Endoplasmic reticulum (ER) stress is of considerable interest to plant biologists because it occurs in plants subjected to adverse environmental conditions. ER stress responses mitigate the damage caused by stress and confer levels of stress tolerance to plants. ER stress is activated by misfolded proteins that accumulate in the ER under adverse environmental conditions. Under these conditions, the demand for protein folding exceeds the capacity of the system, which sets off the unfolded protein response (UPR). Two arms of the UPR signaling pathway have been described in plants: one that involves two ER membrane–associated transcription factors (bZIP17 and bZIP28) and another that involves a dual protein kinase (RNA-splicing factor IRE1) and its target RNA (bZIP60). Under mild or short-term stress conditions, signaling from IRE1 activates autophagy, a cell survival response. But under severe or chronic stress conditions, ER stress can lead to cell death.

The endoplasmic reticulum (ER) has a sophisticated quality control (QC) system to eliminate improperly folded proteins from the secretory pathway. Given that protein folding is such a fastidious process and subject to adverse environmental conditions, the ER QC system appears to have been usurped to serve as an environmental sensor and responder in plants. Under stressful conditions, the ER protein folding machinery reaches a limit as the demands for protein folding exceed the capacity of the system. Under these conditions, misfolded or unfolded proteins accumulate in the ER, triggering an unfolded protein response (UPR). UPR mitigates ER stress by upregulating the expression of genes encoding components of the protein folding machinery or the ER-associated degradation system. In Arabidopsis thaliana, ER stress is sensed and stress signals are transduced by membrane-bound transcription factors, which are activated and mobilized under environmental stress conditions. Under acute or chronic stress conditions, UPR can also lead to apoptosis or programmed cell death. Despite recent progress in our understanding of plant protein QC, discovering how different environmental conditions are perceived is one of the major challenges in understanding this system. Since the ER QC system is one among many stress response systems in plants, another major challenge is determining the extent to which the ER QC system contributes to various stress responses in plants.

Adverse environmental conditions produce endoplasmic reticulum (ER) stress in plants. In response to heat or ER stress agents,Arabidopsisseedlings mitigate stress damage by activating ER-associated transcription factors and a RNA splicing factor, IRE1b. IRE1b splices the mRNA-encoding bZIP60, a basic leucine-zipper domain containing transcription factor associated with the unfolded protein response in plants. bZIP60 is required for the up-regulation ofBINDING PROTEIN3(BIP3) in response to ER stress, and loss-of-function mutations inIRE1bor point mutations in the splicing site ofbZIP60mRNA are defective inBIP3induction. These findings demonstrate thatbZIP60in plants is activated by RNA splicing and afford opportunities for monitoring and modulating stress responses in plants.

Summary: We describe a signaling pathway that mediates salt stress responses in Arabidopsis. The response is mechanistically related to endoplasmic reticulum (ER) stress responses described in mammalian systems. Such responses involve processing and relocation to the nucleus of ER membrane‐associated transcription factors to activate stress response genes. The salt stress response in Arabidopsis requires a subtilisin‐like serine protease (AtS1P), related to mammalian S1P and a membrane‐localized b‐ZIP transcription factor, AtbZIP17, a predicted type‐II membrane protein with a canonical S1P cleavage site on its lumen‐facing side and a b‐ZIP domain on its cytoplasmic side. In response to salt stress, it was found that myc‐tagged AtbZIP17 was cleaved in an AtS1P‐dependent process. To show that AtS1P directly targets AtbZIP17, cleavage was also demonstrated in an in vitro pull‐down assay with agarose bead‐immobilized AtS1P. Under salt stress conditions, the N‐terminal fragment of AtbZIP17 tagged with GFP was translocated to the nucleus. The N‐terminal fragment bearing the bZIP DNA binding domain was also found to possess transcriptional activity that functions in yeast. In Arabidopsis, AtbZIP17 activation directly or indirectly upregulated the expression of several salt stress response genes, including the homeodomain transcription factor ATHB‐7. Upregulation of these genes by salt stress was blocked by T‐DNA insertion mutations in AtS1P and AtbZIP17. Thus, salt stress induces a signaling cascade involving the processing of AtbZIP17, its translocation to the nucleus and the upregulation of salt stress genes.

Stresses leading to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) elicit a highly conserved ER stress response in plants called the unfolded protein response (UPR). While the response itself is well documented in plants, the components of the signaling pathway are less well known. We have identified three membrane-associated basic domain/leucine zipper (bZIP) factors in Arabidopsis thaliana that are candidates for ER stress sensors/transducers. One of these factors, bZIP28, an ER-resident transcription factor, is activated in response to treatment by tunicamycin (TM), an agent that blocks N-linked protein glycosylation. Following TM treatment, bZIP28 is processed, releasing its N-terminal, cytoplasm-facing domain, which is translocated to the nucleus. Expression of a truncated form of bZIP28, containing only the cytoplasmic domain of the protein, upregulated the expression of ER stress response genes in the absence of stress conditions. Thus, bZIP28 serves as a sensor/transducer in Arabidopsis to mediate ER stress responses related to UPR.

Stress agents known to elicit the unfolded protein response in Arabidopsis thaliana upregulate the expression of a constellation of genes dependent on the membrane-associated basic domain/leucine zipper (bZIP) transcription factor, bZIP28. Among the stress-activated genes, a consensus promoter sequence corresponding to the endoplasmic reticulum (ER) stress-responsive element I (ERSE-I), CCAAT-N10-CACG, was identified. Disruption of either the CCAAT or CACG subelement in ERSE-I resulted in reduction of the transcriptional response to ER stress. bZIP28 forms homo- and heterodimers with other bZIP TF family members (in subgroup D) and interacts with CCAAT box binding factors, heterotrimeric factors composed of NF-Y subunits. Arabidopsis encodes 36 NF-Y subunits, and it was found that subunits NF-YB3 and -YC2 interact with bZIP28 and NF-YA4, respectively, in a yeast three-hybrid system. A transcriptional complex containing bZIP28 and the above-mentioned three NF-Y subunits was assembled in vitro on DNA containing ERSE-I. bZIP28, on its own, binds to the CACG subelement in ERSE-I to form a smaller complex I, and in combination with the NF-Y subunits above, bZIP28 assembles into a larger transcriptional complex (complex II). bZIP28 was shown to interact with NF-Y subunits in vivo in bimolecular fluorescence complementation analyses and in coimmunoprecipitation assays. Treatment of seedlings with ER stress agents led to the upregulation of NF-YC2 and the relocation of NF-YB3 from the cytoplasm to the nucleus. Thus, in response to ER stress, bZIP28 is mobilized by proteolysis and recruits NF-Y subunits to form a transcriptional complex that upregulates the expression of ER stress-induced genes.