The membrane-associated transcription factor NAC089 controls ER-stress-induced programmed cell death in plants.

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.

A plasma membrane-tethered transcription factor, NAC062/ANAC062/NTL6, mediates the unfolded protein response in Arabidopsis.

The 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.

Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling.

The lumen-facing domain is important for the biological function and organelle-to-organelle movement of bZIP28 during ER stress in Arabidopsis.

The membrane-associated transcription factor, bZIP28, is relocated from the endoplasmic reticulum (ER) to the Golgi and proteolytically released from the membrane mediated by two proteases, S1P and S2P, in response to ER stress in Arabidopsis. The activated N-terminal domain recruits nuclear factor Y (NF-Y) subunits in the nucleus to regulate ER stress downstream genes. Little is known about the functions of the bZIP28 C-terminal lumen-facing domain. Here, we provide novel insights into how the ER lumen-facing domain affects the biological function and organelle-to-organelle movement of bZIP28 in the ER stress response. First, we demonstrated the functional redundancy of bZIP28 and bZIP60 by generation and analysis of the bZIP28 and bZIP60 double mutant zip28zip60. Subsequent genetic complementation experiments in zip28zip60 background with deletions on bZIP28 lumen-facing domain highlighted the importance of lumen-facing domain for its in vivo function of bZIP28 in the ER stress response. The protein subcellular localization and Western blotting results further revealed that the bZIP28 lumen-facing domain contains ER retention signal which is important for the proteolytic activation of bZIP28. Thus, the bZIP28 lumen-facing C-terminus plays important roles in the ER-to-Golgi movement of bZIP28, which may contribute to the sensing of the ER stress.