC103 relays ER stress signals from bZIP60 to UPR downstream genes through a newly identified ER stress cis-element (UPRE-III) and transcriptional activation activity of its encoded protein NAC103.

SUMMARYThe accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) triggers a well conserved pathway called the unfolded protein response (UPR) in eukaryotic cells to mitigate ER stress. Two signaling pathways, S2P-bZIP28 and IRE1-bZIP60, play important roles in transmitting ER stress signals from the ER to the nucleus in Arabidopsis (Arabidopsis thaliana). It is not known whether other components in the secretory pathway also contribute to the alleviation of ER stress. Here we report the identification of a plasma membrane-associated transcription factor, NAC062 (also known as ANAC062/NTL6), as another important UPR mediator in Arabidopsis plants. NAC062 relocates from the plasma membrane to the nucleus and regulates the expression of ER stress responsive genes in Arabidopsis. Knock-down of NAC062 in the wild-type background confers ER stress sensitivity, while inducible expression of a nucleus-localized form of NAC062, NAC062D, in the bZIP28 and bZIP60 double mutant (zip28zip60) background increases ER stress tolerance. Knock-down of NAC062 impairs ER-stress-induced expression of UPR downstream genes while over-expression of NAC062D-MYC induces the expression of UPR downstream genes under normal growth condition. CHIP-qPCR reveals that NAC062D-MYC is enriched at the promoter regions of several UPR downstream genes such as BiP2. Furthermore, NAC062 itself is also up-regulated by ER stress, which is dependent on bZIP60 but not on bZIP28. Thus, our results have uncovered an alternative UPR pathway in plants in which the membrane-associated transcription factor NAC062 relays ER stress signaling from the plasma membrane to the nucleus and plays important roles in regulating UPR downstream gene expression.

Highlights d BBX18 and BBX23 positively regulate temperature-induced hypocotyl elongation d PIF4 is required for BBX18-mediated plant thermomorphogenesis d BBX18 and BBX23 interact with ELF3 and negatively control ELF3 protein accumulation d BBX18 and BBX23 protein levels are increased in response to elevated ambient temperature